scholarly journals Ex Vivo T-Cell Receptor αβ +/CD19 +depletion of Peripheral Stem Cell Grafts for Pediatric Patients with Bone Marrow Failure (BMF) Undergoing Unrelated Donor Transplantation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 171-171
Author(s):  
Joseph H Oved ◽  
Caitlin W Elgarten ◽  
Yongping Wang ◽  
Stephan Kadauke ◽  
Dimitri S. Monos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Graft Rejection ◽  
Pediatric Patients ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Cell Receptor ◽  
Peripheral Stem Cell ◽  
Unrelated Donors

Abstract Background: Success of alternative donor stem cell transplantation (SCT) for acquired and inherited bone marrow failure (BMF) syndromes has previously been limited by significant risks of severe graft versus host disease (GvHD) and graft failure, particularly for patients who lack fully HLA-matched donors. The development of post-transplant cyclophosphamide (ptCy)-based haploidentical donor SCT has mitigated but not eliminated these risks, and the relatively slow engraftment seen with ptCy bone marrow grafts may not be ideal given high rates of infectious disease and bleeding concerns in patients with BMF. Partial T cell depletion of mobilized peripheral stem cell (PSC) grafts can greatly reduce risks of graft versus host disease while facilitating rapid and robust engraftment by providing a high stem cell dose. We previously reported excellent outcomes with minimal GVHD and no graft rejection using peripheral stem cell transplant (PSCT) combined with ex vivo CD3 +/CD19 +depletion and low dose CD3+ T cell addback from matched unrelated donors (MUD) and mismatched unrelated donors (MMUD) in pediatric patients with BMF. Here, we describe the use of selective ex vivoT cell receptor (TCR)αβ +/CD19 +depletion of mobilized PSC from MUD and MMUD for pediatric patients with BMF, which has the advantage of retaining TCRgd +T cells which may help facilitate engraftment and decrease infections. Methods: We report the outcomes of 26 pediatric patients with BMF (excluding MDS-defining clonal evolution) who underwent MUD/MMUD PSCT with TCRαβ +T cell/CD19 +depletion using CliniMACS at The Children's Hospital of Philadelphia from 2017 to 2021. Patients were enrolled on a prospective clinical trial for patients with BMF (NCT03047746, n=21)or on an expanded access study (NCT03145545, n=5). Conditioning regimens consisted of thymoglobulin (9mg/kg), cyclophosphamide (100mg/kg), fludarabine (150mg/m 2), and low dose TBI (200-300 cGy) for patients with acquired BMF disorders, thymoglobulin (9mg/kg), busulfan (PK-adjusted), fludarabine (150mg/m 2), and thiotepa (10mg/kg) for patients with single lineage BMF or thymoglobulin. One patient with Fanconi Anemia received thymoglobulin and fludarabine, with reduced dosing of busulfan and cyclophosphamide. Patients undergoing MSD-BMT for BMF over a similar time period served as a comparison group for engraftment kinetics, rates of GVHD, donor chimerism, immune reconstitution, and overall survival. Results: Subjects included 18 with severe acquired aplastic anemia (SAAA), 4 with SAAA and concurrent paroxysmal nocturnal hemoglobinuria (PNH), 2 patients with acquired BMF not otherwise specified, 1 patient with DBA, and 1 patient with Fanconi Anemia. 11 patients with SAAA underwent SCT as initial therapy while 11 patients had SCT after failing previous medical therapies. Median age at diagnosis was 10.3 years (0.1-20.6) and at transplant 11.1 years (0.9-21). HLA match of unrelated donors was either 10/10 (n=15), or 9/10 (n=11). Median CD34 +and TCR αβ +T cell dose was 12.0x10 6cells/kg (3-22.6) and 0.1x10 5cells/kg (0.0-4.2). Median times to neutrophil and platelet engraftment per CIBMTR criteria were 15 days (10-22) and 15 days (13-19), respectively, both significantly earlier than engraftment following MSD-BMT (Fig 1a). At a median follow-up of 727 days (39-1498), 25 of 26 patients are alive with resolved hematologic disease (Fig 1b). One patient with SAAA+PNH who failed prior IST achieved trilinear engraftment without GVHD, but died on Day+95 due to acute disseminated toxoplasmosis. No patients exhibited immunologic graft rejection. 2/26 patients had grade II acute GVHD that responded to steroids and none developed Grade III-IV acute or chronic extensive GVHD (Fig 1c) CMV viremia/reactivation occurred in 5 subjects, all responding to antiviral pharmacotherapy, and none developed end-organ CMV disease (Fig 1d). One patient developed recipient-derived EBV post-transplant lymphoproliferative disorder requiring multimodal treatment. Only one patient developed BK cystitis. Total peripheral chimerism exceeded 90% in all patients. Immune reconstitution kinetics were similar to that seen in MSD-BMT. Conclusion: MUD/MMUD PSCT with TCRαβ +T cell/CD19 +depletion in patients with BMF enables rapid, durable engraftment with minimal risk of GVHD and immunologic graft rejection. Figure 1 Figure 1. Disclosures Monos: Omixon: Consultancy, Patents & Royalties. Grupp: Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy.

Haploidentical Stem Cell Transplantation with CD3 Depleted Mobilized Peripheral Blood Stem Cell Grafts for Children with Hematologic Malignancies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2910-2910 ◽  
Author(s):  
Gregory A. Hale ◽  
Kimberly A. Kasow ◽  
Kwan Gan ◽  
Edwin Horwitz ◽  
Joseph P. Woodard ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Graft Rejection ◽  
Peripheral Blood ◽  
Acute Gvhd ◽  
Ex Vivo ◽  
Hematologic Malignancies ◽  
Infectious Complications ◽  
Persistent Disease

Abstract Allogeneic hematopoietic stem cell transplantation is the only curative therapy for patients with high-risk or recurrent hematologic malignancies. As only 25% of patients have matched siblings and not all have unrelated donors, haploidentical HSCT using mismatched related donors is the only option for many patients. However, historically the risks of GVHD, graft rejection, and prolonged immunocompromise have made this donor option rather limited. More recently, highly purified CD34+ hematopoietic cells have been used with decreased GVHD rates, but at the risk of graft rejection and prolonged immunodsuppression with infectious complications. In an attempt to obtain a PBSC graft with higher T-cell content to maintain acceptable GVHD rates while promoting more rapid immune reconstitution, we initiated a prospective clinical trial for patients with hematologic malignancies who lacked a matched related donor or unrelated donor using a novel method of graft processing. The conditioning regimen consisted of TBI (12 Gy in 8 fractions over 4 days), cyclophosphamide (60 mg/kg/day for 2 days), thiotepa (10 mg/kg/day for 1 day), and rabbit ATG (10 mg/kg/course over 4 days). GVHD prophylaxis consisted of cyclosporine initiated at day -2. G-CSF mobilized PBSC grafts from mismatched related donors were infused after ex vivo T-cell depletion using OKT3 on the CliniMACS device. Patients had weekly peripheral blood analysis for evidence of EBV, CMV, or adenovirus DNA by PCR. If positive, pre-emptive therapy was administered. Twenty patients were enrolled with a median age of 11.9 yrs (range, 2.7–22.1). Diagnoses included ALL (2-CR1, 5-CR2, 3-CR3), AML (2-CR1, 1-CR2, 1-persistent disease), MDS (1-CR1, 2-persistent disease), CML (2- first chronic phase) and NHL (1-CR2). Donors and recipients were matched at 3 (n=11), 4 (n=8) or 5 (n=1) of 6 HLA loci. Of the 19 evaluable patients (one patient died prior to engraftment), the median time to attain ANC > 500/mm3 was 13 days (range, 10–19) and the median time to attain a transfusion-independent platelet count of 50,000/mm3 was 18 days (range, 8–37) post-HSCT. Only 3 patients developed grade 1–2 acute GVHD and none developed grade 3–4 acute GVHD. One patient developed limited chronic GVHD. Complications included post-transplant lymphoproliferative disorder (PT-LPD, n=3), VOD (n=2), BOOP (n=1), CMV retinitis (n=1), and adenovirus reactivation (n=7). No patient died of infectious complications or PT-LPD. 6 patients have died of regimen-related toxicities (n=4), or disease recurrence (n=2) at a median of 160 days (range, 4–208) post-HSCT. Fourteen patients remain alive in remission at a median of 162 days (range, 49–947) post-HSCT. OKT3 depleted PBSC grafts from haploidentical donors depleted of T-celss ex vivo results in favorable outcomes and acceptably low rates of GVHD and infectious complications for children undergoing HSCT from parental donors.

Incidence, Clinical Characteristics, and Prognostic Relevance Of Clonal T-Cell Receptor Positive (TCR+) Populations In Patients With Myelodysplastic Syndrome (MDS)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5231-5231
Author(s):  
Preetesh Jain ◽  
Hagop Kantarjian ◽  
Keyur P. Patel ◽  
Elias Jabbour ◽  
Naval Daver ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
T Cell Receptor ◽  
Clinical Characteristics ◽  
Bone Marrow Failure ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Receptor ◽  
Response To Therapy ◽  
Hypomethylating Agents ◽  
Prognostic Relevance

Abstract Introduction Monoclonal or oligoclonal T cell receptor positive populations (TCR+) can be observed in elderly individuals with viral infections, patients (pts) with pancytopenia, bone marrow failure syndromes, and in large granular lymphocytic leukemia. Expansion of these T cell clones may indicate that an antigen driven myelosuppressive mechanism is active in pts with MDS. However the clinical significance of clonal T cell population/TCR+ in MDS is not clear. In the present study we have reported on the clinical characteristics and prognostic relevance of monoclonal or oligoclonal TCR+ cells in pts with MDS. Methods  A retrospective chart review was performed for all pts with a diagnosis of MDS in whom TCR clonal assessment was done at our institution between 2005 and 2013. Using labeled V-primers, TCR (beta and gamma) were assessed in the bone marrow (BM) samples by semi quantitative polymerase chain reaction (PCR) assay with a sensitivity of 1:100 to 1:10,000 lymphocytes. Results The clinical characteristics and outcomes of 50 pts, with a median age of 67 yrs (range 35-85) were analyzed. 17 (34 %) pts had TCRβ+ clones, 22 pts (44 %) had TCRγ+ clones.  Pts with any TCR+ clones (n=27; 54 %) were compared with those pts who were negative for TCR clonality (n=23; 46 %).  The median follow-up for the pts with or without TCR+ clones was 17 months vs. 19 months, respectively.  Clinical characteristics of the pts with and without TCR+ clones are outlined in Table 1.  Overall, the groups were similar with regards to blood counts, blast percentages, bone marrow cellularity, and karyotype.  There were trends towards higher percentages of males, lower neutrophil count, higher IPSS category, and more pts with >10% BM blasts in the TCR+ group, but these did not reach statistical significance.  Three pts (6%) transformed to acute myeloid leukemia and two of these pts had TCR+ clones in the BM. The majority of these pts were previously untreated 34/50 (68%).  We then looked at response to therapy with hypomethylating agents in those pts who were previously untreated and/or who had previously received one type of therapy (N=42). In 20 pts with TCR+ clones, 8 (40%) received hypomethylating agents and 2 went into complete remission (25%), 2 had stable disease and 4 (20%) were non responders.  In 22 pts without TCR+ clones 4 (18%) received hypomethylating agents and there were no (0%) responders. The estimated 3 year overall survival was 21% vs 47% between pts with andwithout TCR+ clones, respectively (P=0.67). In a subset analysis restricted to pts with available TCRγ assessment (n=67), the estimated 3 year OS for pts with TCRγ+ vs. those without TCRγ clones was 17% vs 57%, respectively (P=0.08) (Figure 1). No difference in OS was observed when pts with or without TCRβ clones were compared. Conclusions We report our experience of the clinical impact of clonal TCR+ populations in pts with MDS. TCR+ clonal T-cells were present in about half of pts with MDS.  Although the study is limited with number of pts, those with MDS and clonal TCR+ population tend to have higher proportions of poor risk features and exhibit a trend of inferior outcome as compared to those pts without TCR+ clones. Larger prospective studies are needed to better define the clinical and mechanistic relevance of clonal TCR+ populations in pts with MDS. Disclosures: No relevant conflicts of interest to declare.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1735-1738 ◽  
Author(s):  
Stephanie Bridenbaugh ◽  
Linda Kenins ◽  
Emilie Bouliong-Pillai ◽  
Christian P. Kalberer ◽  
Elena Shklovskaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Cd8 Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

Overcoming Rejection of Bone Marrow Allografts by Treg Cells in a Stringent Mouse Model for T Cell Mediated Rejection: Tolerance Induction by Third Party “Off-the-Shelf” Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 576-576
Author(s):  
David Steiner ◽  
Noga Brunicki ◽  
Esther Bachar-Lustig ◽  
Yair Reisner
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Graft Rejection ◽  
Ex Vivo ◽  
Tolerance Induction ◽  
Treg Cells ◽  
Third Party ◽  
Donor Type

Abstract Recent reports have shown that donor or host CD4+CD25+ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. More recent data suggests that in the context of T cell depleted BM allografting, engraftment was only mildly improved by Treg cells alone, or by Rapamycin (RAPA) alone, but it was markedly enhanced by using Treg cells in conjunction with RAPA. These studies were carried out in a mouse model specifically designed to measure T cell mediated graft rejection. In this model, lethally irradiated (11Gy) C3H mice were infused with 1x104 purified host type T cells (HTC) and were transplanted one day later with 2x106 BM cells from Balb-Nude donors, which are markedly depleted of T cells and do not induce GVHD. Rejection mediated by the HTC is manifested by severe aplasia and lethality within 21 days posttransplant. In 10 independent experiments none of the mice in the irradiation control survived (0/62), the majority of the mice receiving BM survived (58/63) while marked rejection, associated with poor survival (2/62) was found in the group receiving purified HTC prior to the BM transplant. In the present study we further tested in this model whether third party Treg cells could be used instead of donor or host Treg cells to overcome rejection of BM allografts. We initially tested freshly isolated lymph node CD4+CD25+ cells. C3H (H2k) recipients received BM from Balb- Nude (H2d) donors and the Treg cells were obtained from Balb/c or FVB (H2q) donors. As in our previous study, while none of the recipients survived upon treatment with RAPA alone, using third party or donor type Treg cells in conjunction with RAPA led to survival of 9 of 13 and 7 of 10 mice respectively. Thus, the third party fresh Treg cells were as effective as the donor type cells in preventing graft rejection (P>0.05). Considering the low levels of CD4+CD25+ cells in peripheral blood or spleen, new strategies for growing these cells ex-vivo have been developed. Although, Treg cells exhibit low proliferative potential in-vitro upon TCR stimulation, the feasibility of growing mouse or human regulatory cells has been demonstrated mainly using the combination of TCR stimulation (either with an anti-TCR antibody or with allogeneic stimulator cells), costimulatory signals and high doses of IL-2. When tested in the same model, Treg cells ex-vivo expanded by stimulation against 4th party allogeneic cells, exhibited effective enhancement of engraftment of Balb-Nude BM. Thus, in four independent experiments, when assessing treatment with expanded Treg cells, of third party or donor type origin, the survival rate was 19 of 35 (54%) and 25 of 40 (62%) mice, respectively. Again, in both instances the marked potential of Treg cells to overcome T cell mediated rejection was exhibited only when co-administered with RAPA. In conclusion, our data strongly indicate that, at least in the bone marrow transplantation setting, third party Treg cells could afford a new viable ‘off-the-shelf’ source for tolerance induction. The use of third party Treg cells in contrast to donor type cells could allow advanced preparation of a large bank of Treg cells, with all the appropriate quality controls required for cell therapy. Further studies with human Treg cells in-vitro are required to ascertain the potential of third party cells as a valuable source for clinical transplantation.

Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1677-1677
Author(s):  
Zejin Sun ◽  
Yanzhu Yang ◽  
Yan Li ◽  
Daisy Zeng ◽  
Jingling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.

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