Simultaneous secretion of a PD-L1 x 4-1BB bispecific antibody improves antileukemic efficacy of STAb-T cells secreting a CD19-specific T-cell engager

Abstract

Adoptive therapy with CAR-T cells and systemic administration of bispecific T-cell engagers (TCE) have achieved unprecedented success in the treatment of relapsed/refractory (R/R) B-cell malignancies. However, high relapse rates remain a major challenge. STAb (Secretion of T cell-redirecting bispecific Antibodies)-T-cell immunotherapy represents a promising alternative by enabling both polyclonal T-cell recruitment and sustained bispecific antibody release. Here, we describe an evolution of STAb-T19 therapy, which has demonstrated superior outcomes to those of CAR-T-19 cells in preclinical models of B-ALL, on the basis of the simultaneous secretion of two bispecific antibodies: a CD19 × CD3 TCE and a PD-L1 × 4-1BB bsAb. The combined approach aims to increase the antitumor efficacy of STAb-T19 cells by blocking the PD-1/PD-L1 axis with conditional 4-1BB costimulation to ensure the long-term persistence of STAb-T cells. Preclinical data show that this combination improves cytotoxic activity and prolongs antileukemic efficacy compared with low-dose single STAb-T therapy. Our findings suggest that the integration of PD-L1 × 4-1BB bsAbs into STAb-T19 therapy may maximize efficacy, thereby opening a promising avenue to address resistance and relapse in B-ALL. Moreover, this approach may pave the way for the development of next-generation cell-based therapies for hematologic and solid malignancies.

Keywords: 4-1BB costimulation; PD−1/PD-L1 blockers; STAb-T cells; bispecific antibodies.

Figure 1.

Characterization of the novel PD-L1 × 4-1BB bsAb. (A, B) Schematic diagrams showing the genetic (A) and domain (B) structures of the anti-CD19 × anti-CD3 bsAb, which bears a signal peptide from the human K light chain (grey box), the anti-CD19 scFv (A3B1) gene (blue boxes), the anti-CD3 (OKT3) scFv gene (grey boxes) and the His tag (yellow box). (C, D) Schematic diagrams showing the genetic (C) and domain (D) structures of the anti-PD-L1 × anti−4-1BB bsAb, which bears the oncostatin M signal peptide (grey box), the anti-PD-L1 scFv (atezolizumab) gene (red boxes) and the anti−4-1BB (SAP3.28) scFv gene (brown boxes). N-terminal FLAG-Strep and C-terminal Myc-His tags (yellow boxes) were appended for purification and detection purposes. (E–I) HEK293 cells were stably transfected with a plasmid encoding the PD-L1 × 4-1BB bsAb. The recombinant antibody was purified from the conditioned medium by affinity chromatography using Strep-Tactin® columns and subjected to characterization assays. (E) Western blot detection of the PD-L1 × 4-1BB bsAb purified from the supernatant of stably transfected HEK293T cells. Conditioned media from nontransfected HEK293T cells (NTf) were used as a negative control. One representative of three experiments is shown. (F) Detection of purified PD-L1 × 4-1BB bsAb by ELISA against a plastic-immobilized human PD-L1-Fc chimera (hPD-L1-Fc), a human 4-1BB-Fc chimera (h4-1BB-Fc) or BSA. PBS was used as a negative control. The data are presented as the mean ± SD (n = 3). (G) The functional binding of purified PD-L1 × 4-1BB (5 μg/mL) to human PD-L1 and 4-1BB expressed on the cell surface of PD-L1⁺4-1BB^- Nalm6^PD-L1 and PD-L1^-4-1BB⁺ Jurkat^{4-1BB/PD-L1-KO} cells, respectively, was demonstrated by flow cytometry using an APC-conjugated anti-His mAb. PD-L1 and 4-1BB IgGs (5 μg/mL) served as positive controls and were detected with a PE-conjugated antihuman Fc antibody. (H) For PD−1/PD-L1 blockade bioassays, Jurkat^PD-1 cells were cocultured with CHO^APC/PD-L1 in the presence of 10-fold increasing concentrations of PD-L1 × 4-1BB bsAb. After 6 hours at 37 °C, luminescence was determined. The Y-axis represents the reporter gene fold induction relative to the values obtained from Jurkat^PD-1 cells alone. HER2 IgG was used as a negative control, and PD-L1 IgG was used as a positive control. The results are expressed as mean ± SD (n = 3). Significance was calculated by an unpaired Student’s t test. (I) Antigen-dependent Jurkat 4-1BB activation assay with 10-fold increasing concentrations of PD-L1 × 4-1BB protein against plastic-immobilized human PD-L1-Fc (hPD-L1) or BSA. 4-1BB IgG was used as a negative control. The data are presented as the fold induction relative to the values obtained from unstimulated Jurkat^4-1BB cells. The results are expressed as mean ± SD (n = 3). Significance was determined by an unpaired Student’s t test. Significance is defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2.

STAb-T19 and STAb-TP4 cells efficiently secrete CD19 × CD3 and PD-L1 × 4-1BB, respectively, and synergize to induce potent specific cytotoxicity. (A, B) Western blot detection of secreted CD19 × CD3 (A) or PD-L1 × 4-1BB (B) in the conditioned media from lentivirally transduced human primary T cells (STAb-T19 or STAb-TP4, respectively). Conditioned media from nontransduced T cells (NT-T) was used as a negative control. (C, D) Detection of soluble functional CD19 x CD3 (C) or PD-L1 x 4-1BB (D) in the conditioned media from STAb-T19 or STAb-TP4 by ELISA against plastic-immobilized human CD19-Fc chimera (hCD19-Fc), human PD-L1-Fc chimera (hPD-L1-Fc), human 4-1BB-Fc chimera (h4-1BB-Fc) or BSA. Conditioned media from NT-T cells was used as a negative control. The data are presented as mean ± SD (n = 3). (E) Representative analysis of intracellular and cell surface-bound (decoration) CD19 × CD3 and PD-L1 × 4-1BB in NT-T, STAb-T19 and STAb-TP4 cells by flow cytometry. One representative experiment out of four independent experiments is shown. The numbers represent the percentage of cells that stained positive for the His tag. (F) Cell cytotoxicity assay. Decreasing numbers of STAb-T19 cells were cocultured with 5 × 10⁴ CD19⁺PD-L1⁺ (Nalm6^PD-L1/Luc) or CD19^-PD-L1^-(K562^Luc) luciferase-expressing target cells in the presence or absence of a constant number of STAb-TP4 cells. NT-T cells were added as indicated to maintain a constant 1:1 effector:target ratio. Cocultures of target cells with STAb-TP4 cells were used as controls. The percentage of cytotoxicity was calculated after 48 h and normalized to the target cell death observed in cocultures with NT-T cells. The data are shown as mean ± SD (n = 3). Significance was determined by two-way ANOVA with Sidak’s multiple comparisons test. Significance is defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (G) Nalm6^PD-L1 cells were cocultured with 1:1 mixtures of STAb-TP4 + NT-T, STAb-T19 + NT-T or STAb-T19 + STAb-TP4 cells at the indicated E:T ratios, and the relative percentages of T cells (CD2⁺CD10^-) and leukemic cells (CD2^-CD10⁺) were analyzed by flow cytometry after 0, 2, 5, and 9 days.

Figure 3.

PD-L1 × 4-1BB potentiates and prolongs the antitumor effect of CD19 × CD3. Long-term cocultures of Nalm6^PD-L1 target cells with reporter gene-expressing STAb-T cells were generated. (A) Percentage of reporter protein expression in primary STAb-T19 or STAb-TP4 cells (GFP or tdTo, respectively). (B, C) Percentages of CD4⁺ and CD8⁺ T cells (B) and percentages of naïve (TN), central memory (TCM), effector memory (TEM) and effector memory RA⁺ (TEMRA) T cells (C) among nontransduced (NT-T), STAb-T19-transduced and STAb-TP4-transduced T cells. The transduced populations were further classified into bsAb-secreting (+) or nonsecreting (−) cells on the basis of reporter gene expression. The data are shown as mean ± SD (n = 3). (D) Nalm6^PD-L1 target cells were cocultured with STAb-TP4 + NT-T or STAb-T19 + NT-T or STAb-T19 + STAb-TP4 cells at the indicated E:T ratios, and the relative percentages of CD2⁺CD10^- and CD2^-CD10⁺ cells were analyzed by flow cytometry at days 4, 12, 20, and 28. On day 16, the cocultures were split into two wells, and 5,000 target cells were added to one of them; the relative percentages of T cells and tumor cells were analyzed at days 20 and 28. One of two similar experiments is shown.

Figure 4.

Simultaneous secretion of CD19 × CD3 and PD-L1 × 4-1BB by STAb-T19-P4 cells improves the antitumor efficacy of STAb-T19 cells in vitro. (A) Percentage of reporter protein expression in primary STAb-T19, STAb-TP4, and STAb-T19-P4 cells (GFP or tdTo). (B, C) Percentages of CD4⁺ and CD8⁺ T cells (B) and percentages of naïve (TN), central memory (TCM), effector memory (TEM) and effector memory RA⁺ (TEMRA) T cells (C) among nontransduced (NT-T), STAb-T19-, STAb-TP4-, or STAb-T19-P4-transduced T cells. The transduced populations were further classified into bsAb-secreting (+) or nonsecreting (−) cells on the basis of reporter gene expression. Data are shown as mean ± SD (n = 3). (D) Nalm6^PD-L1 target cells were cocultured with STAb-TP4, STAb-T19, or STAb-T19-P4 cells at the indicated E:T ratios, and the relative percentages of CD2⁺CD10^- and CD2^-CD10⁺ cells were analyzed by flow cytometry at days 4, 12, 20, and 24. On day 16, the cocultures were split into two wells, and 5,000 target cells were added to one of them; the relative percentages of T cells and tumor cells were analyzed on days 20 and 24.

Figure 5.

Simultaneous secretion of 19- and P4-bsAbs by STAb-T19-P4 cells improves the antitumor efficacy of STAb-T19 in vivo. Xenograft murine model of B-ALL. NSG mice received 1 × 10⁶ Nalm6^PD-L1/Luc cells intravenously followed 2 days after by intravenous infusion of T cells (4 × 10⁶): STAb-TP4 (2 × 10⁶ STAb-TP4 + 2 × 10⁶ NT-T), STAb-T19 (2 × 10⁶ STAb-T19 + 2 × 10⁶ NT-T), or STAb-T19-P4 cells (4 × 10⁶ STAb-T19-P4). (A) Timeline of the experimental design. (B) Total radiance quantification at the indicated time points. Statistical significance was calculated by two-way ANOVA test corrected a Tukey’s multiple comparison test. Significant differences between STAb-TP4 (red asterisks) or STAb-T19 (blue asterisks) and STAb-T19-P4 are indicated. (C) Bioluminescence images showing disease progression. (D) Changes in body weight over time. (E–H) Percentages of B-ALL cells (CD19⁺) and T cells (CD3⁺) in the peripheral blood (PB) at day 14 (E) and at the time of euthanasia in the PB (F), in bone marrow (BM) (G) and spleen (H), as assessed by flow cytometry. Statistical significance was calculated one-way ANOVA test corrected with a Tukey’s multiple comparison test. Significance is defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.