Immunodepletion of MDSC by AMV564, a novel bivalent, bispecific CD33/CD3 T cell engager, ex vivo in MDS and melanoma
Authors: Pingyan Cheng, Xianghong Chen, Robert Dalton, Alexandra Calescibetta, Tina So, Danielle Gilvary, Grace Ward, Victoria Smith, Sterling Eckard, Judith A. Fox, Jeanmarie Guenot, Joseph Markowitz, John L. Cleveland, Kenneth L. Wright, Alan F. List, Sheng Wei, Erika A. Eksioglu
Journal: Molecular Therapy (2022)

We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors. We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors. IntroductionCD33 is a member of a family of sialic acid-binding immunoglobulin-like lectins (SILGECs; SIGLEC3) and has become a de facto target in many therapeutic applications for myeloid leukemias.1Walter R.B. Appelbaum F.R. Estey E.H. Bernstein I.D. Acute myeloid leukemia stem cells and CD33-targeted immunotherapy.Blood. 2012; 119: 6198-6208https://doi.org/10.1182/blood-2011-11-325050Crossref PubMed Scopus (221) Google Scholar,2Laszlo G.S. Estey E.H. Walter R.B. The past and future of CD33 as therapeutic target in acute myeloid leukemia.Blood Rev. 2014; 28: 143-153https://doi.org/10.1016/j.blre.2014.04.001Crossref PubMed Scopus (113) Google Scholar The CD33 inhibitory receptor is also a key marker of myeloid-derived suppressor cells (MDSCs), which play critical pro-tumorigenic roles in the tumor microenvironment of many malignancies.3Talmadge J.E. Gabrilovich D.I. History of myeloid-derived suppressor cells.Nat. Rev. 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Nagibin V. Groth C. Altevogt P. Utikal J. Umansky V. Myeloid-derived suppressor cells hinder the anti-cancer activity of immune checkpoint inhibitors.Front. Immunol. 2018; 9: 1310https://doi.org/10.3389/fimmu.2018.01310Crossref PubMed Scopus (272) Google Scholar Resistance to checkpoint inhibitor treatment in individuals with melanoma correlated with increased accumulation of CD33hi MDSCs, suggesting that targeting this immunosuppressive population may improve the outcome of individuals receiving these inhibitors.CD33-targeting treatment modalities have included CD33-specific antibodies, unconjugated or conjugated to cytolytic agents; CD33-targeted chimeric antigen receptor (CAR)-T cells; or, more recently, bispecific CD33/CD3 T cell engagers that stimulate activation of T cells.2Laszlo G.S. Estey E.H. Walter R.B. The past and future of CD33 as therapeutic target in acute myeloid leukemia.Blood Rev. 2014; 28: 143-153https://doi.org/10.1016/j.blre.2014.04.001Crossref PubMed Scopus (113) Google Scholar,18Liu Y. Bewersdorf J.P. Stahl M. Zeidan A.M. Immunotherapy in acute myeloid leukemia and myelodysplastic syndromes: the dawn of a new era?.Blood Rev. 2019; 34: 67-83https://doi.org/10.1016/j.blre.2018.12.001Crossref PubMed Scopus (64) Google Scholar Unconjugated, monospecific anti-CD33 antibodies have had disappointing results clinically, showing only modest activity.2Laszlo G.S. Estey E.H. Walter R.B. The past and future of CD33 as therapeutic target in acute myeloid leukemia.Blood Rev. 2014; 28: 143-153https://doi.org/10.1016/j.blre.2014.04.001Crossref PubMed Scopus (113) Google Scholar The utility of conjugated anti-CD33 antibodies has been hampered by slow internalization, drug efflux transporter activity, and toxicity.19Cowan A.J. Laszlo G.S. Estey E.H. Walter R.B. 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The results provide support for continued clinical investigation of AMV564 to reduce CD33hi MDSCs and boost cytotoxic T cell activity in malignancies where MDSCs are implicated, as a single agent and in combination with checkpoint immunotherapies.ResultsAMV564 reduces CD33hi cells and improves hematopoiesis in primary MDS explantsAMV564 treatment has been shown previously to reduce the burden of CD33+ malignant cells in AML.31Reusch U. Harrington K.H. Gudgeon C.J. Fucek I. Ellwanger K. Weichel M. Knackmuss S.H. Zhukovsky E.A. Fox J.A. Kunkel L.A. et al.Characterization of CD33/CD3 tetravalent bispecific tandem diabodies (TandAbs) for the treatment of acute myeloid leukemia.Clin. Cancer Res.: an official J. Am. Assoc. Cancer Res. 2016; 22: 5829-5838https://doi.org/10.1158/1078-0432.CCR-16-0350Crossref PubMed Scopus (66) Google Scholar Therefore, we tested whether AMV564 could reduce excess MDSCs present in MDS bone marrow (BM) primary explants (see specimen information in Table S1) via flow cytometric analysis of several distinct populations: total live cells in the context of CD33 expression (Figure S1A), proportion of T cells and their subsets (Figure S1B), and MDSC subsets based on CD33 and CD14 expression (Figure S1C). When measured, PD1 positivity was gated as done by us previously32Cheng P. Eksioglu E.A. Chen X. Kandell W. Le Trinh T. Cen L. Qi J. Sallman D.A. Zhang Y. Tu N. et al.S100A9-induced overexpression of PD-1/PD-L1 contributes to ineffective hematopoiesis in myelodysplastic syndromes.Leukemia. 2019; 33: 2034-2046https://doi.org/10.1038/s41375-019-0397-9Crossref PubMed Scopus (41) Google Scholar and is represented in Figure S1D. It is important to mention that, throughout our study, we used immunoglobulin G (IgG) as a control, as done by others.33Liu L. Chen J. Bae J. Li H. Sun Z. Moore C. Hsu E. Han C. Qiao J. Fu Y.X. Rejuvenation of tumour-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells.Nat. Biomed. Eng. 2021; 5: 1261-1273https://doi.org/10.1038/s41551-021-00800-2Crossref PubMed Scopus (7) Google Scholar Extensive work with AMV564 in preclinical studies ahead of clinical use has shown that this molecule does not cross-link T cells in a target-independent manner.24Westervelt P. Cortes J.E. Altman J.K. Long M. Oehler V.G. Gojo I. Guenot J. Chun P. Roboz G.J. Phase 1 first-in-human trial of AMV564, a bivalent bispecific (2:2) CD33/CD3 T-cell engager, in patients with relapsed/refractory acute myeloid leukemia (AML).Blood. 2019; 134: 834https://doi.org/10.1182/blood-2019-129042Crossref Google Scholar,34Mettu N.B. Starodub A. Piha-Paul S.A.A. Abdul-Karim R.M. Tinoco G. Shafique M.R. Smith V. Baccei C. Chun P.Y. Results of a phase 1 dose-escalation study of AMV564, a novel T-cell engager, alone and in combination with pembrolizumab in patients with relapsed/refractory solid tumors.J. Clin. Oncol. 2021; 39: 2555https://doi.org/10.1200/JCO.2021.39.15_suppl.2555Crossref Google Scholar Notably, AMV564 led to a significant decrease in CD33hi MDSC populations in BM mononuclear cells (MNCs) (Figures S1A and S1B). Reductions in MDSCs were not due to masking of the antibody used in the flow cytometry analysis because a consistent effect was seen when assessed with an anti-CD33 antibody, HIM3-4b, that binds in a distinct CD33 domain35Perez-Oliva A.B. Martinez-Esparza M. Vicente-Fernandez J.J. Corral-San Miguel R. Garcia-Penarrubia P. Hernandez-Caselles T. Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells.Glycobiology. 2011; 21: 757-770https://doi.org/10.1093/glycob/cwq220Crossref PubMed Scopus (52) Google Scholar (Figure 1C ). These results confirm that AMV564-directed reductions in MDSCs are specifically due to depletion of CD33+ cells rather than masking of epitopes. These decreases in MDSCs correlated with a significant improvement in overall colony formation capacity, suggesting improvement in normal hematopoiesis (Figure 1D). This was supported by a reduction in genomic instability of CD34+ HSPCs (Figure 1E). Thus, AMV564 successfully targets CD33+ MDSCs and improves hematopoiesis in primary MDS explants.AMV564 augments CD3+ T cells and promotes cytotoxic CD8+ T cell skewing in primary MDS specimensThe effects of AMV564 on T cells in primary MDS specimens were assessed in vitro. AMV564 treatment increased the overall percentage of CD3+ T cells (Figures 2A–2D , gated as in Figure S1B) as well as the proportion of CD4+ andCD8+ cytotoxic T cells. AMV564 increased T cell proliferation and interferon (IFN)γ secretion, an established marker of T cell activation.36Hecht T.T. Longo D.L. Matis L.A. The relationship between immune interferon production and proliferation in antigen-specific, MHC-restricted T cell lines and clones.J. Immunol. 1983; 131: 1049-1055PubMed Google Scholar Increased percentages of CD4+ and CD8+ T cells (Figures 2C and 2D) were matched with significant increases in proliferation, measured by the percentage of cells incorporating bromodeoxyuridine (BrdU) (Figures 2E and 2F). Engagement of AMV564 leads to proliferation of these T cells via activation, as evidenced by increased intracellular IFNγ levels (Figures 2G and 2H). Thus, T cells can be activated in primary MDS specimens by AMV564, which is a critical feature given that T cells are dysregulated/suppressed in the MDS microenvironment.30Mailloux A.W. Sugimori C. Komrokji R.S. Yang L. Maciejewski J.P. Sekeres M.A. Paquette R. Loughran Jr., T.P. List A.F. Epling-Burnette P.K. Expansion of effector memory regulatory T cells represents a novel prognostic factor in lower risk myelodysplastic syndrome.J. Immunol. 2012; 189: 3198-3208https://doi.org/10.4049/jimmunol.1200602Crossref PubMed Scopus (70) Google Scholar Finally, when analyzing the number of cell divisions, it was evident that AMV564 preferentially promoted CD8+ T cell proliferation compared with CD4+ T cells (Figures S2A and S2C), even though the level of intracellular IFNγ was similar (Figures S2B and S2D).Figure 2AMV564 augments T cell numbers and function in primary MDS specimensShow full caption(A) From experiments in MDS primary specimens as in Figure 1, analysis was performed on total CD3+ T cells (from total live cells).(B) CD3+ cells were analyzed for the presence of CD4+ and CD8+ cells to assess changes in their proportions (representative data are shown; quantified in C and D).(C–E) Percentages of CD4+ T cells (C), their level of proliferation as assessed by BrdU incorporation (D), and their activation status as assessed by intracellular IFNγ staining (E).(F–H) Percentages of CD8+ T cells (F), their level of proliferation as assessed by BrdU incorporation (G), and their activation status as assessed by intracellular IFNγ staining (H). (B–H) Data show the percentage from total gated CD3+ cells. Error bars denote the SEM, and significance was assessed by paired Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AMV564 significantly enhances anti-PD1 antibody cytotoxic activityThe immune checkpoint receptor program cell death protein 1 (PD1) and its ligand (PDL1) are induced during inflammation and by malignant cells, impairing the anti-tumor immune response.28Schatton T. Schutte U. Frank N.Y. Zhan Q. Hoerning A. Robles S.C. Zhou J. Hodi F.S. Spagnoli G.C. Murphy G.F. Frank M.H. Modulation of T-cell activation by malignant melanoma initiating cells.Cancer Res. 2010; 70: 697-708https://doi.org/10.1158/0008-5472.CAN-09-1592Crossref PubMed Scopus (205) Google Scholar,29Ozkazanc D. Yoyen-Ermis D. Tavukcuoglu E. Buyukasik Y. Esendagli G. Functional exhaustion of CD4(+) T cells induced by co-stimulatory signals from myeloid leukaemia cells.Immunology. 2016; 149: 460-471https://doi.org/10.1111/imm.12665Crossref PubMed Scopus (41) Google Scholar PD1 and PDL1 are expressed predominantly on the surface of activated T cells and on antigen-presenting cells, respectively, where they serve as important regulators of immune homeostasis.37Staron M.M. Gray S.M. Marshall H.D. Parish I.A. Chen J.H. Perry C.J. Cui G. Li M.O. Kaech S.M. The transcription factor FoxO1 sustains expression of the inhibitory receptor PD-1 and survival of antiviral CD8(+) T cells during chronic infection.Immunity. 2014; 41: 802-814https://doi.org/10.1016/j.immuni.2014.10.013Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar,38Jiang Y. Li Y. Zhu B. 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Allison J. Sharma P. Daver N. The emerging role of immune checkpoint based approaches in AML and MDS.Leuk. Lymphoma. 2018; 59: 790-802https://doi.org/10.1080/10428194.2017.1344905Crossref PubMed Scopus (66) Google Scholar, 42Daver N. Boddu P. Garcia-Manero G. Yadav S.S. Sharma P. Allison J. Kantarjian H. Hypomethylating agents in combination with immune checkpoint inhibitors in acute myeloid leukemia and myelodysplastic syndromes.Leukemia. 2018; 32: 1094-1105https://doi.org/10.1038/s41375-018-0070-8Crossref PubMed Scopus (116) Google Scholar This has led to the suggestion that MDSCs may contribute to reduced responsiveness to checkpoint inhibitors.17Weber R. Fleming V. Hu X. Nagibin V. Groth C. Altevogt P. Utikal J. Umansky V. Myeloid-derived suppressor cells hinder the anti-cancer activity of immune checkpoint inhibitors.Front. Immunol. 2018; 9: 1310https://doi.org/10.3389/fimmu.2018.01310Crossref PubMed Scopus (272) Google ScholarWe have shown previously that aberrant activation of PD1 on HSPCs by MDSC-derived PDL1 triggers HSPC death, contributes to BM failure, and suppresses hematopoiesis.32Cheng P. Eksioglu E.A. Chen X. Kandell W. Le Trinh T. Cen L. Qi J. Sallman D.A. Zhang Y. Tu N. et al.S100A9-induced overexpression of PD-1/PD-L1 contributes to ineffective hematopoiesis in myelodysplastic syndromes.Leukemia. 2019; 33: 2034-2046https://doi.org/10.1038/s41375-019-0397-9Crossref PubMed Scopus (41) Google Scholar Therefore, we hypothesized that combined treatment of anti-PD1 antibody with AMV564 would have enhanced activity in MDS specimens. Although the overall percentage of CD4+ (Figure 3A ) and CD8+ (Figure 3B) T cells within the CD3+ population with the same gating as shown in Figure 2B (note the axis difference between CD4 at 100 and CD8 at 25) as well as their proliferation levels (Figures 3D, 3E, S3A, and S3B) did not change significantly with anti-PD1 antibody treatment, AMV564 alone or in combination with anti-PD1 led to a significant increase in proliferation and activity of T cells in primary MDS specimens, as measured by BrdU incorporation and IFNγ positivity (Figures 3C–3F). However, combination of AMV564 with anti-PD1 did not lead to a significant increase from AMV564 stand-alone treatments in either population, although it did have slight but significant increased activation of CD8+ cytotoxic cells compared with either agent alone (Figures 3D and 3F). Interestingly, although anti-PD1 antibody treatment led to an increase in the overall percentage of PD1+ T cells, treatment with AMV564, alone or combined, reduced the proportion of PD1+ T cells (Figures 3G and 3H). To confirm that these effects were not due to analyzing the CD3+ population, we analyzed the percentages of CD4+ and CD8+ T cells among the total MNC population. The increase induced by the presence of AMV564 remained comparable with what we observed in CD3+ gated cells (Figures 3I and 3J), and treatment with AMV564, anti-PD1, or their combination did not change the ratio of CD4 to CD8 (Figure S3C). Finally, and consistent with previous studies demonstrating AMV564's cytotoxic activity,31Reusch U. Harrington K.H. Gudgeon C.J. Fucek I. Ellwanger K. Weichel M. Knackmuss S.H. Zhukovsky E.A. Fox J.A. Kunkel L.A. et al.Characterization of CD33/CD3 tetravalent bispecific tandem diabodies (TandAbs) for the treatment of acute myeloid leukemia.Clin. Cancer Res.: an official J. Am. Assoc. Cancer Res. 2016; 22: 5829-5838https://doi.org/10.1158/1078-0432.CCR-16-0350Crossref PubMed Scopus (66) Google Scholar increased CD8+ killing was confirmed by elevated CD107a degranulation in CD8+ T cells (Figure 3K), which is well established to correlate with cytotoxicity and Granzyme B release.43Bryceson Y.T. March M.E. Barber D.F. Ljunggren H.G. Long E.O. Cytolytic granule polarization and degranulation controlled by different receptors in resting NK cells.J. Exp. Med. 2005; 202: 1001-1012https://doi.org/10.1084/jem.20051143Crossref PubMed Scopus (356) Google ScholarFigure 3Combined AMV564 and anti-PD1 treatment augments immune cell functions in MDS specimensShow full captionPrimary MDS BM MNCs were treated with 50 ng/mL of AMV564 and/or anti-PD1 antibody, followed by assessment of T cell subset activity.(A–D) Percentages of total CD4+ cells from total CD3+ gate (A) as well as assessment of their proliferation (B), activation (C), and PD1 expression (D).(E–H) Percentages of total CD8+ cells, from total CD3+ gate (E) as well as assessment of their proliferation (F), activation (G), and PD1 expression (H).(I) To confirm the enhanced killing capacity, CD107a mobilization was also assessed (representative figure of n = 5). Error bars denote the SEM, and significance was assessed by Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AMV564 and anti-PD1 combination treatment impairs CD33hi cell proliferation and augments colony formation in MDSsAMV564 in combination with the anti-PD1 antibody also reduced the levels of CD33hi cells in primary MDS specimens in terms of overall percentage (Figure 4A ) and proliferative capacity (Figure 4B). The inhibitory effects on proliferation were confirmed by BrdU analysis