T cell receptor-directed antibody-drug conjugates for the treatment of T cell-derived cancers

Abstract

T cell-derived cancers are hallmarked by heterogeneity, aggressiveness, and poor clinical outcomes. Available targeted therapies are severely limited due to a lack of target antigens that allow discrimination of malignant from healthy T cells. Here, we report a novel approach for the treatment of T cell diseases based on targeting the clonally rearranged T cell receptor displayed by the cancerous T cell population. As a proof of concept, we identified an antibody with unique specificity toward a distinct T cell receptor (TCR) and developed antibody-drug conjugates, precisely recognizing and eliminating target T cells while preserving overall T cell repertoire integrity and cellular immunity. Our anti-TCR antibody-drug conjugates demonstrated effective receptor-mediated cell internalization, associated with induction of cancer cell death with strong signs of apoptosis. Furthermore, cell proliferation-inhibiting bystander effects observed on target-negative cells may contribute to the molecules' anti-tumor properties precluding potential tumor escape mechanisms. To our knowledge, this represents the first anti-TCR antibody-drug conjugate designed as custom-tailored immunotherapy for T cell-driven pathologies.

Keywords: T cell leukemia; T cell lymphoma; T cell receptor; antibody-drug conjugate; idiotype; targeted therapy; yeast surface display.

Graphical abstract

Figure 1

Concept overview Identification and expression of tumor-specific TCR was followed by chicken immunization. Afterward, splenic RNA was isolated, and cDNA was synthesized. Genes encoding the variable antibody domains were amplified and assembled as scFv gene string for yeast surface display and subsequent screening by FACS. TCR-binding scFvs were cloned into bacterial and mammalian vectors for scFv and scFv-Fc expression, respectively. After selection of a lead candidate, the TCR idiotype-specific binder was reformatted as full-length antibody and subsequently characterized. Generation of ADCs by antibody MMAE conjugation was conducted by two different approaches resulting in DARs of 2 and 2–8, respectively. Created with BioRender.com.

Figure 2

Screening of scFv immune library for TCRA6 binders by FACS Sorting rounds 1–3 of the chicken-derived yeast surface-displayed scFv library are depicted in green with respective TCRA6 concentrations and percentages of cells in sorting gates. Negative controls without antigen incubation are shown in gray. Surface presentation was detected by anti-c-myc antibody FITC conjugate, and antigen binding was analyzed using DyLight650-labeled TCRA6. Density plots were created by FlowJo v10 Software.

Figure 3

Binding properties of aTCRA6 scFv-Fc/Fab-Fc (A) Binding kinetics. BLI measurement of aTCRA6 scFv-Fc (left) and aTCRA6 Fab-Fc (right) loaded onto AHC biosensor tips and associated with 3.9–62.5 nM TCRA6. Kinetic parameters were estimated using Savitzky-Golay filtering and 1:1 Langmuir modeling. (B) Cellular binding. Flow cytometry analysis of Jurkat TCRA6 target cells and Jurkat WT off-target T cells incubated with varying concentrations of aTCRA6 scFv-Fc (1–1,000 nM; left) or aTCRA6 Fab-Fc (0.5–500 nM; right) and stained via anti-human IgG Fc-PE secondary detection antibody. On-cell K_Ds were determined using variable slope four-parameter fit. Results are shown as mean RFU, and error bars represent standard deviation derived from experimental triplicates. Data are representative of three independent experiments.

Figure 4

Internalization and cytotoxicity of aTCRA6 ADC variants (A) Schematic representation of aTCRA6 ADCs. In aTCRA6 DAR2 (left), the anti-TCRA6 heavy chains are C-terminally modified with DBCO-PEG₄-Val-Cit-PAB-MMAE. In aTCRA6 DAR5_2-8 (right), the interchain cysteines of anti-TCRA6 mAb are conjugated to maleimide-PEG₄-Val-Cit-PAB-MMAE. DBCO, dibenzocyclooctyne; PEG, polyethylene glycol; Val, valine, Cit, citrulline; PAB, p-aminobenzyl alcohol; MMAE, monomethyl auristatin E. (B) Cytotoxicity was assessed by exposition of Jurkat TCRA6 target cells and Jurkat WT off-target cells to aTCRA6 DAR2 (0.06–400 nM) and aTCRA6 DAR5_2-8 (0.005–30 nM) for 72 h. Cell proliferation was normalized to untreated control cells (0 nM). Internalization was studied by application of pHAb-conjugated aTCRA6 antibody in varying concentrations (0.04–250 nM) to Jurkat TCRA6 target cells and Jurkat WT off-target cells and incubation overnight. Fold internalization was calculated by the ratio of relative fluorescence units (RFU) of the respective antibody sample and the untreated sample without antibody (0 nM). EC₅₀s were determined using variable slope four-parameter fit. Results are shown as mean, and error bars represent standard deviation derived from experimental triplicates. Data are representative of at least two independent experiments.

Figure 5

Apoptosis induction of aTCRA6 ADC variants Jurkat TCRA6 target cells and Jurkat WT off-target cells were exposed to 300 nM aTCRA6 DAR2 and 30 nM aTCRA6 DAR5_2-8 for 72 h. Cells were stained with annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry. Percentage of annexin V-FITC+/PI+ and annexin V-FITC+/PI− (apoptotic cells) is depicted in right bar chart. Significant differences (p < 0.05) between ADCs and 0 nM control were determined using an unpaired, two-tailed t test and are depicted by ∗ (0.1234 (ns), 0.0332 (∗), 0.0021 (∗∗), 0.0002 (∗∗∗), and <0.0001 (∗∗∗∗)). Results are shown as mean, and error bars represent standard deviation derived from experimental triplicates. Data are representative of two independent experiments.

Figure 6

Bystander activity of aTCRA6 ADC variants (A) Direct killing was determined by exposition of Jurkat TCRA6 target cells to aTCRA6 DAR2 (0.05–300 nM) and aTCRA6 DAR5_2-8 (0.005–30 nM) for 96 h. (B) Supernatants of pre-treated Jurkat TCRA6 target cells were transferred to freshly seeded Jurkat WT off-target cells in a ratio of 1:1 and incubated for 96 h. Results are shown as mean, and error bars represent standard deviation derived from experimental duplicates. Data are representative of at least two independent experiments.