Adoptive Transfer of Purified Donor-B-Lymphocytes after Allogeneic Stem Cell Transplantation: Results from a Phase I/IIa Clinical Trial
Authors: Winkler Julia , Tittlbach Hannes , Roesler Wolf , Strobel Julian , Zimmermann Roland , Maas Stefanie , Spriewald Bernd , Wolff Daniel , Repp Roland , Kordelas Lambros , Mach Michael , Mackensen Andreas , Winkler Thomas H. 
Published: October 2019

Abstract
Patients after allogeneic stem cell transplantation (alloSCT) show a long lasting immune deficiency involving both T- and B-lymphocytes. Antibody responses to vaccination, as a measure of functional B-cell immunity, are insufficient in the first months after alloSCT and usually recover within 1-2 years. To improve B-cell immune reconstitution after alloSCT, we developed a novel concept of adoptive transfer of memory B-cells from the original stem cell donor (Klenovsek et al., Blood 110:3472-79, 2007). To this end, we first established the technology for the manufactoring of a GMP-qualified B-cell product and initiated a first-in-man phase I/IIa clinical trial in patients after alloSCT to evaluate safety and tolerability of adoptively transferred donor B-cells.
B-cells were manufactured under GMP conditions from 16 unstimulated leukapheresis products derived from the original stem cell donor. The separation protocol consists of two subsequent separation steps on the CliniMACS® System (Miltenyi Biotec) including depletion of CD3+ T-cells and subsequent positive separation of CD19+ B-cells by magnetic beads. In all of the 16 cell products high purities of CD20+ B-cells (average 96%, range 85-99%) with only low frequencies of contaminating T-cells (average 0.10 %, range 0.01-0.82%) were achieved. After manufacturing, B-cell products were cryopreserved for single administration to 4 groups of patients with escalating doses. To prevent GvH reactions the absolute number of CD3+ T lymphocytes in the B-cell product had to be below 4 x 104 CD3+ cells/kg BW.
The clinical trial was designed as an open label dose escalating study using the traditional 3+3 design with 4 dose levels of 0.5-1-2-4x106 B-cells/kg BW. Patients were enrolled on day +140 ± 20 after alloSCT after tapering of immunosuppression. Patients with acute GVHD grade III/IV, chronic GVHD with intermediate to high risk, EBV reactivation (>10,000 EBV copies/ml) or after therapy with B-cell antibodies (i.e. rituximab) were not eligible. Up to now, 13 patients after alloSCT received donor B-cells in 4 different B-cell dosages (average day +147, range from day +130 to d +160). Three patients received 0.5x106 B-cells per kg BW, 3 patients 1.0x106 B-cells/kg BW, 5 patients 2.0x106 B-cells per kg BW, and 2 patients 4.0x106 B-cells/kg BW, respectively. The median number of infused T cells was 0.06x104 T-cells per kg BW (range 0.01-0.13 x104/kg BW). Regarding primary safety data, the B-cell transfer was well tolerated without any acute adverse reactions. Importantly, neither significant EBV-viremia nor acute or chronic GvHD reactions were observed during the observation period of 4 months after the transfer of B-cells.
In addition to the analysis of the primary safety endpoints of the trial, the activity of infused donor memory B-cells in vivo were tested. A characteristic memory B-cell response is defined as a mobilization of plasmablasts (PBs) 7 days after booster vaccination into the peripheral blood (Odendahl et al., Blood 105: 1614-21, 2005). Preliminary results obtained from patients that received the donor derived B-cells demonstrated significant mobilization of PBs in some of the patients after re-vaccination with a pentavalent vaccine (Pentavac®), suggestive for a memory B-cell response. A detailed analysis of the vaccination data from the study patients in comparison to a control group of transplanted patients without B-cell transfer after routine re-vaccination will be presented. Furthermore, the analysis of immunoglobulin VH-sequences by next-generation-sequencing in memory B-cell populations of two individual patients after B-cell transfer revealed that at least 50% of all memory B-cell clones identified in the recipient are unambiguously contained within the memory B-cell population of the infused B-cell product.
In summary, our data show that the manufacturing of a B-cell product from the original stem cell donor under GMP conditions without significant T-cell contamination is feasible. The transfer of donor-derived B-cells into patients after alloSCT is safe with regard to EBV reactivation and induction of acute or chronic GvHD. Future clinical trials will evaluate whether the adoptive transfer of memory B-cells from the donor can significantly reduce the frequency of infections after alloSCT.
(Supported by the Deutsche Forschungsgemeinschaft through SFB 643 "Strategies of cellular immune intervention")

Disclosures
No relevant conflicts of interest to declare.