Clinimacs device

Preferred haploidentical grafts were related, nonmaternal, and younger donors. Haploidentical donors grafts were harvested after granulocyte colony-stimulating factor mobilization at 5 μg/kg subcutaneously twice a day or 10 μg/kg daily, and CD34⁺ cells were selected by the Isolex 300i CD34 depletion device before April 2010 or the CliniMACS device (Miltenyi Biotec Inc., San Diego, CA) thereafter, aiming for a CD3 cell dose below 1 × 10⁴/kg in the initial phase of the study. As of early 2012 the algorithm was adjusted to also limit the CD34⁺ cell dose of the haploidentical donor to approximately 3 to 5 × 10⁶/kg of recipient weight.
Preferred UCB units were HLA matched using standard UCB matching criteria: HLA-A and -B, by antigen matching, and at DRB1 by allele matching with at least 4/6 match. The minimum cell dose varied by protocol and protocol stage from .5 × 10⁷ total nucleated cells (TNCs)/kg recipient weight to 2.0 × 10⁷ TNCs/kg. Preference was first given to optimal HLA matching once the minimum cell dose requirements were met.
Presence of DSAs against the haploidentical and UCB grafts was evaluated by solid-phase immunoassay (Luminex Corporation, Austin, TX). When DSAs were present, desensitization was performed as previously reported [35 (link)]. Haploidentical cells were infused on day 0, and UCB units were infused either later on the same day or on the following day.

CD4+ and CD8+ T cells were isolated from patient apheresis products using the CliniMACS device (Miltenyi Biotec). A 1:1 mixture of CD4-enriched and CD8-enriched cell fractions were then pooled, suspended in X-Vivo 15 (Lonza) media supplemented with 2% KnockOut SR (Life Technologies), 5 ng/mL rhIL-7 (CellGenix), 0.5 ng/mL rhIL-15 (CellGenix) and 10 ng/mL rhIL-21 (Miltenyi Biotec), initiated into culture in a G-Rex 100MCS vessel (Wilson-Wolf), and stimulated using CD3/CD28 CTS® Dynabeads (Life Technologies). Cell products were then transduced with a GMP-grade SIN (self-inactivating) lentivirus encoding the anti-B7-H3CAR, the methotrexate-resistant human DHFR mutein huDHFRdm, and the cell-surface marker EGFRt. On day 3 of culture, 50nM methotrexate (Mylan) was added to the culture vessel to select for cells possessing DHFRdm. On day 7 of culture, CD3/CD28 CTS® beads were removed from the cell suspension using the Dynamag CTS device. Following 10–13 days in culture, patient cells were harvested and washed using the Sepax 2RM device (Cytiva) and resuspended in CryoStor-CS5 (Biolife Solutions) for cryopreservation in CellSeal closed-system vials (Sexton).

HPCs were obtained via G-CSF mobilization of the haploidentical donor, and collection by leukapheresis on day 5 and 6 of G-CSF. The first HPC product collected on day 5 was T-cell-depleted using the CliniMACS device and CD34 Microbead (Miltenyi Biotec, Auburn, CA, USA). Minimum cell dose required for the CD34+ enriched progenitor cell graft was 2 × 10⁶ CD34+ cells/kg. Maximum CD3+ dose allowed for the CD34+ enriched HPC was 0.1 × 10⁶ CD3+ cells/kg.
The HPC product collected on day 6 was processed for CD45RA+ cell depletion using the CliniMACS device and its “Depletion 3.1” software. There was no target CD34+ dose or CD3+ dose on the CD45RA+ depleted product; however release criteria of the product includes a ≥2 log₁₀ depletion of CD45RA+ cells. Two of the 17 patients did not meet the minimum CD45RA depletion after a single depletion step. Per standard operating procedure, a second run under the same conditions was performed and the requisite level of depletion was achieved in both products.
The NK cell product was collected by leukapheresis 5 days after the second HPC collection. It was a nonmobilized product, and was processed on the CliniMACS device as previously described.^{28 (link), 29 (link)} All three cell products were infused fresh.

Patients received myeloablative, non-myeloablative, or reduced-intensity conditioning (RIC) regimens depending on their underlying disease and human leukocyte antigen (HLA)-matched or mismatched grafts from related or unrelated donors. Center for International Blood & Marrow Transplant Research operational guidelines or MSKCC institutional guidelines were used to classify regimens as myeloablative, reduced intensity or non-myeloablative. Type of grafts included unmodified bone marrow (BM) or peripheral blood stem cells (PBSCs), T-cell depleted (TCD) BM or PBSCs or single umbilical cord blood (UCB). Graft selection depended on disease, donor availability and the discretion of the treating clinician. PBSCs were harvested after mobilization according to National Marrow Donor Program and institutional guidelines. Grafts were infused intravenously 36–48 hours after the last dose of chemotherapy. CD34+ cell selection from PBMSCs was performed using the CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany)^{13 (link)} or the ISOLEX 300i Magnetic Cell Selection System (Baxter Health Care Corporation, Dearfield, IL.^{14 (link)} followed by E-rosetting TCD for BM grafts was performed with sequential soybean lectin agglutination and sRBC rosette depletion of T-cells.^{15 (link), 16 (link)}

CD34⁺ HPCs were isolated from commercially obtained umbilical cord blood products (Key Biologics) with the CliniMACS device (Miltenyi Biotec), yielding >90% purity (Fig. S1).
Pups were exposed to irradiation (Cesium irradiator) with 100 cGy (NSG) or 150 cGy (MISTRG or MITRG) within the first 2 postnatal days. Subsequently, 100,000 CD34⁺ HPCs were injected into the liver. Adult 4- to 6-week-old MISTRG mice were irradiated with 250 cGy and received 100,000 CD34⁺ HPCs as intraperitoneal injection. Successful engraftment was defined as >10% and calculated as: $\%=100\times \frac{\text{human}\text{CD}45+}{\text{human}\text{CD45}++\text{mouse}\text{Cd}45+}$