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Updated: May 25, 2026
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
A recombinant triplebody with specificity for CD19 and HLA-DR mediates preferential binding to antigen
Ingo Schubert1, Christian Kellner, Christoph Stein
1Department of Biology, University of Erlangen-Nuremberg, Erlangen, Germany. ischuber@biologie.uni-erlangen.de
Abstract:
To test the hypothesis that dual-targeting confers the novel ability of selective binding to antigen double-positive over antigen single-positive cells, a single-chain triplebody (sctb), HLA-ds16-hu19, was produced and characterized. The molecule carries three single-chain Fv (scFv) antibody fragments in a single polypeptide chain, the two distal ones specific for the human histocompatibility protein HLA-DR and the B-lymphoid cell surface protein CD19, the central one for CD16, the human low affinity Fc-receptor FcγRIII. For comparison, the bispecific scFvs (bsscFv) hu19-ds16 and HLA-ds16 were also produced. All CD16 binding modules are disulfide-stabilized (ds). The sctb bound simultaneously to both CD19 and HLA-DR on the same cancer cell and, thus, showed functional dual-targeting. In a mixing-experiment with HLA-DR single-positive HUT-78 cells and (HLA-DR plus CD19) double-positive SEM cells, the triplebody showed preferential binding to the double-positive cells, even when the single-positive cells were present in a numerical excess of up to 20-fold. In antibody-dependent cellular cytotoxicity experiments with mononuclear cells as effector cells, the sctb promoted equal lysis of Raji cells, an antigen double-positive cell line, at 130-fold lower concentrations than the bsscFv hu19-ds16, indicating that both distal scFvs of the sctb contributed to tumor cell lysis. A panel of stably-transfected HEK293 cell lines was generated that included CD19- and HLA-DR single-positive and (HLA-DR plus CD19) double-positive lines with antigen-surface densities varying over a broad range. Using a pair of cell lines with matching densities, the sctb eliminated double-positive target cells preferentially single-positive cells. This ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting sctbs in cancer therapy.
Insights
Dual-targeting single-chain triplebodies (sctbs) selectively bind antigen double-positive cancer cells over single-positive cells. This dual-targeting approach demonstrates enhanced efficacy in antibody-dependent cellular cytotoxicity, offering new cancer therapy perspectives.
Area of Science:
- Immunology
- Biotechnology
- Cancer Research
Background:
- Developing targeted cancer therapies requires strategies to specifically eliminate malignant cells while sparing healthy ones.
- Dual-targeting molecules offer potential for enhanced specificity by engaging multiple antigens simultaneously.
- Single-chain variable fragment (scFv) engineering allows for the construction of complex multivalent antibody formats.
Purpose of the Study:
- To investigate the hypothesis that dual-targeting confers selective binding to antigen double-positive cells over antigen single-positive cells.
- To characterize a novel single-chain triplebody (sctb) designed for dual-targeting of cancer cells.
- To evaluate the therapeutic potential of this sctb in preclinical models.
Main Methods:
- Production and characterization of a single-chain triplebody (sctb) with specificity for HLA-DR, CD19, and CD16.
- Comparative analysis using bispecific scFvs (bsscFvs) and the sctb in binding assays.
- Functional assessment through antibody-dependent cellular cytotoxicity (ADCC) experiments.
- Evaluation in mixed cell populations and engineered cell lines with varying antigen expression.
Main Results:
- The sctb demonstrated simultaneous binding to both CD19 and HLA-DR on cancer cells, confirming functional dual-targeting.
- In mixing experiments, the sctb preferentially bound to double-positive cells even when single-positive cells were in 20-fold excess.
- The sctb mediated efficient antibody-dependent cellular cytotoxicity (ADCC) against double-positive target cells at significantly lower concentrations compared to bsscFvs.
- Engineered cell lines confirmed the sctb's ability to preferentially eliminate double-positive cells over single-positive cells.
Conclusions:
- Dual-targeting single-chain triplebodies exhibit selective binding and enhanced efficacy against antigen double-positive cancer cells.
- This selective targeting capability offers promising new avenues for developing more effective and specific cancer immunotherapies.
- The sctb design represents a significant advancement in engineering multivalent antibodies for therapeutic applications.
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