T-cell Depleted Allogeneic Hematopoietic Cell Transplants As A Platform For Adoptive Therapy With Leukemia Selective Or Virus-Specific T-cells

Abstract

Allogeneic hematopoietic cell transplants adequately depleted of T-cells can reduce or prevent acute and chronic GVHD in both HLA matched and haplotype disparate hosts, without post-transplant prophylaxis with immunosuppressive drugs. Recent trials indicate that high doses of CD34+ progenitors from G-CSF mobilized peripheral blood leukocytes isolated and T-cell depleted by immunoadsorption to paramagnetic beads, when administered after myeloablative conditioning with TBI and chemotherapy or chemotherapy alone can secure consistent engraftment and abrogate GVHD in patients with acute leukemia without incurring an increased risk of a recurrent leukemia. Early clinical trials also indicate that high doses of in vitro generated leukemia reactive donor T-cells can be adoptively transferred and can induce remissions of leukemia relapse without GVHD. Similarly, virus-specific T-cells generated from the transplant donor or an HLA partially matched third party, have induced remissions of Rituxan-refractory EBV lymphomas and can clear CMV disease or viremia persisting despite antiviral therapy in a high proportion of cases. Analyses of treatment responses and failures illustrate both the advantages and limitations of donor or banked, third party derived T-cells, but underscore the potential of adoptive T-cell therapy in the absence of ongoing immunosuppression.

Introduction

It is well recognized that allogeneic marrow or PBSC transplants derived from suitable histocompatible related or unrelated donors, administered after immunoablative and either myeloablative or myelosuppressive conditioning can provide a curative approach for the treatment of patients with high-risk forms of ALL and AML and are a potentially curative treatment of choice for patients with high-risk forms of these diseases in first remission.^{1, 2} However, a major limitation to the effectiveness of these transplants is GVHD, the immune response of T-cells in the donor graft against alloantigens principally presented by cells of the lympho-hematopoietic system of the host.^{3, 4} Acute GVHD of grade 2-4 severity and requiring immunosuppressive therapy is observed in up to 40% of recipients of HLA-matched related and over 50% of patients transplanted with HLA-matched unrelated marrow irrespective of the combinations of immunosuppressive drugs administered post transplant to prevent this complication.^{5, 6} Furthermore, an additional 50% of those surviving will go on to develop chronic GVHD. ⁷

T-cell Depletion Techniques for Prevention of GVHD

The role of T-cells in the pathogenesis of GVHD was first elucidated in H2 incompatible murine models. Initial direct demonstrations of the potential of T-cell depletion to prevent GVHD were provided by studies of lethally irradiated mice transplanted with H2 incompatible marrow and spleen cells depleted of T-cells either by their failure to develop following neonatal thymectomy⁸ or by antibody or lectin based depletion of T-cells from spleen cell populations.^{9, 10} Such transplants could engraft and reconstitute hematopoiesis and immunity without induction of acute GVHD.

In 1981, our transplant group introduced the use of T-cell depleted haplotype disparate transplants for the treatment of children with SCID and leukemia lacking a matched related or an unrelated donor.^{11, 12} The approach for T-cell depletion included initial agglutination of mature cells in the marrow by using the lectin, soybean agglutinin, followed by removal of residual T-cells by E-rosette depletion. ¹² The approach has been effective in preventing GVHD in children with SCID who have received such transplants from HLA A, C, B, DR haplotype-disparate parental donors, without post transplant immune suppression. Of 78 patients with classical or “leaky” forms of SCID¹³ who have received such transplants at our center since that time, 70% are surviving at a median follow-up of over 15 years including 76% of patients without infection and 65% who had ongoing infection at time of transplant. A recent analysis of children with classical forms of SCID transplanted between 2000-2009 in North America by the Primary Immune Deficiency Transplantation Consortium ¹⁴ has documented a survival rate at 5 years of 93% in non-infected and 65% in infected patients who have received haplotype disparate T-cell depleted transplants without conditioning. Similar results were also achieved with matched unrelated grafts in patients without infection. Most of the patients who received haplotype-disparate grafts in that series also received transplants depleted of T-cells by the lectin-based technique. Patients with active infection who received unrelated marrow, cord blood grafts or HLA haplotype disparate T-cell depleted transplants with pre-transplant conditioning fared poorly with long-term survival rates ranging from 39 to 53%. These studies underscore the potential of HLA haplotype disparate T-cell depleted transplants to correct these lethal disorders but also underscore the critical role of infection as a determinant of transplant outcome.

Since the introduction of this approach, several different methods of T-cell depletion have been evaluated, including techniques employing several different murine monoclonal antibodies directed against T-cell specific antigens. Most of these techniques achieve a 1.5-2 Log depletion of clonable T-cells and have required post transplant immune prophylaxis for full prevention of GVHD. The CD52 specific monoclonal antibody Campath achieves a 2.5-3 Log depletion of T-cells and has been used either in vivo or in vitro for T-cell depletion.¹⁵ This approach has significantly reduced GVHD in matched donor-recipient pairings but has had variable success in preventing GVHD in HLA disparate recipients.¹⁶ In contrast, the soybean lectin, E-rosette depletion technique, which consistently reduces T-cell by at least 3 Log₁₀ and more recently developed techniques employing positive selection of CD34+ progenitor cells from G-CSF mobilized peripheral blood leukocytes that achieve 4-5 Log₁₀ depletion of T-cells have been administered to HLA matched or mis-matched recipients without post transplant prophylaxis and are effective in radically reducing the incidences of severe acute and chronic GVHD.^17-19

Initial application of T-cell depleted transplants in patients with leukemia were associated with unacceptably high rates of graft failure or rejection. Initial clinical observations identified the emergence of host derived T-cells exhibiting reactivity against donor cells at the time of graft failures in leukemic patients who had received T-cell depleted marrow grafts after 1500 cGy hyperfractionated TBI and cyclophosphamide.²⁰ Subsequent studies of rhesus monkeys that had received similar conditioning demonstrated that while such cytoreduction achieves a 3Log₁₀ reduction of alloreactive CTL precursors in vivo, significant populations of radio-resistant CTL precursors remained, that could mediate a graft rejection.²¹ Further studies conducted by our group identified radio-resistant host CD8+ or CD4+ T-cells directed against class I or II HLA alloantigens unique to the donor in HLA disparate transplants and CD8+ T-cells directed against minor alloantigens presented by class I HLA alleles of an HLA matched donor as the principal effectors of graft rejections.^22-25 Based on these studies, we modified conditioning regimens to include antithymocyte globulin to remove radio resistant residual T-cells.¹⁷ Based on studies in murine models by Terenzi et al.²⁶ the Perugia group also introduced thiotepa for more intensive immunosuppressive conditioning.²⁷ At our center, these modifications led to consistent engraftment of HLA matched related and unrelated T-cell depleted grafts and long-term disease free survival (DFS) rates of over 77% and 50% for patients transplanted for AML in first or second remissions, respectively. ¹⁷ In 1994, Aversa et al²⁸ initiated studies combining T-cell depleted marrow with T-cell depleted G-CSF mobilized PBSCs as an approach to secure consistent engraftment in HLA-haplotypehaplotype disparate patients, demonstrating+ engraftment in 16/17 cases. In 1995, Bachar-Lustig et al²⁹ evaluated graded doses of T-cell depleted marrow and formally demonstrated the potential of megadoses of marrow providing four-fivefold higher doses of progenitor cells to overcome resistance and achieve durable engraftment and full donor chimerism in fully allogeneic mice, even when only sublethal TBI was employed. Additional studies in murine models from Reisner et al. have provided evidence that megadoses of these progenitor cells can directly suppress host anti-donor responses.^{30, 31} Introduction of methods for positively selecting CD34+ progenitor cells from G-CSF mobilized human PBSCs have permitted consistent administration of transplants containing doses of progenitor cells 4–10-fold higher than those achievable with lectin separated, E-rosette depleted marrow grafts (Table 1). Furthermore, the level of T-cell depletion is approximately 1 Log greater than that achievable with the lectin approach. At our center, transplants of CD34+ T-cell depleted PBSC after conditioning with TBI, thiotepa and fludarabine have also induced full chimerism and durable reconstitution in HLA compatible related donors without the requirement of antithymocyte globulin.³² Based on these studies, the Bone Marrow Transplant Clinical Trials Network conducted a study evaluating G-CSF mobilized PBSC transplants from HLA matched related donors depleted of T-cells by positive selection of CD34+ cells by using the CliniMacs (Milteny Biotec, Bergish Gladbach, Germany) device. This study, conducted in 13 centers, demonstrated that such transplants could achieve consistent, prompt engraftment without post transplant immuno prophylaxis. The incidence of acute grade 2-4 GVHD was low.¹⁹ Importantly, the incidence of chronic GVHD was significantly lower than that observed following unmodified transplants performed contemporaneously in a separate Bone Marrow Transplant Clinical Trials Network trial.³³ As a result, the T-cell depleted transplants were associated with a significantly higher cumulative incidence of GVH-free survival.³³

Table 1.

Comparative yields of CD34+ progenitor cells and CD3+ T-cells following T-cell depletion by SBA lectin agglutination and E-rosette depletion, selection of CD34+ cells by Isolex followed by E-rosette depletion or selection of CD34+ cells on the CliniMACS device.

	CD34+ CELL/ KG (× 106/ KG)		CD3+ CELLS/ KG (× 103/ KG)
	MEDIAN	RANGE	MEDIAN	RANGE
SBA−E− BONE MARROW (N= 90) MSKCC	2.0	(0.4- 9.14)	45.7	(8.0- 39.4)
CD34+ (ISOLEX) E− PBSC (N= 95) MSKCC	6.6	(0.7- 29.6)	1.4	(0.0-24.1)
CD34+ (MILTENYI) (N= 44) BMT CTN 0303	7.9	(2.4- 31.3)	6.6	(1.1- 84.9)

A major concern limiting the broad application of T-cell depleted marrow grafts was that by depleting T-cells and abrogating GVHD, the GVL effect of an allo-transplant would be eliminated. Indeed, in early experience with T-cell depleted transplants applied to the treatment of patients with chronic myelogenous leukemia the incidence of relapse following T-cell depleted transplants was approximately twice that observed following unmodified grafts.³⁴ Early experience with marrow grafts depleted of T-cells and certain antibodies also suggested an increased incidence of relapse in patients transplanted for AML.³⁵ A prospective randomized trial evaluating unmodified marrow grafts vs. transplants depleted of T-cells with the T10B9 monoclonal antibody confirmed an increased risk of relapse in patients transplanted for CML. However, the incidence of relapse in patients transplanted for AML or ALL was not different from that observed following unmodified grafts.³⁶ Studies at our own center have consistently failed to demonstrate an increase in the incidence of relapse in patients transplanted for AML or ALL. Furthermore, the study exploring CD34 selected HLA-matched related grafts conducted by the Bone Marrow Transplant Clinical Trials Network also failed to demonstrate an increment in relapse in patients transplanted for AML in first remission.¹⁹ More recently, the Memorial Sloan Kettering and MD Anderson Cancer Centers have compared all patients with AML who received T-cell depleted grafts at MSKCC with AML patients who received unmodified transplants at MD Anderson. In this large comparative retrospective study, the disease free survival rates achieved with unmodified and T depleted transplants were identical, and the incidences of relapse post transplant, super-imposable. However, the T-cell depleted transplants were associated with a significantly reduced incidence of acute grade 2-4 GVHD and chronic GVHD.³⁷ Thus the initial randomized trial and subsequent multicenter studies have failed to document an increase in relapse in patients transplanted for AML or ALL following T-cell depleted allografts.

Because of the late complications of total body radiation administered to young children transplanted for leukemia and the excessive transplant related mortality associated with TBI administered in myeloablative doses to adults over 50, major emphasis has been focused on the development of more tolerable conditioning regimens that can still secure consistent engraftment of T-cell depleted PBSC. In our center, we have been evaluating two such regimens, one combining busulfan, melphalan and fludarabine which has been predominantly applied to patients with AML and myelodysplastic syndromes (MDS) and a second combining clofarabine, melphalan and thiotepa which has been applied more to patients with ALL. Each of these chemotherapy-only regimens has secured consistent engraftment with disease free survival rates exceeding 60% for patients transplanted for AML in first or second remission or high-risk ALL in first remission. The relapse rates at 2 years for these two regimens are 11 and 12%, respectively.³⁸ Based on our cumulative results with CD34 selected T-cell depleted HLA matched related and unrelated transplants using these two conditioning regimens as well as recent studies pioneered by the Johns Hopkins group demonstrating the potential of post transplant cyclophosphamide to reduce risks of GVHD following unmodified HLA matched and HLA disparate transplants of marrow also administered without post transplant drug prophylaxis against GVHD,^{39, 40} the Bone Marrow Transplant Clinical Trials Network is initiating a three-armed prospective randomized phase III trial comparing CD34 selected T-cell depleted PBSC transplants administered after either total body radiation, thiotepa and cyclophosphamide or busulfan, melphalan and fludarabine vs. transplants of unmodified marrow administered with post transplant cyclophosphamide only vs. unmodified transplants using standard post transplant prophylaxis with tacrolimus and methotrexate. The primary endpoints will be survival and relapse-free survival without chronic GVHD. This study should provide important new information regarding the relative efficacy of either in vitro T-cell depletion of PBSC transplants or in vivo depletion by post transplant cyclophosphamide when compared with conventional unmodified grafts.

T-cell Depleted Transplant As a Platform for Adoptive Immunotherapies

The development of techniques for depleting T-cells from allogeneic hematopoietic cell transplants that obviate the need for post transplant immunosuppression to prevent GVHD has also provided a unique platform for exploring adoptive T-cell therapies. In the absence of immunosuppressive drugs, transferred T-cells can proliferate and function without exogenous inhibition. Early post transplant, patients are also markedly lymphopenic, which permits homeostatic proliferation of the adoptively transferred T-cells to levels higher than those achievable in patients with normal lymphocyte populations.

Our transplant program is currently evaluating adoptive immunotherapy with transplant donor-derived effector T-cells for two indications: (1) treatment or prevention of relapses of hematologic malignancies; and (2) treatment of drug-resistant viral infections.

For the treatment and prevention of post-transplant relapse, we are conducting trials evaluating in vitro expanded, transplant-donor-derived T-cells specific for peptide epitopes of the WT-1 protein in patients with WT-1+ hematologic malignancies, specifically AML, advanced forms of MDS and myeloma. In addition, we have initiated trials of transplant donor-derived EBV-specific T-cells transduced to express a CD19-specific chimeric antigen receptor for the treatment of relapses of CD19+ B cell lineage ALL and non-Hodgkins lymphoma. Since our own and other centers have already published several reports regarding the clinical activity of T-cells transduced to express a CD19-specific CAR, I will focus on our studies of WT-1-specific T-cells.

Our choice of WT-1 as a target for adoptive immunotherapy is based principally on five considerations. First, WT-1 is differentially expressed in over 70% of AMLs and myelomas and is also expressed at high levels in advanced forms of MDS.^{41, 42} Indeed, in AML and MDS, high expression of WT-1 has been shown to be a prognostic indicator of poor outcome.^{43, 44} Secondly, several lines of evidence suggest that expression of WT-1 may be essential to the survival of clonogenic AML stem cells.^{45, 46} Third, our group and others have shown that peptides of WT-1 can elicit cytotoxic T-cell responses in up to 75% of normal transplant donors.⁴⁷ Fourth, our own and other groups have found that in transplant recipients treated for relapse of AML or myeloma with unselected donor lymphocytes, expansion of WT-1 specific T-cells is closely correlated with eradication of tumor cells and attainment of CR.^{48, 49} Furthermore, our laboratory and others have shown that T-cells specific for immunogenic peptides of WT-1 exhibit strong, HLA-restricted cytotoxic activity against WT-1+ leukemic cells, but do not lyse normal blood cells from the same individual including normal hematopoietic progenitor cells.^{47, 50} These WT-1 specific cells can prevent growth of WT-1+ human leukemic cells when xenografted in NSG (γc−/−SCID (severe combined immune deficiency)) mice, and can also suppress or eradicate established WT-1+ leukemic xenografts in this model.⁵¹

Over the past 3 years, we have analyzed WT-1 specific T-cells generated from over 100 normal transplant donors by in vitro sensitization with autologous, monocyte-derived dendritic cells loaded with a pool of overlapping 15-mer peptides spanning the sequence of the WT-1 protein. Thus far, we have identified 41 previously unreported immunogenic epitopes and their presenting HLA class alleles, and demonstrated that these HLA class I or II restricted epitope specific T-cells selectively lyse patient derived WT-1+ leukemia cells from the same individual.⁴⁷

In a phase I trial, we have administered WT-1 specific T-cells generated from normal hematopoietic cell transplant (HCT) donors by this approach at dose levels ranging from 1-50×10⁶/m² to a series of 17 patients with WT-1+ leukemias, MDS or myeloma in full relapse of disease post transplant. At low doses, the WT-1 specific T-cells have induced only transient clearance of circulating WT-1+ malignant cells. However, at higher doses, marked increases in the frequencies of WT-1 specific T-cells in the blood, lasting up to 3-4 weeks with induction of CRs have been observed. In one patient relapsing with leukemia cutis, WT-1+ AML cells were eradicated from the skin lesions with T-cells of donor origin selectively infiltrating former sites of disease.⁵² Three of these patients, including 2/4 treated at the highest dose levels thus far administered, have achieved CRs, the latter two extending for >12 months. The T-cell infusions have been well tolerated without significant toxicities. Although WT-1 is expressed at low levels in renal podocytes and in CD34+ progenitor cells, no alterations in kidney function or blood cell counts were observed. Furthermore, no patient developed evidence of GVHD. Based on these studies, we are now conducting studies evaluating these T-cells as prophylaxis against recurrence in patients with a probability of post-transplant relapse exceeding 30%.

Our phases I and II studies of adoptive immunotherapy for viral diseases in HCT recipients have been focused on the therapeutic potential of virus-specific T-cells in (1) patients with pathologically documented EBV+ lymphomas, particularly patients who have failed to respond to Rituxan and (2) patients with overt CMV disease or persistent viremia resistant to at least 2 weeks of conventional antiviral therapy. The T-cells employed in our initial studies have been derived from the donor of each patient's hematopoietic cell graft. The T-cells isolated from each donor's PBMC have been cultured in vitro for periods of 28-35 days after sensitization with either irradiated autologous donor B-cells transformed with the EBV strain B95.8 for EBV-specific T-cells,⁵³ or autologous dendritic cells loaded with a pool of overlapping 15-mer peptides spanning the sequence of CMVpp65 for CMV-specific T-cells.⁵⁴ In vitro culture for this period of time not only selects for immunodominant virus-specific T-cells but also deletes alloreactive T-cells potentially capable of inducing GVHD.

Results of these trials have provided evidence supporting the potential of this approach. First, the three weekly infusions of T-cells are safe, without acute toxicities or instances of denovo acute or chronic GVHD or flares of pre-existing GVHD at doses of 1-2×10⁶ virus-specific T-cells/kg/dose. Second, they have been effective in clearing EBV lymphomas and CMV infections that have failed conventional therapies, inducing durable complete remissions in 68% of patients with EBV lymphomas that had failed to respond to Rituxan,⁵³ and over 86% of patients with CMV infection or persistent viremia that had failed to respond to ganciclovir, foscarnet and/or cidofovir.⁵⁵

We have also observed that when virus-specific T-cells are effective in controlling disease they invariably expand by 2-4 log₁₀ in the patient within the first two weeks following an adoptive transfer. Indeed, this expansion of virus-specific T-cell populations has consistently heralded reductions or clearance of viremia and both clinical and radiological resolution of disease. In contrast, lack of such proliferation in vivo has been the hallmark of treatment failures. Failure of the adoptively transferred T-cells to expand in vivo and induce clearance of disease may be due to several factors of which but a few have been identified and characterized. Concurrent treatment of the patient with glucocorticosteroid is widely accepted as a major deterrent to the effectiveness of adoptive therapy.⁵⁶ However, the upper limit of acceptable doses is still poorly defined. Strikingly, concurrent treatment with calcineurin inhibitors has not been shown to interfere with T-cell expansion or function, reflecting their limited activity against memory T-cells.⁵³ In our own and other series, disease burden, reflected, for example, by multiorgan involvement with EBV lymphomas, has been associated with a lower incidence of CR. However, this limitation is not absolute; responses have still been seen in over 50% of patients with widespread disease, including disease of the CNS.⁵³ Furthermore, extent of disease has not been correlated with delays or impairments of T-cell proliferation in vivo. Rather, failure of T-cells to proliferate in vivo has been most consistently correlated with failure of the adoptively transferred T-cells to recognize virus infected or transformed cells in the host, either because the endogenous virus in the host did not express an antigenic epitope recognized by the T-cells,^{53, 57} or the peptide was presented by an HLA allele expressed by virus infected or transformed cells of the host not shared or recognized by HLA-restricted virus-specific T-cell of the donor.⁵³ In the latter case, it is important to recognize that the HLA restriction of the virus-specific donor T-cells is determined not only by the HLA genotype of the donor but also by the specific HLA allele(s) of the donor that present the immunodominant viral epitopes recognized by the donor T-cells that are preferentially maintained in latently infected individuals in vivo and selectively expanded in vitro.

This constraint imposed by the immunodominance of certain epitopes is a particular concern in recipients of HLA haplotype disparate transplants and is illustrated in Figure 1, in this case, the donor inherits the HLA haplotypes HLA A0201, B0702, C0401, DRB₁ 0401 and HLA A0101, B0801, C0701, DRB₁ 0301. However, T-cells generated against CMVpp65 from this or any donor that inherits HLA B0702 will almost invariably be specific for one of two immunodominant epitopes presented by this allele.⁵⁸ Adoptive transfer of CMVpp65-specific T-cells from this donor will proliferate and be effective in an HLA-matched recipient or a haplotype disparate recipient that also inherits HLA B0702, but will be ineffective in the HLA haplotype disparate recipient that does not inherit this allele. We have recently reported such an instance, a patient who developed an EBV lymphoma involving the stomach wall following a transplant from his HLA haplotype disparate mother.⁵³ This case differed from most cases of EBV lymphoma complicating cord blood or T-cell depleted marrow or PBMC grafts in that the lymphoma proved to be of host rather than donor origin. Infusion of maternal, EBV-specific T-cells that were selectively reactive against an EBV epitope presented by HLA A1101 failed to control the growth of the lymphoma and also failed to expand in vivo. Subsequent treatment with EBV-specific T-cells from an unrelated partially HLA-matched ‘third party’ donor that were restricted by an HLA A2601 shared by the donor and the patient was followed by expansion of the T-cells in vivo and induction of a durable CR of disease.⁵³

Figure 1.

Constraints imposed by the immunodominance of CMV-specific T-cells, in this case, T-cells specific for CMVpp65 peptides presented by HLA B0702.

Since CMV infections predominantly affect host tissues and CMV infections are also associated with a threefold higher incidence of mortality in recipients of HLA haplotype disparate T-cell depleted transplants⁵⁹ than observed in similarly conditioned recipients of HLA-matched T-cell depleted grafts,⁶⁰ we have been exploring whether the constraints of immunodominant, HLA-restricted T-cell response might be a factor contributing to this higher incidence of life-threatening CMV infections. T-cell depleted grafts, which usually transfer 2-20×10³ mature T-cells/kg to the host would be expected to co-transfer only a small number of the immunodominant virus-specific CTL precursors that are detected at frequencies of 1/10³-1/10⁴ T-cells in the blood of latently infected seropositive donors. Indeed, our preliminary results indicate that CMVpp65-specific T-cells generated from HLA haplotype disparate donors may be restricted by HLA alleles not shared by the host in up to half of the cases, and, further, that severe disease is encountered almost exclusively in this fraction of recipients.⁶¹

To address these constraints and to more effectively control viral infections following transplantation, both in recipients of HLA disparate T-cell depleted HCT and in patients receiving HLA partially matched cord blood transplants or matched marrow or PBSC transplants from seronegative donors, our group has been exploring the use of HLA partially-matched third party donor derived virus-specific T-cells previously characterized as to their epitope specificity and HLA restriction.

We currently have established banks of over 300 EBV-specific T-cell lines and 125 T-cell lines specific for CMVpp65 for adoptive immunotherapy. Each T-cell line in these banks is GMP grade and separately consented for third party use. All CMVpp65 specific T-cells and EBV-specific T-cells are HLA typed at high resolution and characterized as to virus specificity and HLA restriction. All CMVpp65-specific T-cell lines, and a growing proportion of the EBV-specific T-cell lines are also characterized as to their epitope specificities. All lines have been tested and shown to lack alloreactivity or non-specific cytotoxic activity. They are also microbiologically sterile and contain <5 IU of endotoxin/ml dose of T-cells. The T-cells are cryopreserved in dosings permitting rapid ‘off the shelf’ use for adoptive immunotherapy. Based on requests for use of these cells, the bank of EBV-specific T-cells has been able to provide appropriately HLA restricted EBV-specific T-cells that are matched with the patient for at least two HLA alleles for over 98% of patients referred to our center for treatment. The bank of CMVpp65-specific T-cells is also diverse and has permitted selection of appropriately HLA restricted CMVpp65-specific T-cells for 85-90% of the patients referred.

Our clinical experience with the use of appropriately HLA restricted third party EBV and CMV specific T-cells matched with the patient at 2-6 HLA alleles at high resolution now includes over 70 patients. The doses employed (1-2×10⁶ virus-specific T-cells/kg recipient weight weekly × 3) have been identical to those used for infusions of transplant donor-derived T-cells. Like the infusions of transplant donor-derived T-cells, the third party donor derived T-cells have been well tolerated, and have not been associated with either graft suppression or clinical evidence grade ≥2 of acute or chronic GVHD.

The therapeutic potential of the third party donor-derived virus-specific T-cells has been striking. Of 25 patient recipients of cord blood or T-cell depleted marrow or PBSC transplants treated for an EBV+ lymphoma, 12 have achieved a complete and three a durable partial remission following adoptive therapy with third party EBVCTLs. The 60% response rate is only slightly lower than the 69% response rate observed following adoptive immunotherapy with transplant donor-derived T-cells.^{62, 63} Similarly, of 26 patients treated for CMV disease or persistent viremia refractory to treatment with antiviral drugs, 61% have cleared infection and or viremia or experienced a sustained 2log₁₀ reduction in the level of CMV DNA in the blood. Again, as in recipients of transplant donor-derived virus-specific T-cells, responses have been consistently associated with increases in the frequency of virus-specific T-cells in the circulation. In contrast, however, the increments in circulating virus-specific T-cells observed are of shorter duration, usually ranging from 7 to 28 days after each dose administered, reflecting the fact that these third party donor-derived T-cells only transiently engraft in the host. In our series, these third party donor T-cells are usually not detected beyond 28-35 days post infusion. However, in our own and other series, the T-cells have been detected in certain patients for periods as long as 90-194 days post infusion.^{63, 64} Nevertheless, in most patients, 1-2 courses of three weekly infusions of these T-cells have been sufficient to induce sustained remission of disease.

The mechanisms contributing to the sustained clearances of clinical disease and viremia achieved with transiently engrafted third party virus-specific T-cells are still poorly understood. The initial contribution of adoptively transferred T-cells to disease resolution is consistent with the close correlation observed between expansion of the third party T-cells in vivo and both clinical and virological responses. Accumulations of these T-cells identifiable by their distinctive genetic markers, are also detected at sites of disease. In some cases, these responses may be sufficient to reestablish a healthy equilibrium between the host and these latent viruses. However, the endogenous T-cell responses are likely essential to sustaining such responses since in patients with genetic deficiencies of T-cells that we have treated for EBV lymphomas or persistent CMV infection with third party T-cells, clinical and/or virologic responses have been sustained only as long as the T-cells have been detected in the blood. Whether and to what degree these third party donor-derived T-cells, which are themselves allogeneic to the previously engrafted host, recruit transplant donor T-cells to disease sites, and thereby potentiate in vivo sensitization of endogenous T-cells to viral epitopes released from cells targeted by the third party effectors remains to be determined.

Although the mechanisms whereby adoptive transfer of these third party T-cells induce durable regressions of EBV lymphomas and CMV disease and reestablish control of these latent viral infections are still unclear, these and other reported studies of their in vivo activity,^62-65 have underscored several potential advantages of such banks of pre-generated, fully characterized, GMP grade virus-specific T-cells in the treatment of allogeneic HCT recipients. Their immediate, ‘off the shelf’ availability and the fact that they can be rapidly selected on the basis of their HLA-restriction and level of compatibility are key assets for the treatment of the rapidly progressive EBV lymphomas and CMV infections that can complicate allogeneic HCT, particularly in recipients of HLA haplotype disparate grafts. The degree of depletion of alloreactive T-cells achieved by extended culture, and the clinically demonstrated lack of any suppressive effect on the patient's transplant or either acute or chronic GVHD associated with the use of these third party virus-specific cells also illustrate their safety. The other advantage that is emerging derives from the diversity of epitope-specific T-cell lines represented in these banks. It is well recognized that different strains of EBV may express lytic and/or latent proteins that may contain sequence deletions or variations that can alter the repertoire of peptide epitopes processed and presented by HLA alleles expressed on infected cells.⁶⁶ Alternatively, for both CMV and EBV immunoevasive viral proteins may interfere with the presentation of antigenic peptides by certain HLA alleles.^{67, 68} As a result, T-cells sensitized with cells infected with a standard strain of virus may respond to peptide epitopes not expressed by the virus in the patient. As a result, adoptive transfer of such T-cells would be expected to fail to control the patient's infection. In fact, this scenario has been documented in at least four patients treated for EBV lymphomas.^{53, 57} In such cases, we have found that treatment with a second T-cell line selected from our bank that is specific for a different epitope and restricted by another HLA allele expressed by virus-infected cells in the patient, can induce disease remission.⁶³ Based on these considerations, our group⁶³ and The Baylor Group (Baylor University Medical Center, Houston, TX, USA)⁶⁵ are establishing consortia to permit multicenter trials of banked third party T-cells for EBV, CMV and other life-threatening viral infections complicating transplants.

In conclusion, allogeneic HCT rigorously depleted of T-cells and administered after tolerable myeloablative chemotherapy can secure consistent engraftment and lower incidences of acute and chronic GVHD without an increase in relapse in patients transplanted for acute leukemias. Such transplants provide a promising platform for tumor and pathogen-specific cellular immune therapies. Adoptive transfer of leukemia reactive T-cells can induce remissions in patients who relapse post transplant and are now being evaluated for prevention of disease recurrence. Adoptive transfer of virus-specific T-cells generated from the transplant donor or selected from banked third party donors can also induce durable remissions of EBV lymphomas and drug resistant CMV infection in a high proportion of patients. However, their consistent successful application in HLA disparate recipients necessitates careful selection of virus-specific T-cells that are both restricted by HLA alleles expressed by infected cells and specific for epitopes presented by those alleles.