Preclinical characterization of ISB 1342, a CD38 × CD3 T-cell engager for relapsed/refractory multiple myeloma

Abstract

Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study.

Graphical abstract

Figure 1.

ISB 1342 properties and binding. (A) Schematic 3D representation of ISB 1342, a bispecific antibody based on the BEAT technology with a Fab targeting CD38, an scFv targeting CD3ϵ, and a Fc carrying the LALA (L234A, L235A) mutation. The model was generated using the BioLuminate software (Schrödinger, New York, NY). (B) Mean ± SD of K_D determined either on CD38^- T cells (n = 12 donors in 3 independent experiments) and MM cell lines, KMS-12-BM (20 measures from n = 14 independent experiments), NCI-H929 (12 measures from n = 5 independent experiments) and MOLP-8 (9 measures from n = 3 independent experiments), or recombinant human proteins CD3ϵδ (n = 5 independent experiments) and CD38 (n = 3 independent experiments). (C) Representative binding of ISB 1342 on CD38^– human healthy T cells (mean ± SD of 4 donors) and KMS-12-BM MM cell line (1 representative measurement from 1 experiment). (D) Epitope mapping of daratumumab and ISB 1342 on CD38. Residues in dark red represent the CD38 residues in a 4 Å radius from the daratumumab chain in the crystal structure of 7DHA. Linear peptide mapping by SPR as well as site-directed mutagenesis were used to determine the binding epitope of ISB 1342 on CD38, shown in green on the CD38 chain of crystal structure 7DHA (in beige color). (E) ISB 1342 does not compete with daratumumab and can engage CD38 prebound by daratumumab. Biotinylated human CD38 protein was loaded on a streptavidin SA biosensor. The biosensor with immobilized CD38 was then dipped in a solution of daratumumab in kinetic buffer to reach saturation of the surface. Then, a saturated biosensor was dipped into a premixed solution of daratumumab + ISB 1342 at equimolar concentrations (red curve) or daratumumab only (blue curve). Plots show binding to the sensor tip as a wavelength shift (response, in nm; y-axis) vs time (in sec; x-axis).

Figure 2.

ISB1342 induces the killing of MM cell lines in vitro via T-cell engagement. (A) Representative confocal image of ISB 1342 (white) at the synapse between T cell (green) and KMS-12-BM MM cell line (blue) acquired with Zeiss LSM 800 inverted confocal microscope, magnification ×40. (B-F) Cytotoxicity of KMS-12-BM MM cell line (B) and T-cell activation (C), proliferation (D), and degranulation (E) after treatment with ISB 1342 or control molecules in the presence (F) or absence (B-E) of soluble CD38 (sCD38) and healthy PBMC (E:T, 5:1) for 72 hours. Data represent the mean ± SD from 3 PBMC donors performed in 2 independent experiments (B-E) or mean ± SD of EC₅₀ from 3 donors that were compared with the condition without sCD38 using a 1-way ANOVA followed by a Dunnett post hoc comparison (F). (G-H) Cytotoxicity of the RPMI8226 MM cell line (G) and cytokine release (H) in the presence of ISB 1342 ± dexamethasone and healthy PBMC (E:T, 10:1) for 48 hours. Data represent mean ± SD of EC₅₀ compared using a paired t test (G) and mean ± SD at the maximum dose of ISB 1342 tested compared using a paired t test (H) from 6 PBMC donors and performed in 2 independent experiments. ∗∗P < .01, ∗∗∗P < .001. ns, not significant. ANOVA, analysis of variance.

Figure 3.

ISB 1342 induces potent killing of cell lines showing reduced sensitivity to daratumumab. (A) Absolute number of specific antibody bound per cell (sABC) indicating the relative CD38 density on MM cell lines. Data represent mean ± SD and were compared using a 1-way ANOVA followed by a Kruskall-Wallis post hoc comparison using KMS-12-BM as a reference. (B-D) Cytotoxicity of MM cell lines in the presence of daratumumab in CDC (B), ADCP (C), and ADCC (D) assays. Data represent mean ± SD of the maximum response from 4 donors in 2 independent experiments (B), from up to 10 donors in 3 independent experiments (C), and from 5 donors in 3 independent experiments (D), which were compared using a 1-way ANOVA followed by a Tukey post hoc comparison. (E) Cytotoxicity of MM cell lines in the presence of ISB 1342 and healthy PBMCs (E:T, 5:1) for 72 hours in a RDL assay. Data represent mean ± SD of EC₅₀ from 6 PBMC donors, compared using a 1-way ANOVA followed by Dunnett post hoc comparison to KMS-12-BM. (F-H) Schematic representation depicting the MMoAK assay, including 4 MoA: T-cell RDL, ADCC, ADCP, and CDC (F). Cytotoxicity of various MM cell lines in the presence of ISB 1342 or daratumumab, healthy PBMC (E:T, 5:1), normal human serum, and rhIL-2 for 48 hours in a MMoAK assay. Data represent mean ± SD of duplicates from 1 representative donor using nonlinear regression analysis (F), the mean ± SD of EC₅₀ from up to 10 PBMC donors per treatment, and the cell line from 6 independent experiments that were compared using a two-way ANOVA and Sidak post hoc test (G-H). ∗P < .05, ∗∗P < .01, ∗∗∗P < .0001, ∗∗∗∗P < .00001.

Figure 4.

ISB 1342 in vitro potency is not affected by the concomitant or pretreatment with daratumumab. (A) Schematic representation depicting the MMoAK assay with concomitant treatment with ISB 1342 and daratumumab. (B-C) Cytotoxicity of the NCI-H929 MM cell line (B) and CD8⁺ T-cell response (C) after treatment with increasing doses of ISB 1342 and a fixed dose of daratumumab or monoclonal antibody control (mAb) in the presence of healthy PBMC (E:T, 5:1), normal human serum, and rhIL-2 for 48 hours. Data represent mean ± SD of EC₅₀ or the maximum response, from 12 donors in 3 independent experiments that were compared using an unpaired t test. (D) Schematic representation depicting the MMoAK assay of pretreatment with daratumumab followed by ISB 1342. (E-F) Cytotoxicity of the NCI-H929 MM cell line (E) and CD8⁺ T-cell response (F) in the presence of healthy PBMC (E:T, 5:1), normal human serum, and rhIL-2 with increasing doses of ISB 1342 for 48 hours after pretreatment with a fixed dose of daratumumab or monoclonal antibody control for 24 hours. Data represent mean ± SD of EC₅₀ or the maximum response from up to 9 donors in 3 independent experiments that were compared using an unpaired t test; ns, not significant.

Figure 5.

ISB 1342 maintains high potency to kill tumor cells from patients previously treated with daratumumab. (A-C) Representative cytotoxicity curves of CD138⁺ MM cells by ISB 1342 and daratumumab at 18 to 24 hours in samples from patients not previously treated with daratumumab (dara-naïve, patient sample 4) (A), previously treated with daratumumab (dara-exposed, patient sample 15) (B), and dara-naïve plasma cell leukemia (PCL; patient sample 1) (C). Data are mean (A-B) or mean ± SEM of replicates (C) analyzed using nonlinear regression analysis. (D) Maximal cytotoxicity of CD138⁺ tumor cells with ISB 1342 (10-100 nM) or daratumumab (100 nM) in samples from dara-naïve patients (filled symbols) vs dara-exposed (open symbols). Dots represent individual samples, and data are mean ± SD compared using 1-way ANOVA followed by Dunnett multiple comparison analysis to daratumumab on dara-naïve samples. (E) Radar plot of average values for CD8⁺ T cells, NK cells, and monocytes/macrophages ratio to CD138⁺ MM cells in samples from dara-naïve patients (blue) vs dara-exposed ones (pink). (F) Representative CD8⁺ T-cell activation with ISB 1342 and isotype control measured by flow cytometry with expression of CD69 (blue) and CD25 (pink) in PCL. Data are mean ± SEM of replicates analyzed using nonlinear regression analysis. (G) Maximum T-cell activation (CD25 and CD69) and degranulation (CD107a) on dara-naïve vs dara-exposed patient samples with ISB 1342. Data are mean ± SD compared using unpaired t test.

Figure 6.

ISB 1342 controlled tumor growth in vivo in the KMS-12-BM xenograft hPBMC-transferred NSG mouse model. (A-B) Experimental design (A) and measurement of tumor growth (B) in the KMS-12-BM subcutaneously xenograft hPBMC-transferred NSG mouse model. In vivo activity was followed for ISB 1342 at 0.5 mg/kg injected IV once per week and daratumumab at 16 mg/kg injected IV twice per week, both for 3 weeks with 8 mice per group. Data are mean (mm³) ± SD determined by caliper measurements. Data were compared for both models using 2-way ANOVA followed by Tukey post hoc comparison. ∗Significant differences between ISB 1342 and vehicle control; # shows differences between daratumumab and ISB 1342. (C-D) Infiltration of hCD45⁺ cells (defined as live hCD45⁺mCD45^–) (C) and T cells (hTCRαβ⁺CD14^–CD19^–CD56^–CD45⁺) (D) in tumors of KMS-12-BM xenografted mice after vehicle, ISB1342 or daratumumab treatments. Data are mean ± SD for 5 mice compared using 1-way ANOVA followed by Dunnett post hoc test to ISB 1342. (E) Representative dot plots showing activation profile (CD25 and CD69 expression) on tumor-infiltrating T-cell activation in vehicle, daratumumab, and ISB 1342-treated mice. (F) CD38 expression on MM cells in tumors (KMS-12-BM model). Data are mean ± SD compared using 1-way ANOVA followed by Tukey post hoc test; ∗P ≤ .05.

Figure 7.

Impact of ISB 1342 on circulating leukocytes and systemic soluble factors in cynomolgus monkeys. (A) Expression profile of CD38 on leukocyte populations from cynomolgus monkeys. Dots represent data the relative fluorescence intensity from each measurement and bars represent mean ± SD from 4 animals. Data were compared using a 1-way ANOVA followed by Dunnett post hoc comparison, ∗P ≤ .05. (B) Representative binding of ISB 1342 or isotype control on cynomolgus monkey B cells. (C-H) Cynomolgus monkeys (1 male and 1 female) were injected with 3 consecutive doses of ISB 1342 IV (1, 100 and 1000 μg/kg) at days 1, 29, and 57 respectively. Levels of peripheral B cells (C), monocytes (D), CD8⁺ T cells (E), activated CD8⁺ CD69⁺ T cells (F), CD4⁺ T cells (G), and activated CD4⁺ CD69⁺ T cells (H) were measured using flow cytometry. Data are mean ± SD of 10³ counts/μL normalized to baseline counts for 2 animals. (I-J) Levels of circulating IFN-γ (I) and ISB 1342 (J) were measured using ELISA. Data represent levels per animal and LLOQ is the lower limit of quantification for the assay. ELISA, enzyme-linked immunosorbent assay; LLOQ, lower limit of quantitation.

Figure 7.

Impact of ISB 1342 on circulating leukocytes and systemic soluble factors in cynomolgus monkeys. (A) Expression profile of CD38 on leukocyte populations from cynomolgus monkeys. Dots represent data the relative fluorescence intensity from each measurement and bars represent mean ± SD from 4 animals. Data were compared using a 1-way ANOVA followed by Dunnett post hoc comparison, ∗P ≤ .05. (B) Representative binding of ISB 1342 or isotype control on cynomolgus monkey B cells. (C-H) Cynomolgus monkeys (1 male and 1 female) were injected with 3 consecutive doses of ISB 1342 IV (1, 100 and 1000 μg/kg) at days 1, 29, and 57 respectively. Levels of peripheral B cells (C), monocytes (D), CD8⁺ T cells (E), activated CD8⁺ CD69⁺ T cells (F), CD4⁺ T cells (G), and activated CD4⁺ CD69⁺ T cells (H) were measured using flow cytometry. Data are mean ± SD of 10³ counts/μL normalized to baseline counts for 2 animals. (I-J) Levels of circulating IFN-γ (I) and ISB 1342 (J) were measured using ELISA. Data represent levels per animal and LLOQ is the lower limit of quantification for the assay. ELISA, enzyme-linked immunosorbent assay; LLOQ, lower limit of quantitation.