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. 2021 Feb 22:12:618081.
doi: 10.3389/fimmu.2021.618081. eCollection 2021.

An Antibody-Drug Conjugate That Selectively Targets Human Monocyte Progenitors for Anti-Cancer Therapy

Yuta Izumi  1 , Masashi Kanayama  1 , Zhongchuzi Shen  2 , Masayuki Kai  2 , Shunsuke Kawamura  1 , Megumi Akiyama  1   3 , Masahide Yamamoto  3 , Toshikage Nagao  3 , Keigo Okada  3 , Norihiko Kawamata  3 , Shigeo Toyota  4 , Toshiaki Ohteki  1
Affiliations

An Antibody-Drug Conjugate That Selectively Targets Human Monocyte Progenitors for Anti-Cancer Therapy

Yuta Izumi et al. Front Immunol. .

Abstract

As hematopoietic progenitors supply a large number of blood cells, therapeutic strategies targeting hematopoietic progenitors are potentially beneficial to eliminate unwanted blood cells, such as leukemic cells and immune cells causing diseases. However, due to their pluripotency, targeting those cells may impair the production of multiple cell lineages, leading to serious side effects such as anemia and increased susceptibility to infection. To minimize those side effects, it is important to identify monopotent progenitors that give rise to a particular cell lineage. Monocytes and monocyte-derived macrophages play important roles in the development of inflammatory diseases and tumors. Recently, we identified human monocyte-restricted progenitors, namely, common monocyte progenitors and pre-monocytes, both of which express high levels of CD64, a well-known monocyte marker. Here, we introduce a dimeric pyrrolobenzodiazepine (dPBD)-conjugated anti-CD64 antibody (anti-CD64-dPBD) that selectively induces the apoptosis of proliferating human monocyte-restricted progenitors but not non-proliferating mature monocytes. Treatment with anti-CD64-dPBD did not affect other types of hematopoietic cells including hematopoietic stem and progenitor cells, neutrophils, lymphocytes and platelets, suggesting that its off-target effects are negligible. In line with these findings, treatment with anti-CD64-dPBD directly killed proliferating monocytic leukemia cells and prevented monocytic leukemia cell generation from bone marrow progenitors of chronic myelomonocytic leukemia patients in a patient-derived xenograft model. Furthermore, by depleting the source of monocytes, treatment with anti-CD64-dPBD ultimately eliminated tumor-associated macrophages and significantly reduced tumor size in humanized mice bearing solid tumors. Given the selective action of anti-CD64-dPBD on proliferating monocyte progenitors and monocytic leukemia cells, it should be a promising tool to target cancers and other monocyte-related inflammatory disorders with minimal side effects on other cell lineages.

Keywords: chronic myelomonocytic leukemia; common monocyte progenitor; leukemia; monocyte; tumor-associated macrophage.

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Conflict of interest statement

MKai and ZS are employees of Kyowa Kirin Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Screening of cell surface molecules to target monocytic progenitors. (A) Screening scheme for molecules highly restricted in monocytic progenitors. The expression of 361 cell surface molecules on HSPCs derived from UCB were examined using FCM. Expression of candidates identified from HSPC screening was further assessed on mature hematopoietic cell lineages. (B) Gating strategies for hematopoietic progenitors in UCB. Lineage- cells (Lin- cells; CD2-CD3-CD11b-CD14-CD16-CD19-CD56-CD235ab- cells) were pre-gated. Gating strategies of other HSPCs are shown in Supplementary Figure 1A . (C–F) Expression of CD64 on mature hematopoietic cells and their progenitors. CD64 expression was evaluated on monocytic progenitors (C), monocytes (D), non-monocytic immune cells (E) and the other hematopoietic stem and progenitor cells (F) by FCM. Each population was identified by the following gating strategies: B cell, FSCloSSCloCD3-CD56-CD19+; T cell, FSCloSSCloCD3+; NK cell, FSCloSSCloCD56+CD3-; Neutrophil, SSChiHLA-DR-CD66b+CD16+; pDC, CD3-CD14-CD19-CD56-CD11c-HLA-DR+CD123+; cDC, CD3-CD14-CD19-CD56-CD123-HLA-DR+CD11c+. Blood monocytes (HLA-DR+CD14+) were subdivided into three populations based on their expression of CD14 and CD16 as shown in (D, left panel). The data are representative of three independent experiments.
Figure 2
Figure 2
Selection of antibodies and payloads for ADC construction. (A) Competitive inhibition of anti-CD64 antibodies. Three clones of anti-CD64 antibodies (32.2, 611 and H22) were assessed for their capacities of epitope recognition in the presence of other clones. THP-1 cells were pretreated with blocking antibodies and then stained with Alexa Fluor 647 (AF647)-labeled anti-CD64 antibody in the presence or absence of human IgG. To estimate binding capacity, the signal intensity in the presence of the competitors (red dots) was compared with that in the absence of competitors (blue dots). (B) CD64 expression on THP-1 and Ramos cells. (C) Cytotoxicity assays with H22-dPBD. THP-1 and Ramos cells cultured with different ADCs composed of dPBD and an antibody, such as the anti-DNP antibody humanized with IgG1 (DNP(IgG1)-dPBD), H22 humanized with IgG1 (H22(IgG1)-dPBD), or H22 humanized with nullbody, an IgG4 analogue [H22(Null)-dPBD], in the presence of 20% human serum for 4 days. The relative viability of cells was estimated based on the quantification of ATP. (D) Cytotoxicity assay with H22-MMAE. THP-1 cells were cultured with ADCs composed of MMAE and an antibody, such as the anti-DNP antibody humanized with IgG1 [DNP(IgG1)-MMAE], H22 humanized with IgG1 (H22(IgG1)-MMAE) or H22 humanized with IgG4 nullbody (H22(Null)-MMAE), in the presence of 20% human serum for 4 days. Relative viability was estimated based on the quantification of ATP. (E) Internalization of H22 nullbody by U937 cells. Cells were pre-cultured with Alexa Fluor 488-labeled H22 antibody for the indicated times. The cells were then incubated with a quenching antibody on ice for 1 h and the signal intensity of Alexa Fluor 488 (AF488) was measured by FCM. (F) Internalization of H22 nullbody into primary monocytes. CD14hiCD16- monocytes sorted from peripheral blood were stained with the AF647-labeled H22 nullbody. The cells were then cultured for 23 h after reaction with the H22 nullbody and stained with LysoTracker (100 nM) for 1 h. Cell images were obtained by confocal microscopy. Scale bar, 20 µm. (G) A schematic illustration of the structure of H22-dPBD. Data are representative of two (A–E) or three (F) independent experiments.
Figure 3
Figure 3
Preferential cytotoxic activity of H22-dPBD against monocytic progenitors, but not monocytes. (A) Evaluation of cytotoxic activity of H22-dPBD against THP-1 cells. THP-1 cells were cultured for 6 days in the presence of DNP-dPBD or H22-dPBD and their relative viability was estimated based on the quantification of ATP (n=3). (B) Induction of apoptosis by H22-dPBD. THP-1 cells were cultured with ADCs (0.04 µg/ml) for 3 days and the frequency of apoptotic cells was evaluated by FCM (n=4). (C) Killing activity of H22-dPBD against PMA-treated THP-1 cells. THP-1 cells were stimulated with 40 ng/ml PMA for 24 h prior to culture with the ADC and the susceptibility of cells to H22-dPBD was examined as shown in (A) (n=3). (D) Killing efficacy of H22-dPBD treatment against THP-1 cells and PMA-treated THP-1 cells. Normalized reduction of cell viability was calculated from the data of treatment with 0.04 μg/ml ADCs in (A) and (C) (n=3). (E) Experimental scheme of the killing assay against myeloid progenitors. Lin-CLEC12A+ myeloid progenitors were sorted from UCB and were cultured with ADCs and cytokines (100 ng/ml SCF, 50 ng/ml TPO and FLT3L). The number on the histogram indicates the mean frequency of CD64+ progenies yielded through 6-day culture (n=3). (F) Cytotoxic activity of H22-dPBD against cells generated from Lin-CLEC12A+ progenitors at day 6. Relative viability was assessed based on the quantification of ATP (n=3). (G–I) FCM analysis of CD64+ cells from Lin-CLEC12A+ myeloid progenitors. Cells were cultured with cytokines and 0 or 0.008 µg/ml ADC for 6 days and the numbers of CD14+ monocytes (H) and CD14-CD64+ monocytic progenitors (I) were determined. (J) Cytotoxic activity of H22-dPBD against mature monocytes. CD14hiCD16- monocytes were sorted from the peripheral blood of healthy donors and were cultured for 6 days in the presence or absence of ADC (n=4). The data were pooled from three (A, D) or four (B, J) independent experiments or are representative of two (F) or three (C, E, G–I) independent experiments. Multiple t-test (A, C, F, J), Student’s t-test (B, D) and one-way ANOVA (H, I) were used to assess statistical significance. Error bars represent standard deviation of the mean. *p < 0.05, **p < 0.01, ***p < 0.001; n.s, not significant.
Figure 4
Figure 4
H22-dPBD-mediated elimination of monocytes and their progenitors without severe side effects in hematopoiesis. (A–G) BM-humanized NOG mice were generated as shown schematically in Supplementary Figure 3 . Seven days after intravenous administration of DNP-dPBD or H22-dPBD (0.5 µg/mouse), hematopoietic cells in the BM and blood were analyzed by FCM. The number of BM monocytes and frequency of blood monocytes (A), cell numbers of monocytic progenitors in the BM (B), concentration of platelets (TER119-CD235ab-hCD41a+hCD41b+) in the blood (C), numbers of neutrophils and cDCs in the BM (D), and numbers of Lin-CD34+CD38- HSPCs, CMPs, and MEPs in the BM (F) are shown. Ratios in cell numbers of mature immune cells in the BM and results of their statistical analyses between DNP-dPBD- and H22-dPBD-treated mice are summarized in (E). Ratios in cell numbers of HSPCs and results of their statistical analyses between DNP-dPBD- and H22-dPBD-treated mice are summarized in (G). Each point in the bar graphs shows the value for an individual mouse (n=6 per group). Error bars represent standard deviation of the mean. Student’s t-test (A–D, F) was used to assess statistical significance. **p < 0.01, ***p < 0.001; n.s, not significant. Data were pooled from two (C) or three (A, B, D, F) independent experiments.
Figure 5
Figure 5
H22-dPBD eliminates patient-derived leukemic monocytes in vivo. (A) Experimental scheme showing the treatment of PDX mice with ADCs. CMML patient-derived Lin-CD34+ BM cells were transferred to sublethally irradiated NOG mice 8 weeks before the administration of DNP-dPBD or H22-dPBD (0.5 µg/mouse). The mice were analyzed 7 days after the ADC treatment. (B, C) Frequency of hCD45+ cells (B) and cell morphology (C) of BM CD14hiCD16- monocytes obtained from NOG mice humanized with normal UCB or CMML patient-derived BM cells. Cells were sorted and stained with Diff-quik (scale bars, 5 µm). (D) Representative FCM plots of hCD45+ cells in the peripheral blood (upper panels) or BM (lower panels) from DNP-dPBD- or H22-dPBD-treated PDX mice. (E–G) Impact of ADC-treatment in hematopoiesis of PDX mice. Cell numbers of monocytes, monocytic progenitors and Lin-CD34+CD38- cells are shown in (E), (F), and (G), respectively. Each point in the bar graphs shows the value for an individual mouse (n=4 per group). Error bars represent standard deviation of the mean. Student’s t-test was used to assess statistical significance. *p < 0.05, ***p < 0.001; n.s, not significant. Data are representative (C, D) or pooled (B, E–G) from four independent experiments.
Figure 6
Figure 6
H22-dPBD eliminates TAMs and inhibits the development of solid tumors. (A) Experimental scheme of the generation and therapeutic treatment of solid tumor-bearing humanized mice with ADCs. At day 0, HSC4 cells (1.5×106 cells/mouse) were subcutaneously transplanted in hIL-6 Tg NOG mice humanized with UCB-derived Lin-CD34+ cells. DNP- or H22-dPBD (0.5 µg/mouse) were intravenously injected to the mice once a week from day 7 and tumor sizes were measured every 4 days from day 7. The mice were sacrificed at day 28 and tumor weights and cells infiltrating the tumors were evaluated. mo: months. (B) Ratio of hCD45+ and mCD45+ cells in total leukocytes in the tumor. (C) Frequency of monocytes in circulating hCD45+ cells at day 28. (D) Representative FCM plots of tumor-infiltrating hCD45+ cells. Frequencies of CD163hiCD14+ TAM-like cells are shown. (E) Histological evaluation of tumor-infiltrating CD163+ cells in DNP-dPBD- or H22-dPBD-treated mice. Scale bars: 200 μm. (F) Number and frequency of TAMs in DNP-dPBD- or H22-dPBD-treated mice. (G) Numbers of total hCD45+ cells and hCD45+CD3-CD19-CD56-CD14- (non-lymphoid, non-monocytic) cells in the tumor. (H) Time-course analysis of tumor development. Tumor volumes were calculated according to the following formula; 0.5 × length × (width)2. Statistical analysis was performed on data at day 27. Averages of tumor size in each group were shown. (I) Tumor weights of DNP-dPBD- or H22-dPBD-treated mice at day 28. Error bars represent standard deviation of the mean. Each point in the bar graphs shows the value for an individual mouse (DNP-treated group: n=8, H22-treated group: n=6). Student’s t-test was performed to assess statistical significance. *p < 0.05, **p < 0.01; n.s, not significant. Data are representative of three independent experiments (D, E) or are pooled from two independent experiments (B, C, F–I). Data points more than two standard deviations from the mean were excluded as outliers (H, I).
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