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2200 Selection for an HIV-Resistant Immune System By Base-Edited CD45 CAR-T Cell Therapy

Program: Oral and Poster Abstracts
Session: 801. Gene Therapies: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Nils Wellhausen, BSc1, Yuqi Zhou2*, Michael S Bowman, PhD3*, Kusala Anupindi, BS2*, Abhimanyu Gowda2*, Nils Engel, MD2*, Carl H. June, MD4, Robert L. Bowman, PhD3, James Riley, PhD2* and Saar Gill, MD, PhD5

1Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA
2University of Pennsylvania, Philadelphia
3Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA
4Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA
5University of Pennsylvania, Philadelphia, PA

Introduction

Eradicating cellular reservoirs of human immunodeficiency virus (HIV) in patients on antiretroviral therapy (ART) is critical to curing HIV, since integrated provirus remains in CD4+ cells even in patients without detectable viral load on anti-retroviral therapy (ART). There are rare but compelling reports of HIV cures in patients who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) from CCR5Δ32/Δ32 donors, wherein myeloablative chemotherapy and donor alloreactive T cells completely eradicated the recipient’s immunohematopoietic system and allowed it to be replaced by donor-derived hematopoiesis lacking the HIV co-receptor CCR5. While applying this strategy systematically as a treatment option is not feasible due to the paucity of HLA-matched CCR5Δ32/Δ32 donors for HIV-infected patients and the high-risk nature of alloHSCT, these reports highlight that immunologic depletion of HIV reservoirs (via potent T cell alloreactivity) in combination with an HIV-resistant HSCT (via CCR5 deficiency) can achieve HIV eradication. To exert potent yet broad immune pressure against the cellular components of the viral reservoir we harnessed an HIV- and fratricide-resistant CAR-T cell therapy against the pan-leukocyte antigen CD45.

Results

Fratricide resistance of the anti-CD45 immunotherapy is achieved by epitope editing CD45 using an adenine base editing (ABE) as we recently described [PMID: 37651540]. At the same time, HIV-resistance is accomplished by either base editing the CCR5 start codon to silence CCR5 expression or mutating tyrosine residues (Y14 and Y15) in the N-terminal domain of CCR5 that are known to bind HIV gp120. Base editing CCR5 in primary human CD4 T cells is highly efficient with ~84% A to G editing at the CCR5 start codon mutation, successfully silencing CCR5 protein expression. Similarly, a single gRNA achieved ~90% editing for A5 (Y14H) and ~38% for A2 (Y15H), accomplishing mutagenesis of the sulfotyrosine binding residues for HIV gp120 docking while preserving CCR5 expression. To test whether CCR5 base-edited cells (CCR5BE) are resistant to HIV infection, we infected CCR5BE or unedited CD4 T cells with the CCR5-tropic HIV stain BaL. Both groups of CCR5BE CD4 T cells showed reduced susceptibility to HIV BaL infection both in vitro and in vivo, whereas unedited control cells were readily infected. Resistance to HIV infection translated to a ~4-fold higher preservation of peripheral blood T cell numbers and a ~10-fold lower vRNA copy number in mice that received CCR5BE T cells compared to unedited T cells. Furthermore, we observed a strong enrichment of CCR5 edited cells from ~60-80% to >99% upon HIV infection, suggesting that CCR5BE T cells are protected from infection-induced cell death.

To generate CD45 CAR-T cells that are resistant to fratricide and HIV infection, on-target base editing must co-occur biallelically at both the CCR5 loci and the CD45 epitope with high efficiency. Using single-cell DNA sequencing, we show that biallelic, multiplex base editing is efficient in primary human T cells with editing efficiencies of ~75% (CCR5 start codon + CD45BE and ~85% (CCR5 Y14/15 + CD45BE) respectively.

Lastly, we demonstrate that multiplex base editing CD45 with CCR5 generates fratricide-and HIV-resistant CART45 cells that can eliminate HIV-infected CD4 T cells in xenografted mice while preventing the spreading of HIV particles from infected CD4 T cells to the CD4 positive CAR-T cells. This facilitated the persistence of HIV-resistant CART45, whereas CD45BE only edited CAR-T cells were unable to persist due to HIV-induced T cell apoptosis.

Conclusion

Overall, our study demonstrates the potential of anti-CD45 immunotherapy to eradicate HIV infected cells. We speculate that when combined with an engineered autologous HSCT, this CD45/CCR5 multiplex engineering approach can regenerate an HIV- and anti-CD45 resistant hematopoietic system free of latent HIV infected cells.

Disclosures: Wellhausen: Flagship Pioneering: Ended employment in the past 24 months, Other: Intern. Gowda: Yellowstone Biosciences: Current equity holder in private company, Patents & Royalties. Gill: Novartis: Patents & Royalties, Research Funding; Asher Biotherapeutics: Research Funding; Carisma: Current holder of stock options in a privately-held company; Interius: Current holder of stock options in a privately-held company, Research Funding; Mission Bio: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH