Stem Cell

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model – Nature Communications

Time Stamp: October 11, 2023 8:00 PM
Source Node: 124494

Antibody-drug-conjugate (ADC)

The variable regions in heavy and light chains (VH/VL) of an anti-human CD117 antibody (cross-reactive to cynomolgus macaque and rhesus macaque CD117) were grafted onto a human immunoglobulin G1 (IgG1) Fc region mutated in the heavy chain to abolish neonatal Fc receptor (FcRn) binding to enhance in vivo clearance³². Cysteine residues³³ incorporated in the Fc region for site-specific conjugation to the PBD linker payload tesirine³⁴ yielded CD117-ADC (Magenta Therapeutics, Cambridge, MA, USA). An anti-hen egg lysozyme isotype control with matching engineering and conjugation was used to generate Isotype-ADC which served as a control (Magenta Therapeutics).

Human and non-human primate CD34+ cell killing assay

Primary human CD34+ cells were purchased from STEMCELL technologies (Vancouver, BC, Canada). The cells were obtained using Institutional Review Board (IRB)-approved consent forms and protocols. The steady-state human bone marrow CD34+ cells (3 donors, STEMCELL technologies) or purified mobilized CD34+ cells from two healthy rhesus macaques were cultured (2500 cells/well in 384-well plate) in serum-free StemSpan SFEM media (45 μl, STEMCELL Technologies) supplemented with 100 ng/ml each of Interleukin 6 (IL-6), fms-related tyrosine kinase 3 ligand (FLT3L), and thrombopoietin (TPO). Escalating doses of CD117-ADC as well as IgG isotype control-conjugated ADC were added (5 μl ADC added to 45 μl cells). After 6 days of culture, the number of viable CD34 + CD90+ cells were evaluated by flow cytometry using 7-Aminoactinomycin D (7-AAD as a viability marker, Biolegend, San Diego, CA, USA), CD34 BV785 (clone 561, BioLegend), and CD90 APC (Clone 5E10, BioLegend) detection antibodies. Cells were acquired on a BD Celesta Instrument and analyzed with FloJo. The EC₅₀ and EC₉₀ values were calculated with GraphPad Prism software.

Lentiviral vector design and preparation

For efficient transduction in both human and rhesus CD34+ cells, chimeric human immunodeficiency virus type 1-based lentiviral vectors (χHIV vectors) were prepared in 293 T cells (American Type Culture Collection (ATCC), Manassas, VA, USA) with plasmid transfection and concentrated by ultracentrifugation^22,23. In the β-globin vector, the human β-globin gene is expressed by the β-globin promoter with locus control regions^35,36. In the thEpoR/shmiR-BCL11A vector, a chimeric sequence between thEpoR and shmiR-BCL11A is driven by the erythroid-specific ankyrin-1 promoter with an erythroid-specific BCL11A enhancer, allowing for BCL11A interference and stable HbF induction²⁰. The lentiviral titers were evaluated in transduced HeLa cells (ATCC) with the SIN-LTR probe/primers by quantitative polymerase chain reaction (qPCR, QuantStudio 6 Flex Real-Time PCR System, Thermo Fisher Scientific, Waltham, MA, USA)²⁵.

Depletion of CD34 + CD90 + CD45RA- cells in cynomolgus macaque

Cynomolgus macaques³⁷ were treated with intravenous injection of CD117-ADC (0.1 or 0.3 mg/kg × 1 day) or busulfan (6 mg/kg × 4 days), and bone marrow CD34 + CD90 + CD45RA- cells were quantified by using antibodies against CD34 BV785 (clone 561, BioLegend, 1:300), CD90 APC (clone 5E10, BioLegend, 1:300), and CD45RA VioBlue (clone T6D11, Miltenyi Biotec, 1:50) in flow cytometry 7 days after drug administration (n = 3 per group) and compared to historical phosphate-buffered saline (PBS)-treated naïve animals. Bone marrow cellularity was assessed by complete blood count analysis to determine white blood cells per ml of marrow. Flow cytometry was used to determine the frequency of CD34 + CD90 + CD45RA- cells of total CD45+ cells. The absolute number of CD34 + CD90 + CD45RA- was determined by multiplying the frequency of CD34 + CD90 + CD45RA- cells (out of total CD45 cells) with the bone marrow cellularity in cells per ml.

Autologous CD34+ cell transplantation with lentiviral transduction in a non-human primate

We had developed an autologous CD34+ cell transplantation model with lentiviral transduction in rhesus macaques^22,23, following the guidelines set out by the Public Health Service Policy on Humane Care and Use of Laboratory Animals under a protocol (H-0136) approved by the Animal Care and Use Committee of National Heart, Lung, and Blood Institute (NHLBI). Rhesus CD34+ cells were mobilized with granulocyte-colony stimulating factor (G-CSF, 20 μg/kg for 5 days, Amgen, Thousand Oaks, CA, USA) and plerixafor (1 mg/kg on day 5 of G-CSF, Amgen) and isolated by magnetic separation. The mobilized rhesus CD34+ cells (4e6 cells/ml) were pre-stimulated in X-VIVO10 culture media (Lonza, Basel, Switzerland) including 100 ng/ml each of SCF (R&D Systems, Minneapolis, MN, USA), FLT3L (R&D Systems), and TPO (R&D Systems) for 1 day and transduced with lentiviral vectors encoding either human β-globin gene (13U047 and 12U032)³⁵ or thEpoR/shmiR-BCL11A cDNA (12U018, 12U020, ZL13, ZJ62, H635, and H96G)²⁰ at a multiplicity of infection (MOI) 50 in the fresh culture media supplemented with the same cytokines as well as 10 μ M prostaglandin E2 (R&D Systems) and 100 μ g/ml poloxamer 407 (Sigma-Aldrich, Saint Louis, MO, USA)³⁵. The next day, transduced rhesus CD34+ cells were cryopreserved by Recovery Cell Culture Freezing Media (Thermo Fisher Scientific). After conditioning with either a single intravenous injection of CD117-ADC (0.2 mg/kg in 13U047 and 12U032, 0.3 mg/kg in ZL13 and ZJ62, and 0.4 mg/kg in H635 and H96G) or four-dose intravenous injections of busulfan (5.5 mg/kg × 4 days in 12U018 and 12U020), the frozen CD34+ cells with lentiviral transduction were intravenously infused into autologous macaques (6 days post-ADC at 0.2-0.3 mg/kg, 10 days post-ADC at 0.4 mg/kg, and 1 day post-busulfan). In addition, following G-CSF and plerixafor-based mobilization and the harvest of CD34+ cells, CD117-ADC was intravenously injected into rhesus macaques (0.2 mg/kg in JJ50, 0.3 mg/kg in ZJ10, and 0.6 mg/kg in ZI07) without autologous CD34+ cell transplantation. The plasma concentrations of ADC were measured by enzyme-linked immunosorbent assay (ELISA) using a human therapeutic IgG1 ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) following a single CD117-ADC administration at 0.2 mg/kg (JJ50), 0.3 mg/kg (ZJ10), 0.4 mg/kg (H635), and 0.6 mg/kg (ZI07). Pharmacokinetic parameters were estimated with Phoenix pharmacokinetic software (Certara, Princeton, NJ, USA) using a non-compartmental approach consistent with the intravenous route of administration. Half-life (T½) in hours, maximal concentration (Cmax) in ng/ml, area under the curve (AUC) in ng*hr/ml, clearance (Cl_obs) in ml/hr/kg, and volume of distribution (Vz_obs) in ml/kg parameters were calculated.

During transplantation, complete blood counts (white blood cells, granulocytes, lymphocytes, red blood cells, hemoglobin concentrations, hematocrit, reticulocytes, and platelets) and biochemistry tests (AST, ALT, LDH, total bilirubin (TBIL), albumin (ALB), blood urea nitrogen (BUN), creatinine (CREA), and potassium (K)) were evaluated in peripheral blood samples. Based on blood counts, transplanted animals were maintained by whole blood transfusion and platelet-rich plasma transfusion. Aspiration samples from bone marrow were evaluated by hematoxylin and eosin staining. After engraftment of gene-modified cells, VCN was evaluated in granulocytes and lymphocytes with the SIN-LTR probe/primers by qPCR²⁵. The percentage of F-cells (clone 2D12, BD Biosciences, 1:30) was evaluated by flow cytometry (FACSCanto, BD Biosciences) (Supplementary Fig. 8A)²⁰. Live subsets of peripheral blood mononuclear cells were gated using 7-AAD (BD Biosciences, 1:20) staining and analyzed by using antibodies against CD4 PE (clone L200, BD Biosciences, 1:20), CD8 BUV737 (clone G42–8, BD Biosciences, 1:20), CD11b BUV395 (clone D12, BD Biosciences, 1:40), CD14 FITC (clone M5E2, BD Biosciences, 1:10), CD16 PE-Cy7 (clone 3G8, BD Biosciences, 1:40) CD18 R718 (clone 6.7, BD Biosciences, 1:40), CD20 APC-H7 (clone 2H7, BD Biosciences, 1:20), CD45 V450 (clone D058-1283, BD Biosciences, 1:40), and CD56 APC (clone B159, BD Biosciences, 1:10) (Supplementary Fig. 8B). The amounts of γ-globin production in rhesus red blood cells were evaluated by reversed-phase HPLC^38,39. Serum Epo levels were evaluated before and after transplantation (14–22 months post-transplant) by human Epo ELISA kit (STEMCELL Technologies). The Epo levels were multiplied by 2.5, since the sensitivity of rhesus Epo is ~40% in human Epo ELISA⁴⁰.

Mating was performed among the colony at the Alpha Genesis Primate Research Center (Yemassee, SC, USA). Estradiol, progesterone, and androstenedione were analyzed at the Wisconsin National Primate Research Center (Madison, WI, USA).

The animals are specific pathogen-free. They are individually housed while in the intensive care unit (ICU) during transplantation, pair-housed upon recovery, and group-housed for breeding. When euthanasia is required, the animal is sedated with ketamine and/or telazol, and receives an overdose of pentobarbital (80 mg/kg IV or IC), followed by potassium chloride (100 mg/kg IV or IC).

Integration site analysis

Integration site analysis was performed as previously described⁴¹. In brief, DNA was extracted from the granulocyte fraction of peripheral blood 1 year post-transplantation with CD117-ADC conditioning (0.3 mg/kg in ZL13 and ZJ62, 0.4 mg/kg in H635 and H96G) and myeloablative busulfan conditioning (12U018 and 12U020). The DNA sequences around lentiviral integration sites were linearly amplified and analyzed by next-generation sequencing. Integration sites were mapped to the rhesus genome assembly rheMac10. Due to limited annotation in rheMac10, human gene names and RefSeq ID were used, which are from the Xenogene track from the rheMac10 genome annotation of the University of California, Santa Cruz (UCSC, Santa Cruz, CA, USA).

Statistical analysis

Statistical analysis was performed using the JMP 16 software (SAS Institute Inc., Cary, NC, USA). Two averages were evaluated by the student’s t-test. The averages in various conditions were evaluated by one-way analysis of variance. The correlation was evaluated by R² and p-value for coefficient of correlation. A p-value of <0.01 or <0.05 was deemed significant. The standard deviation was shown as error bars in all figures.