Enhancing donor cell engraftment in prenatal hematopoietic stem cell transplantation with novel antibody-drug conjugates

Abstract In-utero hematopoietic stem cell (HSC) transplantation (IUTx) has been safely performed in humans for decades, but clinical efficacy has only been achieved in fetal recipients with X-SCID. We hypothesize that IUTx can deliver curative levels of HSC engraftment in other genetic disorders after the selective depletion of highly proliferative fetal HSC and/or immune cells. We developed novel antibody-drug conjugates (ADCs) using the ribosomal toxin, saporin, to selectively deplete NK cells expressing CD335, immune cells expressing IL2RG, and fetal HSCs expressing GPI-80. Using a pH-dependent fluorescent reporter and cytotoxic assays, we showed that the maximum internalization and endocytosis of antibodies targeting GPI-80, IL2RG, and CD335 occurred at 6, 18, and 12 hours, and that 150nM of GPI-80, IL2RG, and CD335 killed BM or PB mononuclear cells at 50%, 40%, and 10%, respectively. To evaluate the safety profile and compare GPI-80-, IL2RG- and CD335-ADCs' off-target effects, such as targeting of other cell populations besides hematopoietic cells, with other more studied ADCs such as c-kit, immunohistochemistry was performed on fetal sheep tissues. Fetal kidney, brain, and testes contained 10±3.5%, 25±7.0% and 10±3.5% c-kit positive cells, respectively while GPI-80+, IL2RG+, and CD335+ cells were negligible in these organs. However, IL2RG expression was identified in specific areas of microvascular endothelial cells within the kidney and testes. In contrast, in the bone marrow (BM), levels of GPI-80+, IL2RG+, CD335+, and c-kit+ cells were 46.6±1.9%, 47.9±1.6%, 6.5±1.6%, and 17.7±2.8%, respectively. In the thymus, 20±0.9% of cells were IL2RG+. Having confirmed ADC's functionality and safety profile, human adult BM-derived CD34+ HSC (n=2) at a dose of 108/kg were injected (IP) into fetal sheep at 62-65 gestation days, 1 week after administration of 50ug of GPI-80-ADC (n=4) , IL2RG-ADC (n=5), CD335-AD (n=4), or saline (n=3) (non-ADC control). Prior to IUTx, flow cytometric analysis showed that 97±0.9% of the transplanted cells were CD45+CD34+, and of these, 34±3% and 39±5% were CD38- and CD133+, respectively. CFU-assays confirmed their robust clonogenic potential. Engraftment levels of human donor cells were determined in PB and BM at 60 days post-IUTx by flow cytometry and Digital PCR. The latter was performed using primers specific to the human GAPDH and RPLPO genes. Copies/ml of human GAPDH and RPLPO genes were: for GPI-80 (BM:305±132; 539±50; PB:110±46; 162±48), for IL2RG (BM:18±5; 53±16; PB:10±3; 44±7), for CD335 (BM:80±10; 64±10; PB:21±2; 43±6), and for No-ADC (BM:15±3; 34±19; PB:3±1; 13±3). Flow cytometric analysis of fetal sheep BM showed that the engraftment in BM and PB was multilineage with the presence of lymphoid and myeloid cells. Human CD45+ engraftment was significantly higher in both GPI-80 (BM:19.4 ± 1.8%, PB:11.8 ± 2.1%) and CD335 (BM:16.4± 4.7%, PB:4.3 ± 1.2%) groups when compared with IL2RG (BM:7.5 ± 2.3%, PB:3.4 ± 1.0%) and No-ADC (BM:2.3 ± 1.1%, PB:8.0 ± 2.6%). All ADCs also significantly increased the engraftment of human CD34+HSC when compared to the No-ADC control (BM:0.4±0.2%); (GPI-80:BM:3.4±0.6%, PB:2.0±0.6%); (IL2RG:BM:2.1±0.8% PB:1.8±0.7%); (CD335 5.6±2.2%, PB:0.8 ± 0.2%). GPI-80 also significantly increased the engraftment of CD45+CD33+ myeloid cells when compared to No-ADC control: (BM:GPI-80:39.3±9.8%, PB:19.4±5.0%), (BM:IL2RG:4.8±0.8%, PB:8.8±3.0%), (BM:CD335:7.3±2.5%, PB15.8±5.1%), (BM:No-ADC:7.1±6.7%, PB:7.7±3.5%). Additionally, to determine whether conditioning caused hematologic or metabolic alterations, complete blood cell counts (CBC) and liver enzymes (AST and ALT) were also evaluated. All ADC groups displayed similar CBC counts and AST levels compared to the No-ADC group. However, ALT was elevated in the IL2RG and CD335 ADC groups when compared to the No-ADC group. Histopathological analyses of fetal tissues in transplanted animals demonstrated no evidence of ADC-associated toxicity in any of the examined tissues. In summary, while all selected ADCs displayed minimal off-target effects and selective ablation of endogenous HSCs, GPI-80 conditioning tends to skew engraftment toward the myeloid lineage, while IL2RG and CD335 conditioning tend to skew engraftment toward the B lymphocyte lineage, with GPI-80 thus far being the optimal drug candidate to achieve therapeutic levels of HSC engraftment after IUTx.