Patient-Specific Targeting of the T-Cell Receptor Variable Region as a Therapeutic Strategy in Clonal T-Cell Diseases

Abstract

Purpose: Targeted therapeutics are a goal of medicine. Methods for targeting T-cell lymphoma lack specificity for the malignant cell, leading to elimination of healthy cells. The T-cell receptor (TCR) is designed for antigen recognition. T-cell malignancies expand from a single clone that expresses one of 48 TCR variable beta (Vβ) genes, providing a distinct therapeutic target. We hypothesized that a mAb that is exclusive to a specific Vβ would eliminate the malignant clone while having minimal effects on healthy T cells.

Experimental design: We identified a patient with large granular T-cell leukemia and sequenced his circulating T-cell population, 95% of which expressed Vβ13.3. We developed a panel of anti-Vβ13.3 antibodies to test for binding and elimination of the malignant T-cell clone.

Results: Therapeutic antibody candidates bound the malignant clone with high affinity. Antibodies killed engineered cell lines expressing the patient TCR Vβ13.3 by antibody-dependent cellular cytotoxicity and TCR-mediated activation-induced cell death, and exhibited specific killing of patient malignant T cells in combination with exogenous natural killer cells. EL4 cells expressing the patient's TCR Vβ13.3 were also killed by antibody administration in an in vivo murine model.

Conclusions: This approach serves as an outline for development of therapeutics that can treat clonal T-cell-based malignancies and potentially other T-cell-mediated diseases. See related commentary by Varma and Diefenbach, p. 4024.

Figure 1.. Rapid Generation of αVβ13 antibodies.

(A) Patient J’s T-cells were sequenced and Vβ13.3 expression was identified on the malignant T-LGL clone. (B) A protein antigen composed of the extracellular domain of patient J’s Vβ13.3 chain fused to mouse IgG1 Fc (mFc) was used for mouse immunization to obtain αV13 antibody candidates for screening. (C) ELISA binding to patient Vβ13.3, a CDR3-scrambled mutant (Mt), and a control antigen (Ctrl_Ag) was used to identify αVβ13 and anti-idiotype antibodies. (D) Flow cytometry of αVβ13A binding (red population) of the T-LGL clone characterized by CD3⁺CD8⁺CD4⁻CD2^dimCD16^variableCD56⁻ immunophenotype. (E) EL4 and Jurkat T3.5 cell lines were transduced to express Patient J’s Vβ13.3, titled EL4-V13 and Jurkat-V13, respectively. (F) αVβ13A was generated in either human IgG1 (αVβ13A) or murine IgG2a (αVβ13A-mIgG2a). Flow cytometry of αVβ13A binding to Jurkat T3.5 and αVβ13A-mIgG2a binding to EL4 are shown in grey peaks. Binding of αVβ13A to Jurkat-V13 and αVβ13A-mIgG2a to EL4-V13 are shown in black peaks.

Figure 2.. αVβ monoclonal antibodies bind with specificity to the target T-cell clone.

(A) Binding of αVβ candidates to a proportion of healthy control (HC; n=3) CD3+ T-cells consistent with a diverse T-cell repertoire. (B) Schematic of αVβ antibodies binding the endogenous TCR Vβ on target T-cell lines. (C) Flow histograms illustrating binding of αVβ antibodies to target cell line (black peak) compared to isotype control (grey peak).

Figure 3.. αVβ monoclonal antibodies induce antibody-dependent cellular cytotoxicity (ADCC) in target T-cell clone.

(A) Patient primary T-LGL cells undergo increasing cell death when incubated with the high affinity binders αVβ13A, αVβ13E and αVβ13D and varying ratios of haNK NK-92 cells expressing CD16 in a 4-hour ADCC assay. (B-C) The TCR null Jurkat T3.5 line transduced with Vβ13.3 (Jurkat-V13) and EL-4 murine NK lymphoma line (EL4-V13) undergo cell death when incubated with αVβ13A. Wild-type (WT) lines have no significant cell death relative to isotype control. (D-E) Jurkat, HPB-ALL undergo increasing cell death when incubated with αVβ8 and αVβ5, respectively, at varying ratios of effector cells. (F-H) Healthy control CD3+ T-cells incubated with αVβ5 and αVβ8 and increasing ratios of hANK cells induce cytotoxicity of the target cell population relative to isotype control, as detected by cytosolic Vβ. (I) haNK cells incubated with target cell lines and corresponding αVβ antibodies exhibit higher CD107a PE median fluorescent intensity (MFI) relative to isotype control. For all cytotoxicity assays, a 2-way ANOVA was used to assess for significant difference in cytotoxicity relative to isotype control. Data points represent the mean of triplicate; error bars represent the SEM. * p < 0.01 to 0.05; ** p < 0.01 to 0.001; *** p < 0.001 to 0.0001; **** p < 0.0001. Trials for T-LGL and cell lines were performed three times, in triplicate for each trial. Representative trials are shown. Three healthy controls each underwent one biological trial, and were plated in triplicate; a representative healthy control is shown.

Figure 4.. αVβ monoclonal antibodies induce Activation-induced cell death (AICD) in target T-cell clone.

(A) Incubation of the Patient J’s PBMCs with αVβ13 high affinity candidates results in increased expression of CD69 on the T-LGL clone, similar to the positive control anti-CD3 (OKT3). (B) Incubation of the Patient T-LGL PBMCs with αVβ13A for up to 96 hours results in cell death that is first detectable at 48 hours, and increased over 96 hours. Statistics relative to isotype control were determined using a 1-way Anova. Error bars represent the SEM of triplicate samples. (C) A 96-hour incubation of the Patient T-LGL PBMCs with αVβ13 high affinity candidates results in high cell death relative to isotype control. Samples compared to isotype control within each condition using a 1-way Anova. Error bars represent the SEM of triplicate samples. Representative trial of three biological trials is provided. (D) The observed cytotoxicity of the T-LGL clone, relative to isotype control, is maintained when the αVβ13 isotype is changed from hIgG1 to hIgG4, the latter of which does not have any effector function. Samples compared to isotype control within each condition using a 1-way Anova. Error bars represent the SEM of triplicate samples. (E) Activation of the patient T-LGL clone by αVβ13, but not anti-CD3, is blunted in the presence of complement supplemented media. (F) αVβ8 induce CD69 expression in Jurkat cells following a 24-hour incubation. (G) Jurkat cells treated with αVβ8 have reduced viability at 72 hours by MTS assay. Samples were compared using an unpaired T-test. Error bars represent the SEM of triplicate samples. (H) Jurkat cells treated with αVβ8 are enriched in G1 phrase and less likely to be in S phase. Samples compared using a 2-way Anova. Error bars represent the SEM of triplicate samples. (I) αVβ8 induce Jurkat annexin expression after 5 minutes incubation, and maintain stable expression over 48 hours, relative to isotype control. Samples compared using a 2-way Anova. Error bars represent the SEM of triplicate samples. (J) Serum starved Jurkat cells incubated for 10 minutes with αVβ8, then with or without addition of 10 ug/mL anti-CD3, induces phosphorylation of ZAP70 and ERK. (K) Tumor volume means of EL4-V13 cells injected into the subcutaneous space of C57BL/6J mice and subsequently treated with αVβ13A-mIgG2a or isotype controls. Error bars represent the SEM of tumor volume in ten mice per arm. * p < 0.01 to 0.05; ** p < 0.01 to 0.001; *** p < 0.001 to 0.0001; **** p < 0.0001.

Figure 5.. Distribution of TCRVB Gene Usage across T-cell NHL patients.

TCR Vβ usage in 314 patients with T-cell NHL. The black bars indicate the percentage of patients with each Vβ, while the orange line indicates the cumulative T-cell NHL percentage.