The IL-7R antagonist lusvertikimab reduces leukemic burden in xenograft ALL via antibody-dependent cellular phagocytosis

Abstract

Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursors (BCP-ALL) or T cells (T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R-targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)-targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that ∼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of BCP- and T-ALL patient-derived xenografts (PDX) in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV-mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.

Graphical abstract

Figure 1.

CD127 is expressed in the majority of BCP-ALL and T-ALL cases. CD127 surface expression was prospectively measured via flow cytometry in 371 diagnostic blood or BM samples of patients with BCP-ALL and T-ALL in accordance with International-BFM-FLOW recommendations.^, CD127 low positivity was defined as ≥10% CD127⁺ ALL cells by flow cytometry and high expression as ≥50% CD127⁺ blasts within the CD45dim/CD19⁺ (BCP-ALL) or CD45dim/CD7⁺ (T-ALL) cell population, respectively. (A-C) Pie charts depicting the ratio of patients with CD127-negative, CD127-low, and CD127-high ALL among all analyzed patient samples (A), BCP-ALL (B), and T-ALL cases only within the cohort (C). (D) Comparison of percentage of CD127⁺ ALL cells in BCP-ALL vs T-ALL cases; ∗∗P = .0023, unpaired 2-sided t test. (E) Ratio of CD127⁺ ALL cells in different BCP-ALL subgroups. The stratification relevant lesions ETV6::RUNX1, BCR::ABL1, TCF3::PBX1, and KMT2A-rearrangements (KMT2A-r) were diagnosed by fluorescence in situ hybridization or RNA sequencing. Further genetic alterations (CRLF2-rearrangements [CRLF2-r], TP53-mutations [TP53-mut], and ZNF384-fusions [ZNF384]) were detected within the “B-others” subgroup (BCP-ALL cases without ETV6::RUNX1, BCR::ABL1, TCF3::PBX1, and KMT2A-r). Blue lines show the cutoffs for low and high CD127-positivity. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 2.

LUSV reduces leukemic burden in MRD in vivo models of BCP-ALL and T-ALL. (A-C) Immunodeficient mice were transplanted with PDX cells from 2 different TCF3::PBX1⁺ patients (63.0% and 89.6% CD127⁺ blasts, respectively) and treated with LUSV (5 mg/kg) or a control vehicle (n = 10, respectively) starting the day after injection, modeling an MRD-like situation (IV treatment on day +1, +3, +7, and +14 and every 14 days thereafter as described previously¹²). (A) PB of both, control and LUSV-treated animals was withdrawn when the first PDX mouse showed signs of overt leukemia and compared for the ratio of hCD45⁺/hCD19⁺/mCD45^– cells in the PB as measured via flow cytometry, unpaired 2-sided t test. Animals not showing clinical signs of overt leukemia or >70% PBBs at this time point received further treatment until reaching termination criteria. (B) Therapy-associated differences in the survival of NSG mice were determined using Kaplan-Meier log-rank statistics. (C) The experiment was terminated after 170 days, and BM samples of euthanized animals were analyzed for MRD by PCR for patient–specific Ig/T-cell receptor rearrangements. Overt ALL = mouse showed clinical signs of leukemia upon euthanization; neg, negative (below detection limit). (D-F) A phase 2-like PDX study was performed using CD127-high (≥50% CD127⁺ cells) T-ALL PDX samples (n = 8 patients) including 3 samples from R/R disease. Two NSG mice per patient were injected with PDX cells, randomly assigned into treatment groups and LUSV therapy was conducted in an MRD-like setting. (D) Experimental setup and therapy scheme. (E) Blood of both, control and LUSV-treated animals bearing the same PDX sample was withdrawn when one of the 2 PDX mice showed signs of overt leukemia and the number of hCD45⁺/hCD19⁺/mCD45^– cells in the PB was measured via flow cytometry. The waterfall plot shows the difference in PBBs between respective control and LUSV-treated mice (sorted from weakest therapy response to highest therapy response). Animals not showing clinical signs of overt leukemia or >70% PBBs at this time point received further treatment until reaching termination criteria. (F) Therapy-associated differences in the survival of NSG mice was determined using Kaplan-Meier log-rank statistics. (G-H) Cells from 1 T-ALL PDX (T-ALL PDX number 1) were injected into replicate mice (n = 3 for no-treatment group [control]; n = 5 for treatment groups), animals were subjected to chemotherapy upon 10% PBBs and LUSV immunotherapy (chemotherapy + LUSV) vs mock treatment (chemotherapy only) was initiated upon reoccurrence of PBB (termed “postchemo-MRD” model). (G) Schematic depiction of the experimental setup of the postchemotherapy MRD model. (H) Therapy-associated differences in the survival of NSG mice was determined using Kaplan-Meier log-rank statistics. One mouse in the chemotherapy only group died due to procedural complications. The experiment was terminated after 130 days. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. n.r., not reached.

Figure 3.

LUSV is effective in overt leukemia PDX models. (A-C) A phase 2-like PDX study was performed using 24 PDX samples of patients with BCP-ALL and T-ALL with different CD127 expression levels including 8 samples from relapsed/refractory (R/R) disease. Two NSG mice per patient were injected with PDX cells, randomly assigned into treatment groups, and LUSV therapy was initiated upon detection of 1% PDX cells in the PB, modeling an overt leukemia situation. (A) Blood of both control and LUSV-treated animals bearing the same PDX sample was withdrawn when 1 of the 2 PDX mice showed signs of overt leukemia, and the number of hCD45⁺/hCD19⁺/mCD45^– cells in the PB was measured via flow cytometry. The waterfall plot shows the difference in PBBs between respective control and LUSV-treated mice (ΔPBB, sorted from weakest therapy response to highest therapy response). Animals not showing clinical signs of overt leukemia or >70% PBBs at this time point received further treatment until reaching termination criteria. The dotted line indicates a ΔPBB of 50%. The white asterisk indicates matched samples obtained from initial diagnosis and relapse from the same patient. (B) Therapy-associated differences in the survival of NSG mice were determined using Kaplan-Meier log-rank statistics. The experiment was terminated after 280 days, and 1 LUSV–treated PDX animal was found free of leukemia. (C) The in vivo response to LUSV therapy as depicted via the ΔPBB value was correlated with the ratio of CD127⁺ cells in corresponding PDX samples determined via flow cytometry, Pearson linear regression, confidence interval (0.4010-0.8565). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 4.

LUSV enhanced activity in combination with polychemotherapy. (A-C) A phase 2-like PDX study was performed using 9 PDX samples of patients with BCP-ALL and T-ALL with high CD127-expression levels (CD127^hi). Mice were left untreated (control) or treated with LUSV, ALL induction-like chemotherapy (Chemo), or the combination (Combi). Chemotherapy comprised vincristine, dexamethasone, and PEG-asparaginase cycles as previously published.^,, (A) Schematic depiction of the study setup. (B) Survival was analyzed using the Kaplan-Meier method and log-rank statistics. The P value was adjusted using the Bonferroni method, considering k = 6 comparisons. Hence, a P value of ($\frac{0.05}{\mathrm{k}=6}$) = .0083 was considered at statistically significant. (C) The experiment was terminated after 260 days, and BM samples of sacrificed animals were analyzed for MRD by PCR for patient–specific Ig/T-cell receptor rearrangements. Overt ALL, mouse showed clinical signs of leukemia upon euthanasia. neg, negative (below detection limit); ns, not significant; n.r. = median survival not reached; PCR, polymerase chain reaction; Pos, MRD positivity (MRD level ≥10^–3); neg, MRD negativity (MRD level <10^–3).

Figure 5.

LUSV exhibits direct antileukemic efficacy in ALL cells. Treatment effects of LUSV and IL-7 on leukemic cell survival (A) and proliferation of 2 representative T-ALL PDX specimens (36.2% and 84.1% CD127⁺ cells, respectively) (B). (C) Effect of LUSV treatment on the level of IL-7 induced STAT5 phosphorylation (P-STAT5) in leukemic cells, identifying IL-7 responsive and unresponsive T- and BCP-ALL cell lines and PDX cells, as indicated (also compare supplemental Table 2). (D) Immunodeficient mice were transplanted with PDX cells from an TCF3::PBX1⁺ patient (83.4% CD127⁺ blasts) and treated with LUSV (5 mg/kg) or a control vehicle (n = 10, respectively) starting when 1% PDX cells were detected in the PB (IV treatment on day +1, +3, +7, +14 and every 14 days thereafter). Survival was analyzed using the Kaplan-Meier method and log-rank statistics.

Figure 6.

LUSV eradicates ALL cells via ADCP. (A) Representative images of an in vitro ADCP assay with primary human macrophages (labeled in green) on T-ALL PDX cells (labeled in red) after isotype control or LUSV treatment. (B) Level of in vitro human primary macrophage-mediated phagocytosis induced by increasing concentrations of LUSV on BCP- and T-ALL cell lines as indicated. Correlation between the level of in vitro THP1–mediated phagocytosis induced by LUSV treatment and (C) the level of CD127 expression on leukemic cells or with (D) in vivo efficacy of LUSV treatment (difference in PBBs between respective control and LUSV-treated mice, ΔPBB). Correlation P value and r were measured through the Pearson method. (E-G) Normalized in vivo leukemic burden after control, LUSV-LALAPG, or LUSV treatments in mice injected with either the T-ALL cell line DND41 (IL-7R GoF mutated) (E), the BCP-ALL cell line NALM6 (IL-7 unresponsive) (F), or the T-ALL cell line HPB-ALL (IL-7 responsive) (G). Differences in leukemic burden were assessed using the unpaired Student t test. ∗P < .05; ∗∗P < .01.