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 which expresses one of 48 TCR variable beta (Vβ) genes, providing a distinct therapeutic target. We hypothesized that a monoclonal antibody 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, TCR-mediated activation-induced cell death, and exhibited specific killing of patient malignant T-cells in combination with exogenous NK 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.

Introduction

T-cells regulate the adaptive immune response and play key roles in diseases such as auto-immunity and the diverse group of malignancies that comprise non-Hodgkin’s lymphomas (NHL). Although individual NHL subtypes are rare, T-cell NHL accounts for 10–15% of new NHL diagnoses, ranging from indolent to aggressive, all of which are associated with morbidity and healthcare utilization.[1] Moreover, outcomes are worse than those with B-cell NHL.[2] As T-cell based cancers are poorly treated with existing therapies, improved targeting of clonal T-cell malignancies is needed.[3,4]

Molecular markers that identify target cells have had a significant impact on the treatment of NHL, and have been explored for autoimmune diseases. Pan-specific B-cell markers such as CD20 (Rituximab) and CD19 have been used to target NHL B-cell cancers, with patients tolerating the associated B-cell aplasia.[5,6] Strategies targeting pan-T-cell antigens, such as the anti-CD52 antibody Alemtuzumab, have proven challenging, however, because the resultant T-cell depletion can lead to opportunistic infections.[7] In contrast, targeted markers found on both T and B-cell such as CD30 (Brentuximab) successfully treat Hodgkin lymphoma as well as Anaplastic Large Cell Lymphoma.[3,8] Furthermore, antibodies targeting CD3 have been developed as general immuno-suppressive agents to treat transplant rejection (Muromonab) and auto-immune diseases such as Type 1 Diabetes (Otelixizumab and Teplizumab).[9] Indeed, the Levy lab first showed the potential of utilizing anti-idiotype antibodies to target the malignant B-cell in NHL, seeing remission responses in a majority patients (66%), with some lasting as long as 10 years.[10,11]

T-cell receptors (TCRs) possess tremendous diversity owing to diverse variable β chain (Vβ) (n=48) and α chain (Vα) (n=46) gene segments, that recombine with D and J gene segments to form a functional TCR.[12] In contrast to B-cell receptors which undergo somatic hypermutation, T-cells express a static TCR. T-cell malignancies involve clonal expansion of T-cells that bear the same Vβ and Vα VDJ rearranged genes,[13–18] and auto-immune/inflammatory diseases frequently result in a predominant expansion of specific T-cell clones.[19–23] Multiplexed PCR of the TCR gene has been used to identify the presence of a distinct malignant clone and facilitate diagnosis of T-cell NHL.[21,24] Thus, the Vβ chain represents a cell surface marker that discriminates disease associated T-cell clones from the majority of normal T-cells. Recent studies have begun to demonstrate the potential of targeting the TCR using bispecific T-cell engagers (BiTEs) and chimeric antigen receptor T-cell (CART) directed approaches.[4,25]

As proof of concept, we identified a 76-year-old male (Patient J) with a history of T-cell granular lymphocytic leukemia (T-LGL). T-LGL is an indolent T-cell clonal lymphoproliferative disorder that can be followed for years without therapy.[26] This allowed for ongoing sample acquisition through refinement of testing over several years. We hypothesized that a monoclonal antibody (mAb) that is exclusive to a specific Vβ would eliminate the malignant clone while having minimal effects on healthy T-cells. We thus sought out to build a panel of anti-Vβ candidates (αVβ) utilizing the sequencing information of Patient J’s T-LGL clone. We demonstrate the development of high affinity antibodies that bind with specificity to the patient’s T-LGL clone and employ cell killing, supporting the clinical efficacy of this approach; furthermore, we show these therapeutic candidates are capable of targeting and eliminating healthy T-cells and cell lines from T-cell acute lymphoblastic leukemia and a murine lymphoblast line. From these findings, we are able to expand the generation of a panel of antibodies capable of treating a majority of patients with other clonal T-cell diseases.

Materials & Methods

Cell lines and primary human cells.

Jurkat (DSMZ), HPB-ALL (DSMZ) and Jurkat J.RT3-T3.5 (ATCC) cells were cultured in RPMI 1640 medium (Gibco, 11875–093) supplemented with 15% FBS (HyClone, SH30396.03) and 1% PenStrep (Gibco, 15140–122). EL4 cells (ATCC) were cultured in RPMI 1640 medium supplemented with 15% horse serum (Gibco 16050122). NK-92 cells expressing the CD16 receptor (haNK; ATCC) were grown in MEM alpha medium (Gibco, 12561–056) with 0.2 mM inositol (Sigma Aldrich, I7508), 0.02 mM folic acid (Sigma Aldrich, F8758), 0.1 mM beta-mercaptoethanol (Sigma Aldrich, M3148), 12.5% FBS, 12.5% horse serum (Sigma Aldrich, H1138), and 200 units/mL human IL-2 (PeproTech, 200–02). The cells did not exceed 20 passages from the time of thaw to the time of use in any given experiment. Cells were tested for mycoplasma once a month throughout use in experimentation using a PCR mycoplasma detection kit (Applied Biological Materials, G238). All patient samples were obtained after oral and written informed consent according to our local Institutional Review Board approval (#4422) following our Hyman Research Protection Program that incorporates the ethical principles described in the Belmont Report and US Common Rule. Healthy control patients were excluded if they had a diagnosis of T-cell lymphoma or myeloproliferative disorders. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient and plated within 24 hours in IMDM with 20% FBS (Gibco, 12440–053). All cells in all experiments were cultured for incubation in a humidified incubator at 37 °C with 5% CO₂ unless otherwise noted.

Generation of TCR cell lines.

Patient J’s TCRVB was cloned into lentiviral vector pLX304 (Addgene 25890) and used to transduce Jurkat J.RT3-T3.5 cells to generate cell line Jurkat-V13. Patient J’s TRB and TRA chain variable regions were cloned into pMIG II by replacing the murine Vbeta5.2 TRB and TRA chain variable regions in Addgene 52111,[27] and used to transduce EL4 cells to generate cell line EL4-V13. Viral particles were generated at VectorBuilder Inc.

Binding.

For patient samples, 100 uL of ACK-lysed blood was stained with non-conjugated anti-hIgG (BD Biosciences, 555784) for 20 min to saturate non-specific binding sites, followed by a 20-minute incubation with 30 nM αVβ, and detection with BV786-conjugated anti-hIgG (BD Bioscience, 564230), all on ice. Cells were stained with fluorophore-conjugated lymphocyte markers to identify the malignant T-LGL clone or target T-cell population, including CD2-PE (Beckman Coulter, IM0443U), CD3-APC-AF750 (Beckman Coulter, A66329), CD4-FITC (Beckman Coulter, IM0448U), CD5-PE-Cy7 (Beckman Coulter, A51075), CD8-APC (Beckman Coulter, IM2469U), CD16-ECD (Beckman Coulter, A33098), and CD56-PE-Cy5.5 (Beckman Coulter, A79388). To determine the percentage of T-LGL bound by the αVβ13, the fraction of αVβ-bound CD8⁺ cells detected by anti-hIgG were divided by the percentage of abnormal CD8⁺ cells demonstrating partial loss of CD2.

Cell line binding was determined by incubation with 10 nM or 30 nM αVβ followed by incubation with anti-hIgG-BV786. Competition studies were accomplished by pre-incubating cell line samples with 10 nM or 30 nM αVβ for 30 min, followed by washing and incubating for an additional 30 min with a commercially available anti-TCR Vβ8 (Biolegend, 348106) or anti-TCR Vβ5a (Thermo Fisher, TCR2642).

All binding incubations were performed on ice.

Antibody-dependent cellular cytotoxicity.

Patient J T-LGL CD3⁺ T-cells, isolated with a magnetic column (Biolegend, 480022), were plated at 3E4 cells/well in a U-bottom 96-well plate (Falcon, 353077).[28] Cells were opsonized with 30 nM αVβ for 10 min at room temperature. haNK cells, prepared in Alpha MEM+ media with 10% FBS, 225 U/mL IL-2, were added at effector-to-target ratios of 1:1, 5:1 or 10:1, followed by incubation for 4 h. Cells were then stained with 7-AAD (Beckman Coulter, A07704). Cell lines were labeled with 0.1 uM calcein and plated at 1.25E4 cells/well. For healthy controls, CD3+ T-cells were isolated and 1.25E5 cells/well were plated and incubated as above. They were then stained with live dead aqua (Thermofisher, L34957) and anti-CD2. Cells were fixed and permeabilized, followed by staining with anti-Vβ8.1 and anti-Vβ5a. Surviving target cell lines were identified as calcein⁺7-AAD⁻; surviving T-LGL cells were identified as calcein+7⁻AAD⁻CD8⁺CD2^dimCD5^dim; healthy controls as aqua⁻CD2⁺, and cytosolic Vβ8⁺ or cytosolic Vβ5a⁺. Lastly, NK activation was measured by the addition of anti-CD56 and anti-Lamp1a-BV650 (Biolegend, 328638). Trials for T-LGL and cell lines were performed three times, in triplicate for each trial. Three healthy controls each underwent one biological trial, and were plated in triplicate.

Complement dependent cytotoxicity and deposition.

Jurkat were plated in U-bottom 96-well plates at 4E5 cells/well in 15% FBS RPMI media supplemented with 2.5% Human Sera Complement (HS, Millipore Sigma, S1764).[29] Cells were incubated for 4 hours with 30 nM αVβ8, and stained with 7AAD. Similarly, CD3+ T-LGL cells were plated at 3E4 cells/well in 20% FBS IMDM media with 2.5 % HS and incubated with 30 nm of αVβ13A, αVβ13E, or αVβ13D. Cells were stained with immunophenotyping antibodies as described above. The surviving T-LGL cells were identified as 7-AAD⁻CD8⁺CD2^dimCD5^dim. Complement deposition was measured following 1-hour incubations at 37 deg C using anti-C3b/iC3b-APC (Biolegend, 846106). Trials were performed three times, in triplicate for each trial.

Activation.

Patient PBMCs were incubated with 30 nM αVβ for 24–96 hours in 20% FBS IMDM at 4E5 cells/well. Activation was measured at 24 hours with anti-CD69 (Beckman Coulter, IM1943U). Cytotoxicity was measured as described above. Trials were performed three times, in triplicate for each trial.

Cytotoxicity calculations.

For all cytotoxicity assays, CountBright counting beads (Thermo Fisher, C36950) were added and cells were analyzed using a Fortessa Flow Cytometer (BD Biosciences). A total of 8,000 bead events were collected for all the samples with the exception of healthy controls for which 30,000 bead events were captured. Percent cytotoxicity was determined by subtracting the ratio of live target cells incubated with αVβ to live target cells incubated with the isotype from 1, and then multiplying by 100. Data manipulation was done using Graphpad Prism software.

Internalization.

First, 10 nm αVβ8, 30 nm αVβ5 and 30 nm αVβ13A was labeled with Zenon pH-Rodo (Thermofisher, Z25612). Jurkat and HPB-All were plated at 2.5E5 cells/well in 15% FBS RPMI and labeled αVβ was added at designated timepoints. For T-LGL cells, 4E5 T-LGL cells/well in 20% FBS IMDM media was incubated for 24 hours. All samples were analyzed on a Fortessa flow cytometer.

MTS.

Jurkat cells in 15% FBS RPMI media were plated at 5E3 cells/well with 10 nM ɑVβ8 for 72 hours. Cells were prepared using an MTS tetrazolium assay kit (Promega, G358) and results were obtained using a BioTech Synergy 2 Reader.[30]

Cell Cycle.

Jurkat cells in 15% FBS RPMI media were plated at 2.5E5 cells/mL with 10 nM ɑVβ8 for 24 hours. The cells were fixed using 70% cold ethanol and stained in 250 uL propidium iodide/RNase staining solution (50 ug/mL PI, 0.1% Tween-20/PBS, 4X of 100 mg/mL RNase A in PBS) for 30 min before analyzing on a Fortessa flow cytometer.

Apoptosis

Jurkat were plated at 1E4 cells/mL in 15% FBS RPMI in a 96-well U-Bottom plate, incubated with 10 nM ɑVβ8 for 48 hours and analyzed using the Annexin V-APC Assay kit (Abcam, 236215) on a Guava Easycyte HT plate reader.

Cell Stimulation & Immunoblotting

5E6 Jurkat cells were serum starved for 18 hours prior to incubation with ɑVβ8 for 10 min at 37 deg C, then addition of 10 ug/mL anti-CD3 or phosphate buffered saline for 5 min. Cells were pelleted and lysed with the RIPA Lysis buffer solution on ice. Supernatants were centrifuged and the protein concentration was quantified with BCA Protein Assay. An equal amount of whole-cell lysate was mixed with 3X SDS sample buffer (75mM Tris (pH 6.8), 3% SDS, 15% glycerol, and 0.1% bromophenol blue) containing 8% β-mercaptoethanol. The mixed samples were boiled for 5min in a 95°C heat block before loading on a 4–15% Tris-HCl gradient gel. The proteins were transferred onto a nitrocellulose membrane and blocked with 5% FBS TBST buffer and incubated with antibodies against phospho-Zap70 (Cell Signaling, #2701T), phospho-AKT (Cell Signaling, #9271), phospho-ERK (Cell Signaling, #9101L). The membrane was then stripped, washed, blocked and probed with antibodies against ZAP-70 (Thermo Fisher, #PA5–28697), AKT (Cell Signaling, #9272S) and ERK (Cell Signaling, #9101L). HRP conjugated secondary antibodies against mouse IgG and rabbit IgG were used.

Murine assay.

Animals were cared for under the OHSU Institutional Animal Care and Use Committee (IACUC) in compliance with the Animal Welfare Act regulations and Public Health Services policy. Eight-week-old female C57BL/6J mice (Jackson Laboratory, 20 kg ± 2) were subcutaneously injected on the left flank with EL4-V13 (3E5 cells) (Day 0) using 28-gauge syringes. On Day 1, half of the specimens received an intraperitoneal injection of 200 ug αVβ13A-mIgG2a; the other half received 200 ug mIgG2a isotype. Two additional doses were given on Day 4 and 8. Tumor volumes were measured (volume=(l * w²)/2)) using a caliper. On Day 12, the mice underwent carbon-dioxide-induced euthanasia.

Data Availability.

The data and protocols that support the findings of this study are available from the corresponding author, PMB, upon reasonable request.

Results

Generation of αVβ13 candidates.

Patient J’s PBMCs were isolated and RNAseq analysis was performed to identify dominant T-cell clones. A T-cell clone expressing TRBV6–1, corresponding to Vβ13.3 in Wei nomenclature, was identified and constituted 95% of the CD8 T-cell population (Figure 1A). Flow cytometric analysis revealed an immunophenotype characterized by CD8⁺CD2^dimCD3⁺CD5^dimCD7^dimCD16^variableCD57⁺ (Supplementary Figure 1). Genomic analysis revealed mutations in known oncogenes: DNMT3A R882S and E907del, TET2 E1144K, STAT3 Y640F.

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.

We sought to develop an antibody specific to Vβ13.3 (αVβ13) for eliminating the malignant T-cells (Supplementary Methods). Protein antigen composed of Patient J’s Vβ13.3 fused to mouse IgG1 Fc (mFc) was generated and used to immunize mice (Figure 1B). A total of 320 single B-cells were isolated for antibody cloning and expression [31] and screened by ELISA binding to patient J’s Vβ13 chain (Figure 1C). Binding was also assessed to patient PBMCs, defined as the proportion of αVβ13 bound T-LGL cells to the total population of T-LGL cells (CD3⁺CD8⁺CD16⁺CD2^dim). Seven αVβ13 antibodies binding >80% of the T-LGL population were identified: αVβ13A-αVβ13G (Figure 1D and Supplementary Figure 2A), consistent with ELISA binding. These αVβ13 mAbs can bind to TCRs in the Vβ13 family and have the potential for general treatment of all Vβ13 clones across patients. As each TCR containing Vβ13 has a unique CDR3 sequence created by VDJ recombination, we also assessed binding to a mutant antigen in which the CDR3 was scrambled to distinguish αVβ13 from anti-idiotype (Figure 1C). Importantly, αVβ13J and αVβ13I do not bind the CDR3 mutated antigen and were identified as anti-idiotype antibodies specific to Patient J (Supplementary Figures 2B–C).

αVβ monoclonal antibodies bind with specificity to Jurkat and EL4 cells transduced to express Vβ13.3.

To our knowledge, there are no commercially available cell lines that express Vβ13.3, the Vβ on Patient J’s T-LGL clone. To further characterize the cell binding and functional activity of αVβ13 candidates, we generated cell lines expressing the patient’s Vβ. We utilized a lentiviral transfection to transduce a stable T-cell receptor complex (TCRβ chain and CD3) characterized by patient TCR Vβ13.3 expression using the NK murine lymphoma line EL4 and the TCR-null Jurkat T-ALL line T3.5 to express Vβ13.3, referred to as EL4-V13 and Jurkat-V13, respectively (Figure 1E).

Transduced cell lines were sorted by fluorescent activated cell sorting (FACS) and confirmed for expression using CD3 and Vβ specific antibodies. We found that αVβ13A, a high affinity binder to primary T-LGL patient cells, bound with specificity to Jurkat-V13, indicating expression of a stable TCR complex (Figure 1F). We also exchanged the hIgG1 of αVβ13A with a mIgG2a Fc region (αVβ13A-mIgG2a). This bound with specificity to EL4-V13 and was detected using a secondary against mIgG2a (Figure 1F). αVβ13A did not bind to the wild type lines.

αVβ antibodies bind to a healthy control T-cells and T-cell lines.

Next, we assessed binding to healthy control PBMCs (n=3). We found that αVβ13A bound 1.89–3.12% of the CD3⁺ population, a proportion in line with healthy T-cell repertoire diversity (Figure 2A).[32] Conversely, the anti-idiotype αVβ13J bound to the patient’s T-LGL clone and Jurkat-V13, but not to CD3⁺ lymphocytes from two healthy controls (Supplementary Figures S2B–D).

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).

Given the heterogeneity of T-cell lymphoma phenotypes, we also analyzed antibodies against Vβ8 (corresponding to TRBV12–3, hitherto αVβ8) and Vβ5.2/5.3 (corresponding to TRBV5–5 and TRBV5–6, hitherto αVβ5) (Figure 2B).[33,34] We found that αVβ8 bound 3–4% of CD3⁺ cells from healthy controls, consistent with published values of 3.5–6.5 % (Figure 2A).[32] Although αVβ5 binding was relatively weak, preincubation with αVβ5 reduced the binding of a commercially available αVβ5-FITC antibody by 2-fold (p = 0.002; Figure 2A, Supplementary Figures S2E–F). We also observed strong binding of αVβ8 to Jurkat cells that express Vβ8, and αVβ5 to HPB-ALL cells, characterized by Vβ5.3 expression (Figure 2C). Pre-incubation with αVβ8 or αVβ5 also competitively inhibited other αVβ8 or αVβ5 antibodies from binding, demonstrating specificity of αVβ candidates (Supplementary Figures S2G–H).

Targeted T-cells undergo antibody dependent cellular cytotoxicity in vitro.

Having established a panel of αVβ13 antibodies, we assessed the capability of mediating antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). αVβ antibodies were formatted as human IgG1 (hIgG1) and mouse IgG2a isotypes (mIgG2a), both competent for Fc-mediated cell death. At baseline, Patient J demonstrated undetectable NK cells, a known sequela of T-LGL.[35] We thus investigated the ability of αVβ to induce ADCC utilizing exogenous NK-92 effector cells engineered to express the Fcγ receptor CD16 (haNK). [36] We mixed target T-LGL patient CD3⁺ cells with effector haNK cells at three different effector-to-target ratios (E:T) of 1:1, 5:1 and 10:1 in a 4-hour cytotoxicity assay.[28]

In Patient J’s CD3+ T-cells, we observed cytotoxicity of the T-LGL clone, identified by CD8⁺CD5^dim, using αVβ13A, αVβ13E, αVβ13D at all E:T ratios, relative to isotype control (Figure 3A). αVβ13A induced target cell death of 37.1% at 1:1 E:T ratio (p=0.0010), increasing to 70.5% at 10:1 E:T ratio (p<0.001). Less than 20% of Patient J’s CD8 positive T-cells are normal T-cells, characterized by CD8⁺CD5⁺. We found no significant change in this healthy CD8 population relative to isotype controls at all E:T ratios (Supplementary Figure 3A).

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.

Similarly, we observed cell death of engineered Jurkat-V13 and EL4-V13 cell lines but not wild-type cell lines when using αVβ13A at E:T ratios of 5:1 and 10:1 (Figure 3B–C). Similar results were seen with αVβ5 and αVβ8 in HPB-ALL and Jurkat cells, respectively (Figure 3D–E).

We then examined the ability of αVβ antibodies to induce target cell death in healthy controls. However, there is no additional unique epitope on the target population. We found that αVβ8-bound cells contain cytosolic Vβ8 that can be detected by a commercially available αVβ8 antibody (Supplementary Figure 3B), and thus cytosolic expression could be used to track the target population. We proceeded with a 4-hour ADCC assay with healthy control CD3+ T-cells and haNK cells. We observed elimination of the target population with both αVβ5 and αVβ8 antibodies at all E:T ratios in three healthy controls (Figure 3F–H, p<0.0001, Supplementary Figures 3C–H). We did not test αVβ13 ADCC activity against healthy controls owing to the lack of a commercially available antibody against the target epitope.

In some cases, we measured the NK activation marker CD107a which correlates to NK cell-mediated lysis of target cells.[37] We found that CD107a was elevated in haNK cells incubated with Jurkat-V13 and Jurkat and their corresponding αVβ antibodies, αVβ13 or αVβ8, respectively, relative to isotype control (Figure 3I).

Target T-cells do not undergo complement mediated cytotoxicity.

Patient J’s CD3+ T-cells were incubated with high affinity αVβ13.3 antibodies for 4 hours in the presence of human complement.[29] No T-LGL cell death was induced by αVβ13 mAbs, while Alemtuzumab resulted in cell death in both the T-LGL cells (17%, p=0.144) and normal CD8 T-cells (71.8%, p=0.0001) (Supplementary Figures 4A–B). Only Jurkat, incubated with ɑVβ8, and human complement had cell death at 24 hours (Supplementary Figure 4C). Complement deposition was assessed and we did not visualize C3 deposition on treated cells. In contrast, a control mAb against CD3, OKT3, showed effective deposition (Supplementary Figure 4D).

Targeted T-cells incubated with αVβ undergo activation induced cell death.

Next, we evaluated additional potential mechanisms that reduce target T-cell viability. Activation-induced cell death (AICD) may occur when repeated stimulations of the T-cell receptor lead to apoptosis.

To test for AICD, Patient J’s PBMCs were incubated with αVβ13 antibodies for 24 hours. This resulted in expression of CD69, a marker of activation, in the T-LGL clone (Figure 4A), but not healthy T-cell subsets (Supplementary Figure 5A). We then incubated the PBMCs for 96-hours with αVβ13A, in the absence of complement or NK effector cells. The target cell underwent cell death at 48–96 hours relative to the isotype control (Figure 4B). Other αVβ13 antibodies behaved similarly after 96-hour incubations (Figure 4C).

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.

To ensure cell death was not due to effector function, the hIgG Fc region of αVβ13A and αVβ13J was exchanged with human IgG4 which lacks effector function, called αVβ13A-hIgG4 and αVβ13J-hIgG4, respectively. There was significant cell death at 96-hours relative to the isotype control irrespective of the Fc isotype (Figure 4D). Interestingly, we found that the presence of complement inhibited cell death. This was accompanied by inhibition of CD69 expression, indicating that a lack of activation was critical for the observed cell death (Figure 4E). In Jurkat cells, complement does not inhibit αVβ8 mediated cell death (Supplementary Figure 4C).

To further explore reduced cell viability, we focused on αVβ8 and Jurkat cells. Similar to T-LGL cells, CD69 expression in αVβ8 incubated Jurkat cells increased by 31-fold relative to isotype following a 24-hour incubation (Figure 4F). Jurkat cells had reduced viability relative to isotype control (81.4%, p>0.0001) in a 96-hour MTS assay (Figure 4G). Analysis of cell cycle progression showed a higher percentage of the αVβ8-treated population in G1 phase (76.0% versus 53.0%, p<0.0001) and less in S phase (16.1% versus 31.0%, p<0.0001) or G2 phase (10.6% versus 16.4%, p=0.03) (Figure 4H). They also exhibited elevated annexin expression, a marker of apoptosis (Figure 4I).

We measured the phosphorylation of ZAP70, a critical component in AICD, and phosphorylation of ERK for MAPK pathway which has been shown to mediate apoptosis.[38,39] As a control, we added soluble anti-CD3 (OKT3), a potent activator and apoptosis inducer, following αVβ incubation.[40] We found that 10 minute incubation of αVβ8 induced phosphorylation of ZAP70 and ERK in serum starved Jurkat cells, similar to OKT3 (Figure 4J). AKT was phosphorylated in all sample, consistent with defective expression of phosphatase and tensin homologue (PTEN) and constitutive activation of the phosphatidylinositol 3-kinase (PI3K)-signaling pathway.[41]

αVβ induce cell death in a murine syngeneic model

To demonstrate effective killing of Vβ13.3 cells by αVβ13A in vivo, we utilized a syngeneic mouse model that allow for both ADCC and AICD as potential mechanisms of cell death. We formatted αVβ13A in mIgG2a, an ADCC competent isotype (Figure 1E,F). We performed subcutaneous injection of EL4-V13 into the flank of C57/BL6 mice and observed decreased tumor growth velocity by αVβ13A-mIgG2A relative to isotype control (p < 0.002) (Figure 4K).

αVβ Differentially Impact TCR Cell Surface Expression

TCR down-modulation following ligation is a pivotal event in T-cell activation.[42] We assessed the ability of αVβ to impact TCR surface expression. The T-LGL clone exhibited a drop in TCR detection, as assessed by CD3 and αVβ detection at 1 hour, and progressively decreased over the following 18 hours, while CD2 remained stable (Supplementary Figure 5B). In Jurkat cells, detectable CD3 and αVβ drops within 5 minutes and is sustained for 24 hours (Supplementary Figure 5C). We next assessed internalization utilizing fluorophore pHrodo-labeled αVβ and found that Jurkat and HPB-ALL cells internalized αVβ8 and αVβ5, respectively, while primary T-LGL cells did not (Supplementary Figures 5D, E).

Vβ usage in T-cell lymphoma is targetable by a small number of antibodies.

Given target cell death induced by αVβ mAb, we sought to derive an efficient path to assemble a panel of antibodies that could target the majority of T-cell NHL patients. We performed a meta-study of TCRBV gene usage and homology in NHL patient data sets to prioritize antibody specificity. We identified 9 NHL datasets in which 500 NHL patient samples were analyzed by RNA sequencing, PCR and/or FACS methods, of which 314 patients had sufficient data to determine the exact gene (Supplementary Table 1A).[13,43–50] Based on TCRVβ gene homology, where 80% identity suggests a high probability of obtaining a cross-reactive antibody, we clustered TCRVβ groups to identify a set of Vβ genes likely to offer optimal coverage of NHL patients (Figure 5, Supplementary Table 1B). Five antibodies could theoretically recognize the majority (54%) of NHL malignant clones, and 70% of malignant clones could be targeted with 9 distinct αVβ antibodies.

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.

Discussion

The development of targeted therapies to treat T-cell based diseases has been a long-term goal of medicine. Most targeted T-cell therapies, however, discriminate poorly between healthy and diseased tissues. Because T-cells express a single rearranged TCR, pursuing Vβ represents a potential route to targeted therapy. Two recent studies using CART and BiTE constructs have shown the ability to kill clonal T-cells.[4,25] The Vogelstein lab paired two existing anti-TCR Vβ antibodies formatted as scFvs with an anti-CD19 directed scFv to target effector T-cells to kill malignant clones. They showed that constructs specifically lysed malignant T-cell lines patient-derived cells in vitro and resulted in tumor suppression in murine models. The use of BiTEs is appealing and could theoretically provide a fast, patient-specific and selective targeting approach that preserves healthy T-cells. BiTEs, however, do possess significant limitations including bi-directional killing and the current need for near continuous infusion due to a short half-life.[51] Use is hampered by cytokine release syndrome (CRS) and effector cell–associated neurotoxicity syndrome.[52] Finally, the manufacture and development of bispecific antibodies is challenging.[53]

While having excellent cytotoxicity, the use of CART presents a technical and logistical burden for T-cell disease. CART strategies require cellular engineering to block target antigen expression by the CART cells to prevent T-cell fratricide and palliative strategies to compensate for loss of T-cell function are challenging. A novel strategy was recently documented that targets the TCR constant region (TRBC1) with a CART approach. This approach destroys about one half of the T-cell repertoire, and while preserving a function T-cell response, would potentially possess other clinical risks such as CRS. The CART targeting format can require autologous CART generation and deployment, a cell population which may be unavailable from immunosuppressed cancer patients. Likewise, the use of allogeneic NK and T-cell lines pose other technical issues.[54]

The use of monoclonal antibodies is an established approach enjoying well-defined clinical and developmental properties. Attributes such as pharmocokinetics, ADCC, CDC, manufacturing, delivery and storage are well understood, and can be easily modulated as part of therapeutic development.[55–57] We found that our antibodies effectively engaged effector cells for target cell cytotoxicity while also inducing downstream apoptosis, partially through T-cell activation. Our murine model demonstrated significant reduction in tumor growth rate using an aggressive T-cell tumor model, however did not completely eliminate the subcutaneous tumors. It is possible that the therapeutic antibody penetration of a solid tumor is impaired. For instance, a clinical trial assessing mogamulizumab in patients with cutaneous T-cell lymphoma showed poorer responses in the skin (ORR 42.1%) and lymph node (ORR 25.0%) compartments compared to the blood (ORR 94.7%).[58] Future studies could assess if tumor growth is controlled with ongoing therapy. Furthermore, effector enhancing Fc formats such as afucosylation and mutations that impact FcGR engagement will be assessed to further optimize cell killing as needed.[59] Recent studies suggest that levels of galactosylation of antibodies produced in HEK instead of CHO cells leads to improved C1q engagement and opsonophagocytic killing.[60] Future development could also focus on enhancing complement fixation.[57] Lastly, in aggressive, refractory or bulky T-cell lymphoma, multiagent chemotherapy regimens have clinical activity but are associated with greater toxicity and possibly risk of death when used earlier in treatment.[61,62] Immunochemotherapy approaches are often more efficacious without significant toxicity compared to straight chemotherapy regimens. In this setting, use of a targeted αVβ therapeutic antibody, much like the use of Bentuximab vedotin in CD30 positive peripheral lymphomas or rituximab in diffuse large B-cell lymphoma, could improve survival and be accompanied by a manageable safety profile.[8]

The exhausted CD8+ T-cell phenotype of T-LGL might influence the clone’s susceptibility to AICD. Other T-cell lymphomas with different subsets of T-cells might behave differently upon targeted Vβ depletion. Collectively, this underscores the reality that T-cell lymphoma represents a heterogeneous disease characterized by varying levels of aggressiveness, location of the T-cell clone, type of T-cell and equitable access to advanced therapies. Thus, multiple modalities to treat this population of patients is likely necessary. Additionally, a lack of effector cells might necessitate administration of allogeneic NK cell lines to support efficacy. Allogenic NK cell therapies are currently in clinical development, and would have potential efficacy advantages relative to cell lines such as haNK cells, which must be irradiated, thus limiting persistence, proliferation and long-term impact on efficacy of co-administered antibody.[63]

We observed with interest the lack of αVβ-induced T-LGL activation and cell death in the presence of complement. It has been shown that C5a enhances T-cell proliferation and diminishes effector T-cell apoptosis via upregulation of the anti-apoptotic protein BCL2, highlighting the importance of complement in T-cell homeostasis.[64] While it is unclear how this might hamper the antibody effector function in vivo, it is reassuring that the syngeneic in vivo murine model showed a significant reduction in tumor velocity growth. We also noted the difference in internalization of the TCR complex upon binding of αVβ. The cell lines had internalization of the antibody while the patient’s primary T-LGL samples did not. The loss of TCR expression in the primary patient cells might be due to prevention of TCR complex recycling over internalization.[42]

We have demonstrated that monoclonal antibodies targeting specific Vβ subsets has the potential to be an effective, off the shelf strategy to eliminate clonal populations of diseased or healthy T-cells while leaving the healthy immune system intact. Moreover, we demonstrate a pipeline for the efficient generation and testing of candidates, and use primary patient data to show that a majority of the T-cell NHL patients may be treated with a handful of antibodies.

Translational Relevance.

We have developed anti-Vβ13.3 antibodies that bind and kill healthy and malignant T-cells expressing this epitope with specificity. These antibodies function by antibody dependent cellular cytotoxicity and TCR-mediated T-cell activation, providing an approach to the targeted elimination of the malignant T-cell clone and an advancement to existing therapies which result in clinically significant immunosuppression. Moreover, we have identified the most highly utilized Vβ subunits in published T-cell lymphoma cohorts to establish a pipeline for off-the shelf therapeutics to treat this population.