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Clinical-scale manufacturing of γδ T cells for protection against infection and disease recurrence following haploidentical peripheral blood stem cell transplantation and cyclophosphamide gvhd prophylaxis

Haploidentical hematopoietic cell transplantation (HAPLO HCT) as therapy for hematopoietic malignancy can result in long-term survival and cure for patients who require allogeneic HCT but lack an HLA-matched donor. [1] Administration of cyclophosphamide to eliminate the alloreactive T cells entering into an activated state 3–4 days post HCT following infusion of either bone marrow or mobilized peripheral blood (PBSC) [2] grafts [3] has been shown be effective GVHD prophylaxis. Although post-transplant cyclophosphamide (PTCy) has generally expanded the use of HAPLO HCT, the success of this procedure has been hindered by high risks of graft rejection, prolonged immune reconstitution with subsequent infectious complications [4], and when combined with immunosuppressive therapy may result in an increased risk of disease relapse. [5]

Preclinical and clinical studies strongly suggest that post-transplant infusion of donor gamma delta (γδ) T cells during this period of slow immune recovery could constitute an effective prophylaxis against infection and relapse in HAPLO HCT. This is because of the graft-versus-leukemia (GvL) effect mediated by the donor immune cells include not only alloreactive αβ T cells but also γδ T cells and natural killer (NK) cells. In contrast to alloreactive αβ T cells that recognize mismatched minor or major HLA antigens, γδ T cells recognize their target cells in a non-HLA restricted manner and do not initiate GVHD [6].. Indeed, γδ T cells directly recognize and respond to a variety of HLA-like stress-induced self-antigens [7, 8] and are known to both facilitate engraftment [9,10,11,12] and exhibit a strong graft-versus-leukemia (GvL) effect [13]. Indeed, a significant subset of HAPLO HCT patients who received an αβ TCD (T-cell depleted) graft was shown to have favorable homeostatic reconstitution of γδ T cells compared to that observed with patients receiving Pan-CD3-depleted grafts, a finding recently confirmed by Airoldi in children receiving haplo HSCT using αβ T-cell/CD19+ B-cell-depleted grafts. [14] Decreased relapse rate and a significant improvement in relapse-free survival has also been noted among haplo HCT patients who recovered with increased peripheral blood γδ T-cell counts, [13] a finding that was found to be durable over 7 years following BMT. [15] As there is not effective in vivo techniques to increase the γδ T-cell number, we developed a graft engineering protocol to provide a post-HAPLO HCT γδ T cell “boost” during the period of early post-HCT immune recovery.

Ex vivo expansion and activation of γδ T cells is required to generate an effective cell dose as they constitute only a minor circulating lymphocyte population ( < 10% of T cells). Previous attempts have shown γδ T cells manufacturing procedures to be cumbersome and generally confined to the research setting. In order to simplify procedures and widen applicability for clinical therapy, we adapted the CliniMACS Prodigy (Miltenyi Biotec; Bergisch Gladbach, GERMANY) for γδ T cells manufacturing in a closed-system cGMP (current Good Manfacturing Practice) compliant process to support our pivotal Phase I clinical trial in HAPLO HCT. An overview of the general procedure is depicted in Fig. 1. A total four healthy volunteer donors were accrued. Approximately 100 mL of apheresis product was phenotyped, and loaded into the CentriCult® chamber. Automated density gradient centrifugation and subsequent washes were performed in closed system (Fig. 1) and resuspended into OpTimizer® cell culture media (Thermo Fisher, Waltham, MA) containing Zoledronate (Novartis: Basel, SWITZERLAND) and recombinant human IL-2 (Miltenyi). Cultivation was continued with media exchanges for 12–14 days at which time αβ TCD was performed using biotinylated anti-αβ monoclonal antibody and streptavidin-coated ferromagnetic microspheres. The product was then washed and harvested in buffer. Product composition was assessed by flow cytometry (BD Biosciences Fortessa® SE) using primary antibodies to CD3, CD4, CD8, TCR-αβ, TCR-γδ, Vδ1, Vδ2, CD45, CD45RA, CD195, CD27, HLA-DR, CD28, CD57, PD-1 (Duraclone®: Beckman-Coulter; Miami, FL), and CD16, CD56, CD19 (BD Biosciences; San Jose, CA). Potency was measured by cytotoxic activity against K562 erythroleukemia target cells using the ImmunoChemistry Technologies (ICT) Basic Cytotoxicity Kit. Sterility was determined by microbiologic assays of 4-day sterility (BAC-T Alert®: BioMerieux; Durham, NC), endotoxin, (Endosafe® Limulus Amebocyte Lysate assay: Charles River; Germantown, MD) and Mycoplasma contamination (Clongen; Gaithersburg, MD) using aerobic and anaerobic cultures of the final cell product on specific agar for 28 days. Following αβ TCD, only trace amounts of αβ T cells (>0.05%) remained in the product. The γδ T cells represented 89%.0 ± 15.1 (range 66.5–90.0) of the remaining CD3+ T cells from an initial 3.6% ± 3.8 (range 1.2–9.2). NK cell expansion was modest at <7% with the exception of one product expanded 12.7% NK cells (31.5% following αβ TCD). B cell content was negligible. As expected, the γδ T cells were overwhelmingly Vγ9Vδ2+ CD4−CD8−CD28+ with incremental expression of CD56, CD57, and PD-1. The first product showed evidence of activation-induced cell death (AICD), a common problem seen when peripheral blood γδT cells are cultured for too long. Expanded/activated γδT cells were overwhelmingly CD45RA+ CD27−CD195− effector and CD45RA+ CD27−CD195 effector/memory phenotype. Final cell viability ranged from 62 to 67%. Cytotoxicity against K562 cells (Fig. 2) showed variability as expected, 52.7 ± 22.4 (range 33.0 to 84.0) but passed predetermined specifications at an effector to target (E:T) ratio of 20:1. Gram stain, 14-day bacterial culture, endotoxin and mycoplasma were negative for organisms in all processing runs.

Fig. 1
figure1

Flow diagrams showing steps involved separation of mononuclear cells, cultivation and enrichment of γδ T cells. Module 1 allows using CentriCult unit for Ficoll based separation of mononuclear cells directly from apheresis product. Module 2 initiates cultivation setup for expansion of γδ T cells till day completion. At the end of the cultivation period, module 3 is initiated for αβ T cell depletion from the expanded product. Interim sampling is done for monitoring cell density, sterility and phenotype of manufactured cells

Fig. 2
figure2

(a) Potency determination for four validations by target killing efficiency (%) was measured for γδ T cells expanded in Prodigy®, post αβ T cell depletion. The T cells were co-cultured with target cell (K562) in increasing E:T ratio and lytic ability was measured using 7 AAD based cytotoxicity assay In-process control (b) Flow cytometric analysis of effector/memory status and PD-1 expression from Validations 1 and 4 at harvest. Note that validation 1 is predominately comprised of effector/memory (E/M) and effector (E) subtypes and high PD-1 expression as well as poor potency indicating over-stimulation. Validation 4 shows strong potency, a central memory (CM)/EM phenotype and low PD-1 expression. (c) γδ T cell percentage and (d) count (bottom) was monitored at Apheresis, completion of culture, and following αβTCD for all validations. Note increasing γδ T cell percentage and further purification with αβTCD. Absolute count parallels percentage, although significant numerical losses of γδ T cells occurs during αβTCD

When extrapolated to a 2 blood volume apheresis, this manufacturing protocol met the predetermined dose level of 3 × 106 γδ+ T cells/kg, our predetermined criteria for initiation of the phase I clinical trial. Depletion of αβ T cells was extremely efficient, delivering a total αβ T cell total dose of <1.0 × 105/kg (assuming a 70-kg recipient) although substantial loss of γδ T cells also occurred (69–78%) during the αβ TCD procedure. Based on the outcome of this manufacturing protocol, we believe that cGMP compliant γδ T cells expansion is feasible, reproducible, and sufficiently reliable for advancement into clinical trials. Indeed, our Phase I trial for expanded/activated γδ T cells “boost” following HAPLO HCT has recently been approved by FDA for our institution. Remaining challenges in process development include excessive loss of γδ T cells in the αβ TCD step and scaling to higher doses across all potential patient weights, however, the automation of closed-system GMP γδ T cells expansion should widen acceptance and applicability of this procedure in HAPLO HCT and other settings.

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Conflict of interest

The translational studies contained were funded by Incysus, Ltd (www.incysus.com). Dr. L.S.L. receives no compensation from Incysus separate from the percentage of effort applied toward his standard compensation as a Professor of Medicine at the University of Alabama at Birmingham (UAB). Dr. L.S.L. and Dr. A.S. share intellectual property developed at UAB surrounding the treatment strategy. Dr. L.S.L. is a founder of Incysus and has founder’s equity that is managed independently. The remaining authors declare that they have no conflict of interest.

Correspondence to Lawrence S. Lamb.

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