A new approach to replacing the hematopoietic system has broad therapeutic potential

Hematopoietic stem cell transplantation (HSCT) is used to treat a broad range of hematological malignancies. However, the need for untargeted chemotherapies to pre-condition patients, plus challenges for treating malignant cells after transplant, limit its utility. Antigen-specific cell-depleting therapies, such as antibody-drug conjugates (ADCs), promise to improve HSCT, as demonstrated by recent advances in clinical practice for B cell malignancies. Yet identifying candidate antigens with a profile as favorable as CD19 is extremely difficult. To circumvent this issue, researchers from Basel University Hospital, Switzerland, combined an ADC targeting the pan-hematopoietic marker CD45 with the transplantation of hematopoietic stem and progenitor cells (HSPCs) engineered to evade ADC-mediated depletion. Their data, published in Nature, show this approach to provide complete and selective depletion of multiple hematological cancer cells in vivo.?

Engineering and characterization of CD45 variants?

Deleting CD45 from HSPCs to protect against CD45-targeting ADC is impossible due to the associated risk of severe combined immunodeficiency. For this reason, Garaudé et al. tested different combinations of base editor mRNAs (made using TriLink’s CleanCap? cap analogs and modified nucleotides) and single guide RNAs to engineer CD45 such that it would not be recognized by anti-CD45 antibodies. Characterization of the CD45 variants included flow cytometry–based analysis of antibody binding, and the use of biolayer interferometry to quantify shielding and determine key biophysical properties. In addition, testing with a surrogate ADC (BC8-Saporin) in human primary T cells helped to reveal two optimal variants, CD45K352E and CD45K352G, which were designated sg49.3HSPCs.?

Shielded HSPCs resist ADC-mediated depletion?

The ADC for in vivo experiments was produced by humanizing and engineering the anti-CD45 antibody MIRG451 (resulting in CIM053) and conjugating it with SG3376, a non-cleavable pyrrolobenzodiazepine drug-linker. CIM053–SG3376 was initially tested in vitro, where it effectively depleted unedited HSPCs and multiple hematological cancer cell lines, while sg49.3HSPCs were more resistant to increasing ADC concentrations.?

In vivo shielding of sg49.3HSPCs from the ADC was investigated by injecting an immunodeficient mouse model (NBSGW) with either unedited HSPCs or sg49.3HSPCs prior to administration of CIM053–SG3376. Flow cytometry analysis of the bone marrow showed the unedited HSPCs to be completely depleted by a single dose of CIM053–SG3376, while mice engrafted with sg49.3HSPCs maintained high human chimerism.?

Selective tumor targeting in vivo?

To determine whether CIM053–SG3376 was capable of selective tumor targeting, without affecting shielded HSPCs, NBSGW mice were xenografted with either unedited HSPCs or sg49.3HSPCs. The mice were then injected with MOLM-14 cells (an aggressive myeloid leukemia cell line), followed by administration of saline (control) or CIM053–SG3376. While the saline-treated animals exhibited rapid tumor growth, tumors were undetectable following CIM053–SG3376 treatment. In addition to tumor cells, CIM053–SG3376 eliminated unedited hCD45+ non-tumor cells, whereas sg49.3HSPCs were found to persist in all tested organs.?

Selective tumor-targeting was also assessed using patient-derived cells. Specifically, mice that had previously been xenografted with unedited HSPCs or sg49.3HSPCs were injected with acute myeloid leukemia (AML) patient-derived xenograft (PDX) cells before receiving CIM053–SG3376. Key findings included the observation that CIM053–SG3376 treatment resulted in a pure population of edited hCD45+ cells in all organs, indicating that the PDX cells were eliminated and only shielded cells remained.?

Conclusion?

This study shows CD45 to be a promising target for antigen-specific therapy of hematological diseases. By combining a CD45-targeting ADC with shielded HSPCs, Garaudé et al. have developed a strategy for replacing the hematopoietic system that has broad therapeutic potential, not only to treat hematological cancers, but also severe autoimmune diseases and possibly even HIV infection.?

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Article reference: Garaudé S., Marone R., Lepore R., et al. Selective haematological cancer eradication with preserved haematopoiesis. Nature. 2024 (630) 728-758. https://doi.org/10.1038/s41586-024-07456-3??