CMPL-Based Purification and Depletion of Human Hematopoietic Stem Cells: Implications for Pre-Transplant Conditioning

Overview

Journal bioRxiv

Date 2024 Mar 11

PMID 38464076

Authors

Daisuke Araki

Sogun Hong

Nathaniel Linde

Bryan Fisk

Neelam Redekar

Christi Salisbury-Ruf

Allen Krouse

Theresa Engels

Justin Golomb

Pradeep Dagur

Diogo M Magnani

Zhirui Wang

Andre Larochelle

Affiliations

Soon will be listed here.

Abstract

The transplantation of gene-modified autologous hematopoietic stem and progenitor cells (HSPCs) offers a promising therapeutic approach for hematological and immunological disorders. However, this strategy is often limited by the toxicities associated with traditional conditioning regimens. Antibody-based conditioning strategies targeting cKIT and CD45 antigens have shown potential in mitigating these toxicities, but their long-term safety and efficacy in clinical settings require further validation. In this study, we investigate the thrombopoietin (TPO) receptor, cMPL, as a novel target for conditioning protocols. We demonstrate that high surface expression of cMPL is a hallmark feature of long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ HSPC subset. Targeting the cMPL receptor facilitates the separation of human LT-HSCs from mature progenitors, a delineation not achievable with cKIT. Leveraging this finding, we developed a cMPL-targeting immunotoxin, demonstrating its ability to selectively deplete host cMPL LT-HSCs with a favorable safety profile and rapid clearance within 24 hours post-infusion in rhesus macaques. These findings present significant potential to advance our understanding of human hematopoiesis and enhance the therapeutic outcomes of autologous HSPC gene therapies.

References

Berckmueller K, Thomas J, Taha E, Choo S, Madhu R, Kanestrom G . CD90-targeted lentiviral vectors for HSC gene therapy. Mol Ther. 2023; 31(10):2901-2913. PMC: 10556220. DOI: 10.1016/j.ymthe.2023.08.003. View

Bankova A, Pang W, Velasco B, Long-Boyle J, Shizuru J . 5-Azacytidine depletes HSCs and synergizes with an anti-CD117 antibody to augment donor engraftment in immunocompetent mice. Blood Adv. 2021; 5(19):3900-3912. PMC: 8679673. DOI: 10.1182/bloodadvances.2020003841. View

Nishino T, Miyaji K, Ishiwata N, Arai K, Yui M, Asai Y . Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL. Exp Hematol. 2009; 37(11):1364-1377.e4. DOI: 10.1016/j.exphem.2009.09.001. View

Chen J, Larochelle A, Fricker S, Bridger G, Dunbar C, Abkowitz J . Mobilization as a preparative regimen for hematopoietic stem cell transplantation. Blood. 2006; 107(9):3764-71. PMC: 1895779. DOI: 10.1182/blood-2005-09-3593. View

Palchaudhuri R, Saez B, Hoggatt J, Schajnovitz A, Sykes D, Tate T . Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nat Biotechnol. 2016; 34(7):738-45. PMC: 5179034. DOI: 10.1038/nbt.3584. View

Shin J, Hu W, Naramura M, Park C . High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias. J Exp Med. 2014; 211(2):217-31. PMC: 3920569. DOI: 10.1084/jem.20131128. View

Haas S, Trumpp A, Milsom M . Causes and Consequences of Hematopoietic Stem Cell Heterogeneity. Cell Stem Cell. 2018; 22(5):627-638. DOI: 10.1016/j.stem.2018.04.003. View

Liu Y, Woo J, Neville D . Targeted introduction of a diphtheria toxin resistant mutation into the chromosomal EF-2 locus of Pichia pastoris and expression of immunotoxin in the EF-2 mutants. Protein Expr Purif. 2003; 30(2):262-74. DOI: 10.1016/s1046-5928(03)00129-3. View

Persaud S, Ritchey J, Kim S, Lim S, Ruminski P, Cooper M . Antibody-drug conjugates plus Janus kinase inhibitors enable MHC-mismatched allogeneic hematopoietic stem cell transplantation. J Clin Invest. 2021; 131(24). PMC: 8670850. DOI: 10.1172/JCI145501. View

10.

Pang W, Czechowicz A, Logan A, Bhardwaj R, Poyser J, Park C . Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice. Blood. 2019; 133(19):2069-2078. PMC: 6509544. DOI: 10.1182/blood-2018-06-858159. View

11.

Alvarado L, Huntsman H, Cheng H, Townsley D, Winkler T, Feng X . Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ. Blood. 2019; 133(19):2043-2055. PMC: 6509545. DOI: 10.1182/blood-2018-11-884486. View

12.

Magnani A, Semeraro M, Adam F, Booth C, Dupre L, Morris E . Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome. Nat Med. 2022; 28(1):71-80. PMC: 8799465. DOI: 10.1038/s41591-021-01641-x. View

13.

Woo J, Liu J, Kang S, Singh R, Park S, Su Y . GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials. Protein Expr Purif. 2007; 58(1):1-11. DOI: 10.1016/j.pep.2007.11.006. View

14.

Hu Y, Smyth G . ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods. 2009; 347(1-2):70-8. DOI: 10.1016/j.jim.2009.06.008. View

15.

Zonari E, Desantis G, Petrillo C, Boccalatte F, Lidonnici M, Kajaste-Rudnitski A . Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy. Stem Cell Reports. 2017; 8(4):977-990. PMC: 5390102. DOI: 10.1016/j.stemcr.2017.02.010. View

16.

Gyurkocza B, Sandmaier B . Conditioning regimens for hematopoietic cell transplantation: one size does not fit all. Blood. 2014; 124(3):344-53. PMC: 4102707. DOI: 10.1182/blood-2014-02-514778. View

17.

Radtke S, Pande D, Cui M, Perez A, Chan Y, Enstrom M . Purification of Human CD34CD90 HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy. Mol Ther Methods Clin Dev. 2020; 18:679-691. PMC: 7424231. DOI: 10.1016/j.omtm.2020.07.010. View

18.

Gurney A, de Sauvage F, Moore M . Thrombocytopenia in c-mpl-deficient mice. Science. 1994; 265(5177):1445-7. DOI: 10.1126/science.8073287. View

19.

Aydin O, Yildiz L, Kefeli M, Kandemir B . CD117 expression in normal, neoplastic, inflammatory, and reactive lesions of the thyroid. Pathol Res Pract. 2008; 204(6):359-65. DOI: 10.1016/j.prp.2008.01.003. View

20.

Masiuk K, Brown D, Laborada J, Hollis R, Urbinati F, Kohn D . Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells. Mol Ther. 2017; 25(9):2163-2175. PMC: 5589063. DOI: 10.1016/j.ymthe.2017.05.023. View