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. 2026 May 26;10(16):5653–5668. doi: 10.1182/bloodadvances.2025019380

A PBD dimer–containing antibody-drug conjugate targeting CCRL2 for high-risk MDS/AML including TP53-mutated disease∗

Nour Sabiha Naji 1, Taha Ahmedna 1,2, Xinghan Zeng 1, Yuju An 1, Yashvi Hemani 1, Brandy Perkins 1, Zanshe Thompson 3, Tushar D Nichakawade 2, Yanyan Wang 1, Bum Seok Lee 2, Evangeline Watson 2, Theodora Chatzilygeroudi 1, Li Luo 3, Bogdan Paun 1, Melanie Klausner 4, Teodora Supeanu 1, Ivana Gojo 1, Gabriel Ghiaur 1, Amy E DeZern 1, Mark J Levis 1, Linda Resar 3, Richard J Jones 1, J David Peske 5, Styliani Karanika 6, Suman Paul 1,2, Theodoros Karantanos 1,∗
PMCID: PMC13499191  PMID: 42160756

Key Points

  • •

    Anti-CCRL2 ADC shows prominent antileukemic effect in high-risk MDS/AML, including TP53-mutated in vitro and in vivo models.

  • •

    Anti-CCRL2 ADC demonstrates no significant toxicity against healthy hematopoietic cells and systemic toxicity in mice.

Visual Abstract

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Abstract

Patients with myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) with high-risk features including TP53 mutations have poor outcomes because of the lack of effective therapies. The atypical chemokine surface receptor C-C motif chemokine receptor-like 2 (CCRL2) is overexpressed in MDS and secondary AML compared with healthy hematopoietic cells, and we recently found that TP53-mutated MDS/AML and AML with erythroid features express the highest levels of this receptor across MDS/AML subtypes. To illustrate the therapeutic potential of CCRL2 as a therapeutic target, we developed an anti-CCRL2 antibody-drug conjugate (ADC) by conjugating an anti-CCRL2 antibody with the cytotoxic drug pyrrolobenzodiazepine (PBD), which causes DNA double-strand breaks, leading to cancer cell death. Anti-CCRL2 ADC demonstrated strong CCRL2-selective cytotoxicity associated with DNA damage against cell lines derived from patients with MDS/AML with TP53 mutations and erythroid features, surpassing the cytotoxic effects observed with gemtuzumab and PBD-conjugated anti-CD33 and anti-CD123 ADCs. It also induced apoptosis and suppressed the clonogenicity of primary MDS/AML bone marrow samples without affecting the survival, differentiation, and clonogenicity of healthy hematopoietic stem and progenitor cells. This agent also suppressed the leukemic growth of TP53-mutated MDS/AML cell line xenografts, improving mice survival and decreasing the leukemic burden in patient-derived TP53-mutated MDS/AML xenografts. In conclusion, our study introduces CCRL2 as a potential new therapeutic target in high-risk MDS/AML, including TP53-mutated subsets.

Introduction

Patients with high-risk myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML) continue to exhibit poor outcomes, especially in the presence of high-risk features, such as TP53 mutations or deletions.1, 2, 3 Thus, the development of effective therapies is urgently needed for these individuals. Antibody-drug conjugates (ADCs)4, 5, 6 show prominent activity in lymphoid cancers7, 8, 9 and good-risk AML10,11; however, they demonstrate relatively limited efficacy in high-risk MDS/AML.12 As a result, there are currently no available effective ADCs to be used as single-agent therapies or incorporated in combinational therapies for patients with these disease subsets.

The atypical chemokine receptor C-C motif chemokine receptor-like 2 (CCRL2) is normally expressed in differentiated myeloid cells, contributing to the regulation of cell migration in inflammation sites.13, 14, 15 This surface receptor is upregulated in MDS and sAML, whereas normal hematopoietic progenitors demonstrate minimal expression.16 CCRL2 silencing suppresses MDS/sAML cell growth and sensitizes them to hypomethylating agents but does not affect the survival and clonogenicity of healthy CD34+ cells.16,17 We recently discovered that across MDS/AML subtypes, TP53-mutated MDS/AML and AML with erythroid features express the highest CCRL2 levels. Intriguingly, TP53 deletion in TP53 wild-type (WT) AML cells caused a direct prominent CCRL2 upregulation.18 The main downstream target of CCRL2 is interferon gamma signaling, which has been associated with AML clonal evolution, TP53 deletion, acquisition of erythroid features, and treatment resistance.18, 19, 20 Thus, CCRL2 is potentially a promising target in high-risk MDS/sAML.

In this study, we show that an ADC targeting CCRL2 exhibits significant antileukemic activity in high-risk MDS/AML, including TP53-mutated cells, but has limited effect against normal hematopoietic cells and no significant systemic toxicity in murine models.

Material and methods

Cell lines and reagents

TF-1, SKM1, SET2, and MV4-11 cell lines were purchased from the American Type Culture Collection. F36P, OCI-AML3, and MOLM13 cells were purchased from Leibniz Institute DSMZ.21 Culture conditions and reagents are described in the supplemental Material and methods.

ADC generation

ADC generation is described in the supplemental Materials and methods. High-performance liquid chromatography methods and drug-antibody ratio calculations were described previously.22,23

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay

TF-1, F36P, SET2, MOLM13, MV4-11, and SKM1 cells were plated in 96-well plates (7500 cells per well) and treated for 6 days with anti-CCRL2, anti-CD33, anti-CD123, gemtuzumab, and immunoglobulin G2a (IgG2a) antibodies. For the pretreatment experiment, cells were treated with 500nM anti-CCRL2 antibody or IgG2a for 2 hours before exposure to conjugated IgG2a or anti-CCRL2 ADC. The MTT assay methodology is described in the supplemental Material and methods.

Flow cytometry analysis

Flow cytometry analysis is described in the supplemental Material and methods.

Immunofluorescence and confocal microscopy

Antibodies are described in the supplemental Materials and methods. Cell fixation, permeabilization, staining methods, and coverslip mounting to slides using Prolong Gold Antifade with DAPI (4′,6-diamidino-2-phenylindole) have been described previously.24, 25, 26 Images were acquired using a Zeiss LSM700 confocal microscope, cropped and adjusted for contrast using the Zeiss Zen software.

Western blotting

Protein collection and western blotting were performed as previously reported18 and are described in detail in the supplemental Materials and methods.

CCRL2 and TP53 knockout (KO)

Transfection and transduction are described in the supplemental Materials and methods.

Colony formation assay

Clonogenic assays were conducted as previously reported and are described in detail in the supplemental Materials and methods.17,18

Patients and sample processing

Specimen collection and processing are described in the supplemental Material and methods.

Treatment of C57BL/6 mice

Treatment of C57BL/6 mice is described in the supplemental Material and methods.

Cell line xenografts

Luciferase+ TF-1, SKM1, and F36P cells were injected into 8- to 10-week-old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (106 cells per mouse).16,17 Disease burden monitoring and treatments are described in the supplemental Material and methods.

Patient-derived multihit TP53-mutated MDS/AML xenografts

Mononuclear cells (MNCs) collected from marrow aspirates of multihit TP53-mutated patients with MDS/AML were injected into sublethally irradiated (1.5 Gy) 8- to 10-week-old female NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloySzJ (NSGS) mice (5 × 105 cells per mouse). Bone marrow aspirates and peripheral blood were collected from mice on day 30, and based on disease burden, engrafted mice (≥2% human CD45+ [hCD45+] cells in bone marrow) were randomized to receive conjugated IgG2a or anti-CCRL2 ADC (1 mg/kg). On day 55, peripheral blood, spleen, liver, and bone marrow were collected from mice to assess disease burden by flow cytometry and immunohistochemical analysis. Institutional animal care and use committee guidelines were followed.

Immunohistochemistry

After preparation of paraffin-embedded bone marrow, spleen, and liver sections, slides were prepared and stained with hematoxylin and eosin stain.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). The Mann-Whitney U test was used to compare the statistical significance of 2 groups. One-way analysis of variance was used for comparisons among 3 or more groups. Kaplan-Meier analysis and the Mantel-Cox log-rank test were used to compare mice survival. Standard deviation was used to assess centrality and dispersion.

Results

Anti-CCRL2 ADC is cytotoxic against MDS/AML cell lines

To identify the best payload for the development of an ADC with high activity against high-risk MDS/AML, 5 commonly used ADC payloads (mertansine, monomethyl auristatin E, exatecan, 7-ethyl-10-hydroxycamptothecin, and pyrrolobenzodiazepine [PBD]) were compared, and PBD showed the highest cytotoxicity against TP53-mutated TF-1, F36P, and SKM1 cells expressing high levels of CCRL2 (supplemental Figure 1A-C). This effect was not affected by CCRL2 expression based on the comparison of CCRL2 WT or KO cells (supplemental Figure 1D). Because our primary objective was the selective killing of CCRL2-expressing malignant myeloid cells with minimal off-target toxicity, we selected the highly potent PBD payload, whose in vitro activity allowed us to attribute cytotoxicity to CCRL2-dependent antibody binding rather than to bystander killing. The PBD dimer SG319927–conjugated ADC loncastuximab has received approval from the US Food and Drug Administration for the treatment of high-grade B-cell lymphomas.28,29 Thus, an anti-CCRL2 ADC was developed by conjugating a monoclonal antihuman CCRL2 antibody with PBD. A PBD-conjugated IgG2a antibody was used as a control. Conjugations were confirmed by high-performance liquid chromatography (supplemental Figure 2A-B), showing a drug-antibody ratio of ∼3.7 (supplemental Table 1).

To assess the selective efficacy of anti-CCRL2 ADC, CCRL2 WT and KO TF-1, F36P, and SET218 cells were treated with 0 to 300 ng/mL of ADCs for 6 days. Anti-CCRL2 ADC had significantly higher cytotoxicity in WT cells compared with KO cells and cells treated with conjugated IgG2a (Figure 1A-B; supplemental Figure 2C). Doxycycline-inducible CCRL2 TF-1 cells, which were genetically engineered to express CCRL2 under doxycycline,17 were used. The activity of anti-CCRL2 ADC was significantly higher in cells treated with doxycycline compared with untreated cells (Figure 1C). Finally, to confirm CCRL2-selective cytotoxicity, TF-1, F36P, and SKM1 cells were pretreated with 500nM anti-CCRL2 antibody or an IgG2a isotype control, followed by exposure to anti-CCRL2 ADC. Pretreatment with anti-CCRL2 antibody significantly reduced the cytotoxic effect of anti-CCRL2 ADC (Figure 1D), providing additional support for the selectivity of anti-CCRL2 ADC for CCRL2-expressing cells.

Figure 1.

Figure 1.

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Anti-CCRL2 ADC is cytotoxic against MDS/AML cell lines. (A-B) CCRL2 WT and KO TF-1 and F36P cells were treated with increasing doses of the ADCs (0 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, and 300 ng/mL) for 6 days and showed higher toxicity in TF-1 WT (IC50 = 15 ng/mL; P < .001) and F36P WT (IC50 = 23 ng/mL; P < .001) cells treated with anti-CCRL2 ADC compared with KO cells (IC50 = 406 ng/mL for TF-1 KO; IC50 = 46 714 ng/mL for F36P KO) or cells treated with the conjugated IgG2a. (C) Doxycycline-inducible CCRL2 TF-1 cells were treated with increasing doses of anti-CCRL2 ADC in the presence or absence of 10 ng/mL doxycycline, showing higher cytotoxicity in cells treated with doxycycline (IC50 = 3 ng/mL vs 74 ng/mL, respectively; P = .008). (D) TF-1, F36P, and SKM1 cells were pretreated with 500nM IgG2a or anti-CCRL2 antibodies for 2 hours, followed by treatment with anti-CCRL2 ADC (0-300 ng/mL). Pretreatment with anti-CCRL2 ADC significantly reduced the sensitivity of MDS/AML cells to anti-CCRL2 ADC (IC50 = 18.66 ng/mL for IgG2a-treated TF-1 cells vs IC50 = 261.7 for anti-CCRL2–treated TF-1 cells, P < .001; IC50 = 18 ng/mL for IgG2a-treated F36P cells vs IC50 = 255 for anti-CCRL2–treated F36P cells, P < .001; IC50 = 9.57 ng/mL for IgG2a-treated SKM1 cells, IC50 = 240.7 ng/mL for anti-CCRL2–treated SKM1 cells). (E) TF-1, F36P, SKM1, and MDS-L cells were treated with 20 or 40 ng/mL of anti-CCRL2 ADC or 40 ng/mL anti-IgG2a ADC. Apoptosis was measured by flow cytometry using 7-AAD staining. Anti-CCRL2 ADC induced apoptosis in TF-1 (P = .002 for 20 ng/mL; P < .001 for 40 ng/mL), F36P (P = .003 for 20 ng/mL; P < .001 for 40 ng/mL), SKM1 (P < .001 for 20 ng/mL and 40 ng/mL), and MDS-L (P = .008 for 20 ng/mL; P < .001 for 40 ng/mL) cells compared with anti-IgG2a ADC after 6 days of treatment. (F) TF-1, F36P, SKM1, and MDS-L cells were treated with 10 and 20 ng/mL of anti-CCRL2 ADC. Anti-CCRL2 ADC suppressed clonogenicity of TF-1 (P = .0013 for 10 ng/mL; P < .0001 for 20 ng/mL), F36P (P = .0336 for 10 ng/mL; P = .0003 for 20 ng/mL), SKM1 (P = .004 for 10 ng/mL; P < .001 for 20 ng/mL), and MDS-L (P < .001 for 10 ng/mL and 20 ng/mL) cells. Each experiment was repeated 3 times. 7-AAD, 7-aminoactinomycin D; IC50, 50% inhibitory concentration.

Treatment with 20 or 40 ng/mL anti-CCRL2 ADC induced apoptosis in TF-1, F36P, SKM1, and MDS-L cells compared with treatment with 40 ng/mL conjugated IgG2a (Figure 1E). Similarly, 10 and 20 ng/mL anti-CCRL2 ADC suppressed clonogenicity in these cells compared with treatment with the same doses of conjugated IgG2a (Figure 1F).

Collectively, anti-CCRL2 ADC shows CCRL2-selective cytotoxicity against MDS/AML cell lines in vitro.28,30,31

Anti-CCRL2 ADC is internalized by MDS/AML cells and induces DNA damage

To elucidate the possible mechanisms implicated in anti-CCRL2 ADC–mediated MDS/AML cell death, internalization and intracellular trafficking of anti-CCRL2 ADC were assessed. Analysis of TF-1 and F36P cells treated with 10 μg/mL anti-CCRL2 by immunofluorescence and confocal microscopy showed that the antibody was taken up 30 minutes after treatment, with prominent lysosomal colocalization at 4 hours (Figure 2A; supplemental Figure 2D). To assess the downstream consequences of anti-CCRL2 ADC, the induction of γH2AX loci was also assessed. Immunofluorescence with confocal microscopy revealed that 20 and 40 ng/mL anti-CCRL2 ADC induces γH2AX loci in SKM1 and TF-1 cells, respectively, after 24 hours (Figure 3B). Given our reported impact of CCRL2 expression on interferon-γ signaling, the effect of anti-CCRL2 ADC on this pathway was assessed. Treatment with 20 ng/mL anti-CCRL2 ADC reduced signal transducer and activator of transcription 1 phosphorylation after 24 hours in TF-1 cells compared with treatment with conjugated IgG2a; however, this effect was not maintained after 48 hours and was not observed in SKM1 cells (supplemental Figure 2E).

Figure 2.

Figure 2.

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Anti-CCRL2 ADC is internalized by MDS/AML cells, inducing DNA damage, and shows higher cytotoxicity in MDS/AML cell lines compared with gemtuzumab and PBD-conjugated ADCs targeting CD33 and CD123. (A) Treatment of TF-1 cells with 10 μg/mL anti-CCRL2 antibody led to antibody internalization after 30 minutes of treatment and lysosomal colocalization after 4 hours of treatment based on concurrent staining with CCRL2, DAPI, and the lysosomal marker LAMP1. Areas of colocalization are shown with white arrows. (B) Treatment of SKM1 cells with 20 ng/mL anti-CCRL2 and TF-1 cells with 40 ng/mL anti-CCRL2 ADC led to induction of γH2AX loci after 24 hours of treatment compared with conjugated IgG2a-treated cells. (C) The IC50 of anti-CCRL2 ADC is similar or higher compared with gemtuzumab and PBD-conjugated anti-CD33 and anti-CD123 ADCs in MOLM13, OCI-AML3, and MV4-11 cells, but lower compared with gemtuzumab and PBD-conjugated anti-CD33 and anti-CD123 in TF-1 and F36P and PBD-conjugated anti-CD33 and anti-CD123 in SKM1 cells. (D) TP53 deletion by CRISPR-Cas9 gene editing in MOLM13 and MV4-11 cells significantly increased the IC50 of gemtuzumab and anti-CD123 and anti-CCRL2 ADCs. (E) The relative increase in IC50 in TP53-deleted cells was significantly lower for anti-CCRL2 ADC compared with anti-CD123 ADC and gemtuzumab. Statistical significance is denoted as ∗P < .010 and ∗∗ P < .001. Each experiment was repeated 3 times.

Figure 3.

Figure 3.

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Anti-CCRL2 ADC induces apoptosis and suppresses clonogenicity in MDS/AML primary cells. (A) Genomic landscape of patients with MDS/AML (de novo TP53 WT, MDS-related TP53 WT, and TP53-mutated) included in the analysis of primary samples. (B) Treatment with 20 ng/mL and 40 ng/mL anti-CCRL2 ADC increased apoptosis in primary MDS/AML cells. Treatment with 20 ng/mL and 40 ng/mL anti-CCRL2 ADC suppressed clonogenicity of the primary MDS/AML samples. Of note, 2 of the TP53-mutated MDS/AML samples did not grow colonies in methylcellulose. (C) CCRL2 expression in blasts gated by high CD45 and low SSC-A by flow cytometry is higher in patients with TP53-mutated acute erythroid leukemia compared with the rest of the groups, and patients with TP53-mutated MDS/AML have overall relatively higher CCRL2 expression compared with de novo AML (P = .063). (D) The effect of anti-CCRL2 ADC in primary samples, defined as the ratio of apoptosis with 40 ng/mL anti-CCRL2 ADC to apoptosis with 40 ng/mL IgG2a, was positively associated with CCRL2 surface expression (R2 = 0.407; P = .004). Statistical significance is denoted as ∗P < .01 and ∗∗P < .001. Each primary sample was analyzed in 3 replicates, and the median value was used for further analysis. SSC-A, side scatter area.

Anti-CCRL2 ADC shows higher cytotoxicity against TP53-mutated MDS/AML cell lines compared with gemtuzumab and PBD-conjugated ADCs targeting CD33 and CD123

Next, to compare the efficacy of ADCs that target CCRL2 with the efficacy of ADCs against other targets, PBD-conjugated ADCs targeting the well-described AML targets CD33 and CD123 were developed (supplemental Figure 3A-B). Cytotoxicity of the calicheamicin-conjugated ADC gemtuzumab and the PBD-conjugated anti-CD33, anti-CD123, and anti-CCRL2 ADCs was assessed in AML cell lines derived from patients with de novo/TP53-WT AML (MOLM13, OCI-AML3, and MV4-11) and MDS/AML cell lines (TF-1 erythroleukemia cells, F36P MDS–related erythroleukemia cells, and SKM1 TP53–mutated MDS/AML cells). Consistent with our previously published data, CCRL2 expression is significantly higher in MDS/AML cell lines with erythroleukemic features and TP53 mutations compared with AML cell lines derived from patients with de novo/TP53 WT AML (supplemental Figure 3C). The expression of CD33 was higher in F36P and TF-1 cells compared with the rest of the cell lines tested (supplemental Figure 3C), whereas the expression of CD123 was overall higher in de novo/TP53 WT AML cell lines compared with those exhibiting erythroleukemic features and TP53 mutations (supplemental Figure 3C).

AML cell lines derived from de novo/TP53 WT AML were overall more sensitive to ADC treatment compared with MDS/AML cell lines (Figure 2C; supplemental Figure 4). Particularly, the activity of gemtuzumab and the PBD-conjugated ADCs targeting CD33 and CD123 was found to be significantly decreased in TP53-mutated MDS/AML cell lines compared with AML cell lines derived from de novo/TP53 WT AML (Figure 2C; supplemental Figure 4). Anti-CCRL2 ADC showed cytotoxicity similar to or lower compared with cytotoxicity of gemtuzumab and PBD-conjugated ADCs targeting CD33 and CD123 in AML cell lines derived from de novo/TP53 WT AML (Figure 2C; supplemental Figure 4). However, within TP53-mutated MDS/AML cell lines, anti-CCRL2 ADC showed the highest cytotoxicity in TF-1 and F36P cells and the second highest cytotoxicity in SKM1 cells (Figure 2C; supplemental Figure 4).

To test the impact of TP53 deletion in the cytotoxic activity of these ADCs, isogenic TP53 WT and KO MOLM13 and MV4-11 cell lines were developed by CRISPR-Cas9 gene editing (supplemental Figure 5A). Treatment of WT and KO lines with gemtuzumab, anti-CD123, and anti-CCRL2 ADCs showed that TP53 deletion significantly decreased their sensitivity to all ADCs (Figure 2D; supplemental Figure 5B-C). However, the relative increase of 50% inhibitory concentration in TP53 KO cells compared with WT cells was significantly higher for gemtuzumab and anti-CD123 ADC compared with anti-CCRL2 ADC (Figure 2E).

Overall, the activity of anti-CCRL2 ADC appears to be higher compared with the other ADCs against TP53-mutated MDS/AML cell lines and appears less affected by TP53 deletion.

Anti-CCRL2 ADC induces apoptosis and suppresses clonogenicity in MDS/AML primary cells

Next, the activity of anti-CCRL2 ADC in primary MNCs from patients with MDS/AML was analyzed. Based on our published data supporting the conclusion of higher expression of CCRL2 in TP53-mutated MDS/AML and in myeloid neoplasms with erythroid features compared with de novo AML,16,18 4 patients with de novo/TP53 WT AML, 4 patients with TP53 WT MDS–related AML, and 10 patients with multihit TP53-mutated (complex karyotype and TP53 mutation with a variant allele frequency of ≥50%) MDS/AML, including 3 individuals with acute erythroid leukemia (supplemental Tables 2 and 3; Figure 3A), were included. Cells were treated with 20 or 40 ng/mL anti-CCRL2 ADC or 20 ng/mL conjugated IgG2a for 5 days. Treatment with 10 and 20 ng/mL anti-CCRL2 ADC increased apoptosis and suppressed clonogenicity compared with treatment with conjugated IgG2a (Figure 3B). Fluorescence in situ hybridization of isolated colonies from multihit TP53-mutated treated samples confirmed the presence of 17p and 5q deletions and trisomy 8 in isolated cells (supplemental Figure 6). To assess if CCRL2 expression is associated with the cytotoxicity of anti-CCRL2 ADC, the expression of CCRL2 in the surface of primary blasts was measured by flow cytometry (Figure 3C). Consistent with our previously reported results,16,18 acute erythroid leukemia blasts expressed the highest levels of CCRL2, and TP53-mutated MDS/AML blasts expressed relatively higher CCRL2 levels compared with de novo AML blasts (Figure 3C). Linear regression showed a modest positive correlation between CCRL2 expression and the apoptotic effect of anti-CCRL2 ADC, defined as the ratio of apoptosis induced by 40 ng/mL anti-CCRL2 ADC to apoptosis induced by 40 ng/mL IgG2a (Figure 3D). No significant correlation was observed between the apoptotic effect of anti-CCRL2 ADC and TP53 variant allele frequency among the 10 samples from patients with TP53-mutated MDS/AML.

Taken together, our results support the conclusion that anti-CCRL2 ADC promotes apoptosis and suppresses the clonogenicity of primary MDS/AML cells. Although de novo AML samples demonstrated similar cytotoxicity compared with MDS/AML and TP53-mutated samples, higher CCRL2 expression was associated with a better response. These findings suggest that the observed antileukemic activity in TP53-mutated MDS/AML and, particularly, erythroid leukemia is likely associated with higher target expression and not cell-intrinsic sensitivity to the ADC.

Anti-CCRL2 ADC does not affect healthy hematopoietic cells and does not cause systemic toxicity in vivo

To assess the effect of anti-CCRL2 ADC on healthy hematopoietic cells, CD34+ cells sorted from the bone marrow of 3 independent healthy donors (supplemental Table 2) were treated with 100 ng/mL conjugated IgG2a or anti-CCRL2 ADC, which is a substantially higher dose compared with the doses selected to treat primary MDS/AML samples for 5 days. Treatment with anti-CCRL2 ADC did not induce apoptosis (Figure 4A), affect differentiation (Figure 4B), or suppress the clonogenicity (Figure 4C) of healthy CD34+ cells. Given that CCRL2 is overexpressed in leukemias with erythroid differentiation and that its deletion decreases the survival and clonogenicity of erythroleukemic cells,18 the effect of anti-CCRL2 ADC on healthy erythroid progenitors was assessed. MNCs from 3 additional healthy bone marrow donors were treated with 100 ng/mL anti-CCRL2 or conjugated IgG2a for 5 days. Treatment with anti-CCRL2 ADC did not alter the percentages of erythroid progenitors CD71+CD235a−, CD71+CD235a+, or CD71−CD235a+ (Figure 4D).

Figure 4.

Figure 4.

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Anti-CCRL2 ADC does not affect healthy hematopoietic cells and does not cause systemic toxicity in vivo. (A-B) Treatment of sorted CD34+ cells from 2 independent healthy bone marrow donors with 100 ng/mL of conjugated IgG2a or anti-CCRL2 ADC for 5 days did not increase apoptosis (A) and did not alter the percentage of CD34+ or CD34+CD38− (B). (C) There was no effect on clonogenicity of healthy CD34+ cells after treatment with 100 ng/mL anti-CCRL2 ADC. (D) Treatment of MNCs from 2 different healthy bone marrow donors with 100 ng/mL anti-CCRL2 ADC resulted in no significant alteration of the percentages of erythroid progenitors (CD71+CD235a−, CD71+CD235a+, or CD71−CD235a+). (E) CBC were tested in 14 C57BL/6 mice, which were then randomized to receive either anti-CCRL2 ADC (7 mice) or conjugated IgG2b (7 mice; 1 mg/kg IV). Mice weight was measured weekly, and CBC and chemistry panels were analyzed 4 weeks after treatment. Mice were harvested, and kidney and spleen weights were measured. (F) Weekly weight changes in mice treated with anti-CCRL2 ADC or conjugated IgG2b. (G) Hgb, HCT, WBC, and PLT count before and after treatment did not differ between mice treated with anti-CCRL2 ADC or conjugated IgG2b. (H) Chemistry panels did not differ between mice treated with anti-CCRL2 ADC or conjugated IgG2b. CBC, Complete blood counts; HCT, hematocrit; Hgb, hemoglobin; PLT, platelets; WBC, white blood cells.

To further characterize the safety profile of this agent in vivo, an antimouse CCRL2 ADC was developed (supplemental Figure 7A). Healthy 6- to 8-week-old female C57BL/6 mice were treated IV with 1 mg/kg anti-CCRL2 ADC or conjugated IgG2b (7 mice per group; Figure 4E). No significant weight changes or systemic toxicities were observed after treatment (Figure 4F), and the weights of the spleen and kidney was not affected by treatment 4 weeks after injection (supplemental Figure 7B). Consistently, no significant alterations were found on the mice complete blood counts (Figure 4G; supplemental Figure 7C) or chemistry panels (Figure 4H; supplemental Figure 7D).

Collectively, our results support the conclusion that anti-CCRL2 ADC has a relatively good safety profile, with no prominent effect on healthy hematopoiesis.

Anti-CCRL2 ADC suppresses leukemic growth and improves the survival in TP53-mutated MDS/AML cell line xenografts

To test the in vivo efficacy of 1 dose of anti-CCRL2 ADC in TP53-mutated MDS/AML models, TF-1 and SKM1 xenografts were treated. Luciferase+ TF-1 cells were injected IV into NSG mice. On day 15, to ensure balanced baseline tumor burdens, mice were stratified into groups with comparable bioluminescence signals and received anti-CCRL2 ADC or conjugated IgG2a (0.75 mg/kg, 1 dose) (Figure 5A). Mice treated with anti-CCRL2 ADC showed suppressed leukemic growth (Figure 5B) and improved survival compared with those treated with conjugated IgG2a (median survival, 47 vs 37 days; Figure 5C). Luciferase+ SKM1 cells were injected IV into NSG mice, which were randomized based on their bioluminescence signal on day 11 to anti-CCRL2 ADC or conjugated IgG2a (1 mg/kg, 1 dose; Figure 5D). Treatment with anti-CCRL2 ADC suppressed leukemic growth (Figure 5E) and significantly prolonged mice survival compared with treatment with conjugated IgG2a (median survival, 39 vs 9.5 days; Figure 5F). Of note, mice engrafted with SKM1 cells exhibited particularly enlarged liver at the time of their death. Mice treated with anti-CCRL2 ADC had significantly lower liver weights compared with those treated with IgG2a (Figure 5G).

Figure 5.

Figure 5.

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Anti-CCRL2 ADC suppresses the leukemic growth of TF-1 and SKM1 xenografts. (A) Luciferase+ TF-1 cells were injected IV into 12 NSG mice (106 per mouse). On day 15 after injection, mice were randomized based on their bioluminescence signal to receive 1 IV dose of 0.75 mg/kg of anti-CCRL2 ADC (6 mice) or conjugated IgG2a (6 mice) and monitored until day 65. (B) Mice treated with anti-CCRL2 ADC showed suppressed leukemic growth by bioluminescence signal. (C) Mice treated with anti-CCRL2 ADC had improved overall survival (median survival, 47 vs 37 days) compared with mice treated with IgG2a (P = .015). (D) Luciferase+ SKM1 was injected IV into 14 NSG mice (106 per mouse). On day 10 after injection, mice were randomized based on their bioluminescence signal to receive 1 IV dose of 1 mg/kg of either anti-CCRL2 ADC (8 mice) or IgG2a (6 mice) and monitored until day 50. (E) Mice treated with anti-CCRL2 ADC showed decreased leukemic growth by bioluminescence signal compared with those treated with IgG2a. (F) Mice treated with anti-CCRL2 ADC showed significantly improved overall survival compared with mice treated with IgG2a (39 vs 9.5 days; P < .0001). (G) Mice treated with anti-CCRL2 ADC had lower liver weights compared with those treated with conjugated IgG2a.

Next, the effect of multiple anti-CCRL2 ADC doses was tested in the least aggressive TP53-mutated MDS/AML F36P xenograft model. On day 15 after IV injection of luciferase+ F36P cells, mice received 1 mg/kg anti-CCRL2 ADC or conjugated IgG2a (Figure 6A; supplemental Figure 8A). On day 35, mice were sacrificed and those treated with anti-CCRL2 ADC had a significantly lower percentage of hCD45+ cells (hCD45+%) in their bone marrow compared with those treated with conjugated IgG2a (Figure 6B). Another group of F36P xenografts received 2 doses of 1 mg/kg of anti-CCRL2 ADC or conjugated IgG2a on day 15 and 35 (Figure 6C). Mice treated with anti-CCRL2 ADC demonstrated suppressed leukemic growth (Figure 6D), improved survival (median survival, 55 vs 38.5 days; Figure 6E), and decreased hCD45+% in bone marrow compared (Figure 6F) with those treated with conjugated IgG2a.

Figure 6.

Figure 6.

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Multiple anti-CCRL2 ADC doses suppress the leukemic growth of F36P xenografts. (A) Luciferase+ F36P cells were injected IV into 10 NSG mice (106 per mouse), and on day 15, they were randomized to receive 1 IV dose of 1 mg/kg of either anti-CCRL2 ADC (5 mice) or IgG2a (5 mice) and were harvested on day 35. (B) Mice treated with anti-CCRL2 ADC had significantly lower percentage of hCD45+ cells in bone marrow (P = .008) compared with those treated with conjugated IgG2a. (C) Luciferase+ F36P cells were injected IV into 8 NSG mice (106 per mouse), and on day 15, they were randomized to receive 1 IV dose of 1 mg/kg of either anti-CCRL2 ADC (4 mice) or IgG2a (4 mice). On day 35, mice received a second dose of 1 mg/kg of anti-CCRL2 ADC or IgG2a and were monitored until day 75. (D) Mice treated with 2 anti-CCRL2 ADC doses showed suppressed leukemic growth by bioluminescence signal. (E) Mice treated with 2 anti-CCRL2 ADC doses had significantly improved overall survival compared with mice treated with conjugated IgG2a (55 vs 38.5 days; P = .0285), with 3 of 4 mice surviving until day 75. (F) Mice treated with 2 anti-CCRL2 ADC doses had decreased percentage of hCD45+ cells in bone marrow compared with mice treated with conjugated IgG2a (P = .029).

Taken together, these results support the conclusion that treatment with anti-CCRL2 ADC suppresses the leukemic growth of TP53-mutated MDS/AML cell line xenografts.

Anti-CCRL2 ADC suppresses leukemic growth in patient-derived multihit TP53-mutated MDS/AML xenografts

Next, the efficacy of anti-CCRL2 ADC in patient-derived xenografts was assessed. MNCs from 5 patients with TP53-mutated MDS/AML (supplemental Table 4) were injected into sublethally irradiated (1.5 Gy) NSGS mice. Engraftment was assessed on day 30 by flow cytometric analysis of peripheral blood and bone marrow aspirate (Figure 7A). Three out of the 5 patient samples were successfully engrafted (≥2% hCD45+ cells in bone marrow) in a total of 11 mice (Figure 7A). To ensure balanced baseline tumor burdens, mice were stratified into groups with comparable hCD45+% in bone marrow, received anti-CCRL2 ADC or conjugated IgG2a (1 mg/kg IV), and harvested on day 55 (Figure 7A). Mice that were treated with anti-CCRL2 ADC showed a reduction or stabilization of hCD45+% in peripheral blood (Figure 7B) and bone marrow (Figure 7C) compared with those treated with conjugated IgG2a. The hCD45+% in bone marrow was not significantly different between the 2 groups (supplemental Figure 8B), but given the significant variation of hCD45+% at baseline, hCD45+% fold change was assessed and found significantly lower in mice treated with anti-CCRL2 ADC compared with those treated with conjugated IgG2a (Figure 7D). Similarly, mice treated with anti-CCRL2 ADC had significantly lower hCD45+% in their spleen compared with those treated with conjugated IgG2a (Figure 7E).

Figure 7.

Figure 7.

Figure 7.

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Anti-CCRL2 ADC suppresses leukemic growth in patient-derived multihit TP53-mutated MDS/AML xenografts. (A) A scheme summarizing the development and treatment of patient-derived multihit TP53-mutated MDS/AML xenografts. MNCs isolated from bone marrow aspirates of patients with TP53-mutated MDS/AML containing leukemic blasts were injected IV into NSGS mice (5 × 105 per mouse). On day 30, peripheral blood and bone marrow aspirates were collected from mice and engraftment was assessed (≥2% of hCD45+ cells in bone marrow). Human cells were almost uniformly CD34+. Mice were then randomized based on their hCD45+% to receive IV 1 dose of 1 mg/kg of either anti-CCRL2 ADC or conjugated IgG2a. Twenty-five days later, mice were sacrificed to assess disease burden. (B) Mice treated with anti-CCRL2 ADC (5 mice) showed a decline or stabilization of hCD45+% in blood as opposed to mice treated with conjugated IgG2a (6 mice), which showed a prominent increase in hCD45+%. Representative peripheral blood smear showing circulating blasts in an IgG2a-treated mouse, in parallel to differentiated leukocytes in mouse treated with anti-CCRL2 ADC. (C) Mice treated with anti-CCRL2 ADC showed a decline or stabilization of bone marrow hCD45+% as opposed to mice treated with conjugated IgG2a, which showed a prominent increase in bone marrow hCD45+% cells before and after treatment. (D) Mice treated with anti-CCRL2 ADC showed a significantly lower fold increase of hCD45+% in bone marrow compared with those treated with IgG2a (P = .004). (E) Mice treated with anti-CCRL2 ADC had a relatively lower percentage of hCD45+ cells in their spleens compared with those treated with conjugated IgG2a (P = .016). (F) Immunohistochemical analysis of spleen, bone marrow, and liver showed lower leukemic involvement in mice treated with anti-CCRL2 ADC compared with mice treated with IgG2a. Representative figures showing diffuse involvement of leukemic blasts (IgG2a) as opposed to patchy involvement (anti-CCRL2 ADC) in mice engrafted with patient 1 MNCs, leukemic blasts (IgG2a) as opposed to normal bone marrow (anti-CCRL2 ADC) in mice engrafted with patient 2 MNCs, and atypical infiltrates in the liver (IgG2a) as opposed to normal liver (anti-CCRL2 ADC) in mice engrafted with patient 3 MNCs. (G) Mice treated with anti-CCRL2 ADC had a lower MDS/AML involvement score (P = .013) compared with those treated with conjugated IgG2a. Pt1, patient 1.

Pathologic analysis of mouse tissue revealed that mice treated with conjugated IgG2a had more advanced disease involvement in bone marrow, spleen, and liver compared with those treated with anti-CCRL2 ADC (Figure 7F). An MDS/AML involvement score was developed (supplemental Figure 8C) and mice treated with anti-CCRL2 ADC had significantly lower scores compared with those treated with conjugated IgG2a (Figure 7G).

Collectively, these results suggest that treatment with anti-CCRL2 ADC suppresses leukemic growth in patient-derived multihit TP53-mutated MDS/AML xenografts.

Discussion

Patients with high-risk MDS/AML, including those with TP53 mutations and deletions, exhibit relatively poor outcomes because of a lack of effective therapies and a high incidence of treatment resistance.1, 2, 3 ADCs, including the PBD-conjugated loncastuximab, have prominent activity in hematologic malignancies,10,11,28,29 but currently available ADCs show relatively limited activity in high-risk MDS/AML and TP53-mutated disease.12 Thus, we developed an anti-CCRL2 ADC using the PBD payload and showed a significant CCRL2-selective, single-agent antileukemic effect in various MDS/AML models. Anti-CCRL2 antibody showed rapid internalization and lysosomal trafficking in MDS/AML cells and the ADC induced DNA damage within 24 hours of treatment.

Our studies demonstrated that cell lines derived from patients with MDS/AML with erythroid features and TP53 alterations are generally less sensitive to gemtuzumab and PBD-conjugated ADCs targeting CD33 and CD123 compared with cell lines from patients with de novo AML or good- to intermediate-risk disease. Consistently, TP53 deletion reduced the sensitivity of MDS/AML cell lines to ADCs. These findings align with the clinical responses observed in patients with high-risk MDS/AML treated with gemtuzumab.12 In contrast, anti-CCRL2 ADC exhibited consistent efficacy across different cell line subtypes, demonstrating higher activity in cells derived from patients with MDS/AML with erythroid features and TP53 mutations compared with gemtuzumab and PBD-conjugated ADCs targeting CD33 and CD123. Similarly, the cytotoxic effect of anti-CCRL2 ADC was less affected by TP53 deletion compared with other ADCs. These results suggest that the marked upregulation of CCRL2 in high-risk MDS/AML and TP53-mutated cells may offset their inherent resistance to drug toxin–mediated cell killing.

Analysis of anti-CCRL2 ADC activity in primary samples confirmed that it induces apoptosis and suppresses the clonogenic potential of MDS/AML cells. MDS/AML and TP53-mutated samples showed overall similar sensitivity to anti-CCRL2 ADC compared with de novo AML samples, with erythroleukemia samples showing the highest sensitivity. Notably, there was a modest positive association between CCRL2 expression and the apoptotic activity induced by the agent, consistent with findings reported for other ADCs targeting CD33.32, 33, 34 Our group recently demonstrated that TP53-mutated MDS/AML and AML with erythroid features exhibited the highest levels of CCRL2 expression across the spectrum of MDS/AML subtypes.18 These findings support the conclusion that the significant antileukemic effect of anti-CCRL2 ADC in TP53-mutated MDS/AML and, particularly, erythroleukemic samples is likely associated with the prominent target overexpression in these subsets and not with their cell-intrinsic sensitivity to the released toxin. On the contrary, de novo AML samples are probably more sensitive to toxin-mediated cell killing based on our cell line data, thereby counterbalancing their relatively low CCRL2 expression.

PBD-conjugated ADCs targeting CD33 and CD123 showed promising preclinical efficacy in high-risk AML.35,36 However, early-phase clinical trials revealed cytopenia and hepatotoxicity, which hindered further clinical development. Notably, not only gemtuzumab but also bispecific antibodies and chimeric antigen receptor T cells targeting CD33 have been associated with cytopenia.37, 38, 39 This suggests that these adverse effects may be related to the expression of CD33 and CD123 on the surface of healthy hematopoietic cells. Although transient elevations in liver enzymes have been observed with PBD payloads, there have been no reports of fatal hepatotoxicity.28,29 On the contrary, healthy stem and progenitor cells expressed minimal CCRL2 levels.16,17 Consistently, anti-CCRL2 ADCs demonstrated minimal impact on healthy hematopoietic cells and did not induce systemic toxicity or significant changes in blood counts or chemistry panels in C57BL/6 mice, similarly to recent findings from our group.40 If cytopenia or hepatotoxicity arise with anti-CCRL2 ADCs, fractionated dosing may be considered because this approach has enabled the safe and effective use of gemtuzumab. It should be noted, however, that activated differentiated cells, such as neutrophils, monocytes, and endothelial cells involved in inflammatory responses, express high levels of CCRL2,13, 14, 15 which could result in on-target, off-MDS/AML toxicities. This possibility should be carefully considered during the development of this therapeutic agent. Finally, although bystander killing is a recognized property of potent payloads because of their lipophilic DNA–crosslinking warheads, allowing diffusion after linker cleavage41 and payload release, clinical trials on PBD-conjugated ADCs support the conclusion that any potential bystander effect is tolerable.7,29,42 Our in vivo studies demonstrated that anti-CCRL2 ADC effectively suppressed leukemic growth in cell line xenograft models, resulting in improved survival of treated mice. Notably, 2 doses of the agent administered 20 days apart were well tolerated by NSG mice and led to a marked reduction in disease burden. Furthermore, bone marrow aspirates collected from NSGS mice engrafted with TP53-mutated MDS/AML cells enabled randomization to treatment with either anti-CCRL2 ADC or an isotype control and comparison of pre- and posttreatment samples. This approach revealed a significant antileukemic activity of anti-CCRL2 ADC. Despite the marked reduction in disease burden in vivo, most treated mice ultimately experienced disease progression and died from leukemia, suggesting that incorporating this agent into combination therapeutic regimens may be a more effective strategy, particularly for TP53-mutated disease.

Further studies in syngeneic MDS/AML models are required to solidify the efficacy and safety of anti-CCRL2 ADC, but this agent appears to be a promising candidate as a single agent or as part of combination therapies for high-risk MDS/AML, including TP53-mutated disease, especially given the lack of effective antibody-based therapies in these disease subsets. Similarly, conjugating PBD to antibodies targeting other surface antigens that are particularly overexpressed in high-risk MDS/AML and that show very low expression in healthy hematopoietic cells may be an attractive strategy to improve the outcomes of patients with these neoplasms.

Conflict-of-interest disclosure: T.K., S.K., R.J.J, and S.P. are named as inventors on the patent applications filed by Johns Hopkins University that are related to technologies described in this study, and Johns Hopkins University may also file additional patent applications related to the work described in this study. S.P. served as a consultant for Merck and Gritstone Bio; is a cofounder, member of the board of directors, and equity holder of Winnow Therapeutics; owns equity in Gilead; and received payments from IQVIA, Reckner Healthcare, and Curio Science. The aforementioned companies, as well as other companies, have licensed previously described technologies from Johns Hopkins University. Licenses to these technologies are or will be associated with equity or royalty payments to the inventors and to Johns Hopkins University. The terms of all these arrangements are managed by Johns Hopkins University in accordance with its conflict-of-interest policies. The remaining authors declare no competing financial interests.

Acknowledgments

T.K. was supported by National Heart, Lung, and Blood Institute grant K08HL168777, the Leukemia Research Foundation New Investigator Research Grant Program, the MacMillan Pathway to Independence Program Award, and the Hematologic Malignancies and Bone Marrow Transplantation (HBMT) pilot grant award from Johns Hopkins University. S.P. was supported by National Cancer Institute (NCI) grant K08CA270403, the Leukemia & Lymphoma Society Translation Research Program award, the American Society for Horticultural Science award, and Swim Across America Translational Cancer Research award. T.D.N. was supported by NCI grant T32 CA153952. The authors thank Dr. Starczynowski for providing MDS-L cells.

Authorship

Contribution: N.S.N., S.P., X.Z., and T.K. conceived and designed the study; N.S.N. and T.K. wrote the manuscript; T.A., B.S.L., X.Z., Y.H., T.D.N., and S.P. performed antibody-drug conjugation experiments; N.S.N., B. Perkins, T.C., Y.A., X.Z., Y.W., and T.K. performed MTT, apoptosis, and clonogenicity assays using cell lines; N.S.N., Y.A., and X.Z. performed CRISPR-Cas9 gene editing; X.Z. and Y.H. performed immunofluorescence and confocal microscopy; X.Z. and Y.A. performed western blot analyses; B.P. and T.K. processed primary samples and performed in vitro studies using primary cells; M.K. and B.P. performed fluorescence in situ hybridization analyses; Z.T., L.L., and L.R. performed safety studies in C57BL/6 mice; N.S.N., E.W., S.K., S.P., and T.K. performed cell line xenograft studies; N.S.N., E.W., G.G., T.S., S.K., and T.K. performed patient-derived xenograft studies; J.D.P. performed immunohistochemical analyses; I.G., A.E.D., M.J.L., and R.J.J. identified clinical samples and provided clinical information; and S.P., T.K., A.E.D., I.G., G.G., M.J.L., R.J.J., and S.K. interpreted the data and edited the manuscript.

Footnotes

∗

N.S.N., T.A., and X.Z. are joint first authors and contributed equally to this study.

The data that support the findings of this study are available from the corresponding author, Theodoros Karantanos (tkarant1@jhmi.edu), on request.

The full-text version of this article contains a data supplement.

Supplementary Material

Supplemental Figures, Methods, and Tables
BLOODA_ADV-2025-019380-mmc1.pdf (1.2MB, pdf)

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Supplementary Materials

Supplemental Figures, Methods, and Tables
BLOODA_ADV-2025-019380-mmc1.pdf (1.2MB, pdf)

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