Cell Therapies for Hematological Malignancies: Don't Forget Non-Gene-Modified T Cells!

Abstract

Cell therapy currently performs an important role in the treatment of patients with various hematological malignancies. The response to the cell therapy is regulated by multiple factors including the patient's immune system status, genetic profile, stage at diagnosis, age, and underlying disease. Cell therapy that does not require genetic manipulation can be mediated by donor lymphocyte infusion strategies, selective depletion in the post-transplant setting and the ex vivo expansion of antigen-specific T cells. For hematologic malignancies, cell therapy is contributing to enhanced clinical responses and overall survival and the immune response to cell therapy is predictive of response in multiple cancer types. In this review we summarize the available T cell therapeutics that do not rely on gene engineering for the treatment of patients with blood cancers.

Introduction

The burgeoning field of immunotherapy for hematological malignancies under active clinical investigation includes adoptive transfer of antigen-specific and specificity-augmented genetically-engineered T cells; T regulatory cell suppression; co-stimulation agonists; and vaccines that enhance antigen processing and presentation effectiveness. Early phase clinical trials utilizing T cells are releasing promising preliminary data. The FDA approval of CD19-directed chimeric antigen receptor (CAR) T cells, which have demonstrated spectacular responses in salvage therapy settings, has revolutionized treatment of CD19+ B cell malignancies. Nevertheless, severe on-target, off-tumor toxicities, including healthy B cell aplasia, cytokine release syndrome and neurotoxicity, means that these studies can currently only be conducted at institutions that can provide intensive care support. While clinicans are learning to better manage these biological limits the current paucity of suitable antigenic targets further limits the broader applicability of this approach to both hematologic and solid tumors. However, while less in the public eye, effective T cell therapies which do not employ gene-engineering are also being developed. Here we review the application of these non-gene-modified T cells in the treatment of hematological malignancies.

Role of the immune system in hematological cancer and immunogenic features of current treatments

Many hematological malignancies arise in B and T lymphocytes and the tumor microenvironment comprises active interactions between tumor and immune cells. CD4+ T cells interact with antigen presenting cells (APC) exhibiting tumor peptides in their MHC Class II (HLA DR, DP, DM, DOA, DOB, DQ) molecules. CD8+ T cells can directly recognize tumor cells displaying peptides in their major histocompatibility (MHC) Class I (HLA A, B, C) molecules, stimulating activation against the neoplastic cell. Dendritic cells (DC)^1–3, B cells^4–7 and macrophages^2,3,8–10 all have the capacity to cross-present tumor-associated peptides on MHC-I and, providing sufficient costimulation is concurrently supplied, can generate a robust T cell activation against the tumor peptide(s). The classification cytotoxic T lymphocyte (CTL) has primarily been applied to CD8+ T cells, however the data shows that more than just “helper cells” who provide “help” for B cells and CD8+ T cells, CD4+ T cells also act as “CTLs” in their own right^11,12. Activated antigen-specific T cells form immunological synapses with target cells, subsequently releasing cytokines such as interferon-gamma (IFN-γ), tumor-necrosing factor alpha (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL). These cytokines, along with cyototoxic perforin and granzyme molecules and the transmembrane protein FAS ligand, lead to lysis and apoptosis of tumor cells. Our current understanding of immune surveillance posits that this T cell-mediated tumor cell killing is carried out during the elimination phase, during which time tumor growth is controlled by the immune system¹³.

It is now understood that beyond elimination of rapidly dividing cells, among the reasons for the success of chemotherapy and radiotherapy is their generation of non-targeted innate and adaptive immune activation by triggering immunologic tumour cell death and disrupting immune suppression and tolerance (reviewed in^14,15). Chemotherapeutic tumour cell death can trigger immunoadjuvant pathways^16–18 while cyclophosphamide-mediated blocking of immune suppression occurs via selective depletion of regulatory T cells (T_REGs)¹⁹. Radiotherapy of cancer tissue also induces immunogenic tumor cell death and destruction of immune barriers, both of which can lead to increased immune cell infiltration, increased tumor-associated antigen (TAA) presentation and T cell activation^20–24. Thus, alongside their roles as debulking and cytotoxic agents, certain chemotherapies and radiotherapy, are increasingly regarded as beneficial primers or adjuncts for cancer immunotherapy^{14,20,25–30}. Unfortunately, chemotherapy and radiotherapy are non-specific treatments that result in substantial bystander tissue toxicities, including late sequelae. Immunotherapies have the potential to deliver on the vision for effective targeted approaches that minimize damage to healthy tissue. The immune system's vital role in control of hematological malignancies is demonstrated in the graft versus tumor (graft versus leukemia; GVT; GVL) effect that is seen in a proportion of patients following allogeneic hematopoietic stem cell transplant (allo-HSCT)^30,31. But this immune-mediated response is insufficient and relapse continues to be the greatest reason for HSCT failure.

Allogeneic bone marrow or peripheral blood stem cell transplant (HSCT)

Through its potent GVL effect HSCT represents the original form of successful cell therapy for hematological malignancy. Despite a huge reduction in transplant-related mortality in this century, the well-recognized complications from the procedure, which include graft-versus-host disease (GVHD), continue to limit its use. In selecting the best treatment for the individual with a hematologic malignancy current disease-free survival estimates following HSCT must be continually balanced against improved outcomes from non-transplant treatments. Detailed risk stratification of individual diseases has improved our ability to select the best treatment approach. For acute myelogenous leukemia (AML) “good-risk” patients receive non-transplant treatments while the preferred treatment option for higher risk AML patients is an allogeneic HSCT in first complete remission³². Indications for HSCT in AML continue to change as more patients achive long-term survival with novel therapies³³. In acute lymphoblastic leukemia (ALL) chemotherapy achieves high cure rates in children and young adults and HSCT is seldom indicated. However in older adults with ALL HSCT is widely indicated³⁴. In all types of acute leukemia HSCT is generally indicated in second or subsequent remission when primary treatment has failed. HSCT is also indicated for many patients with myelodyasplastic syndrome and some patients with myeloproliferative disorders. However the role of HSCT in chronic leukemias continues to dwindle: in chronic myelogenous leukemia (CML), following successful development of tyrosine kinase inhibitors, HSCT is seldom required, except in treatment failure and for selected children with CML^35,36. In chronic lymphocytic leukemia (CLL) new treatments such as bruton kinase inhibitors and novel bi-specific antibodies have also reduced the indications for HSCTin the last decade³⁷. HSCT continues to be used usually as a second line treatment for aggressive lymphomas.

Improving the GVL effect through manipulation of donor immune cells

HSCT as a treatment for a wide range of hematological malignancies continues to improve with a continuing fall in transplant-related mortality extending acceptable transplant safety. However, there remains a substantial risk of leukemic relapse after HSCT, particularly for patients over 60 years of age who can generally only tolerate less intensive conditioning, and for those with no fully compatible donors who require additional pharmacological immunosuppression and/or graft manipulation. In 1990 Kolb and colleagues set the scene for T cell therapy as a means to augment the GVL effect. They used donor lymphocyte infusions (DLI) to achieve sustained remissions in CML patients who had relapsed after allogeneic SCT. Other investigators sought to modify the graft contents to selectively remove GVHD-reacting cells while preserving GVL-reactive cells³⁸. The successful application of this “selective depletion” strategy can permit the infusion of large numbers of leukemia-reactive donor T cells without GVHD or the need for immunosuppression to prevent GVHD resulting in superior GVL effects. In recent years cell-mediated therapeutic approaches have increased in their sophistication, diversity and efficacy. Despite their toxicities, genetically engineered T cells such as CAR T cells and T cells with gene-modified TCRs have caused excitement because of their potency and novelty³⁹ . However less risky but equally specific ways to create leukemia-reactive T cells have been developed following technologies first used to successfully control virus reactivation after HSCT. Furthermore the unique properties of natural killer (NK) cells in specifically targeting malignant cells without risking GVHD have led to studies of the adoptive transfer of NK cells to treat malignant disease^40–42. Table 1 summarizes the various strategies used to augment the GVL effect. Table 2 lists the available cell-based therapies for hematological malignancies and Table 3 summarizes clinical trials that have utilized non- receptor engineered T cells for hematological malignancies.

Table 1. Cell transfer-based approaches to enhance GVL effect of allogeneic HSCT.

Graft manipulation to prevent GVHD while conserving GVL

Selective allodepletion

CD8+ depletion

Naïve T cell depletion

Donor lymphocyte infusion (DLI)

Whole buffy coats

DLI + interferon/IL-2

CD4-selected DLI

T cells genetically engineered for leukemia speficicity

CAR-T cells*

TCR modified T cells*

Antigen-specific T cells

Targeting minor histocompatibility antigens

Targeting tumor antigen

Targeting viral antigens expressed by lymphoma

Ex vivo selected and expanded NK cells*

^*Not discussed in this review

Table 2. Non-gene-modified T cell therapies available for patients with hematological malignancies.

Treatment Approach	Description
Unmodified donor lymphocyte infusion (DLI)30,31,43–50	Infusion of donor lymphocytes to mount GVT response
Modified DLI51–56	Depletion of DLI subpopulations, TREGs, naïve T cells, to select for most potent CTLs
Tumor antigen-targeted cytotoxic T lymphocytes (CTLs)57–63	Infusion of ex vivo expanded tumor-specific allogeneic or autologous CTLs for elimination of tumor targets
Viral antigen targeted T-cells64–66	Infusion of ex vivo-expanded virus-specific allogeneic or autologous T-cells to minimize viral reactivation during immunosuppressive period post transplant
Tumor peptide-loaded DC vaccination67–70	Infusion of peptide loaded DCs for in vivo presentation of antigen to endogenous T cells
NK cells and cytokine-induced killer cells41	Infusion of ex vivo expanded tumor-specific allogeneic or autologous NK cells or CIKs for elimination of tumor targets

Table 3. Summary of Clinical Trials Using Non- Receptor Engineered T cells for Hematological Malignancies.

Trial #	Intervention	Tumor Target	Primary Outcome	Secondary Outcome	Enrollment
NCT01627275	Naive T Cell Depleted DLI	N/A	MTD	Immunological recovery; incidence of aGVHD, cGVHD & opportunistic infections	28
NCT00675831	CD25+ Depleted DLI	N/A	Feasibility of CliniMACS to engineer CD25- product; Safety;	Clinical response; Immunological Impact	24
NCT02203903 (RESOLVE)	Tumor associated antigen lymphocytes (TAA-CTL)	PRAME, WT-1, survivin	Safety	TAA-CTL responses	11
NCT00052598	Allogeneic CD8+CTL clones+aldesleukin (IL-2)	PR3	Toxicity	Persistence; migration to BM; duration of response; proportion of responders	7
NCT00052520	Allogeneic CD8+CTL clones+aldesleukin (IL-2)	WT-1	Toxicity	Relapse	37
NCT00107354	Allogeneic CD8+ C TL clones+aldesleukin (IL-2)	mHAg	Toxicity	Persistence; migration to BM; Antileukemic activity	NP
NCT02895412 (INTACT-WT1)	Allogeneic C TLs	WT-1 & multiple pathogens (CMV,Adv, EBV, VZV, IFV, BKV, fungal infections)	TRAE	N/A	20
NCT02074657 (LANK-2)	Activ ated and expanded natural killer cells (NKAEs)	N/A	Safety	Febrile neutropenia, or infection incidence; hematological recovery; ORR; immune reconstitution	13
NCT00620633	Allogeneic C TLs	WT-1	Toxicity	Disease progression; CTL survival & proliferation	22
NCT00460629	Prophylactic donor CTLs	LAA	Feasibility; kinetics of BCR-ABL load;	Rates of aGVHD, cGVHD & infections	20
NCT00002663	Allogeneic C TLs	EBV	Efficacy; expansion & duration; durability of response (time)	N/A	84
NCT01948180 (CITADEL)	Autologous T cells	EBV	ORR	CRR; Response Duration; time to response; PFS; DFS; OS; AEs	35
NCT00779337	Autologous CTLs	EBV (AdE1- Latent Membrane Protein)	Feasibility; safety; reconstitution of EBV-specific CTL immunity with anti-viral efficacy	Optimal dose; clinical efficacy	8
NCT00005606	Allogeneic CTLs	EBV	NP	NP	10-20
NCT01636388	Allogeneic CTLs	EBV (LMP)	Safety, toxicity	Feasibility	45
NCT01956084	Third party CTLs	EBV (LMP)	DLT	Survival & function of LMP-specific CTLs	24
NCT02057445	Allogeneic CTLs	EBV	Safety; development of bank of third-party CTLs	Response rate	18
NCT01498484	Allogeneic CTLs	EBV	Efficacy	In vivo expansion and duration of EBV-CTLs; durability of response	112
NCT01447056	Allogeneic CTLs	EBV (LMP)	DLT; safety	Safety of dosing; immune function of CTLs; dz response	18
NCT01555892 (GRALE)	CTLs	EBV (LMP, BARF1 & EBNA1)	Toxicity	Survival and immune function of LMP/BARF1/EBNA1-specific CTLs; anti-viral and anti-tumor effects of CTLs	136
NCT00062868	CTLs	EBV (LMP1/2)	DLT	Survival and immune function of LMP1/2-specific CTLs; anti-viral load and anti-tumor effects	89
NCT02287311 (MABEL)	Allogeneic CTLs	EBV (LMP, BARF1 And EBNA1)	DLT	Persistence; proliferation in vivo; CTL response to viral antigens; CR; PR	42
NCT00002663	Allogeneic CTLs	EBV	Efficacy; In vivo ex pansion and duration; durability of clinical responses	N/A	84
NCT02973113 (PREVALE)	CTLs+Nivolumab	EBV	DLT	Duration of response;	36
NCT01333046 (TACTAL)	C TLs+/- 5-azacytidine	NY-ESO-1, MAGEA4, PRAME, Survivin and SSX	Safety (AEs/SAEs)	expansion, persistence and anti-tumor effects of CTLs; epitope spreading;	74

TAAT: Tum or Associated Antigen –specific T cells; DLI: Donor Lymphocyte Infusion; MTD: maximum tolerated dose; BM: bone marrow; CTL: Cytotoxic T Lymphocytes; mHAg: minor histocompatibility antigen; NP: Not Provided; TRAE: Treatment-Related Adverse Events; cytomegalovirus (CMV), Adenovirus (Adv), Epstein Barr virus (EBV), Varicella-Zoster virus (VZV), Influenza, BK virus (BKV), and fungal infections; LAA: Leukemia-Associated Antigens; ORR: Overall Response Rate; CRR: Complete Response Rate; PFS: Progression Free Survival; DFS: Disease Free Survival; OS: Overall Survival; AEs: Adverse Events; DLT: Dose Limiting Toxicity;

Donor lymphocyte infusions (DLI)

Infusions of non-specific, third-party, naïve and antigen-experienced lymphocytes can mount a response against the recipient's tumor and are a common treatment for leukemic relapse after allo-HSCT. When successful DLIs undertake a GVT effect that eliminates neoplastic host cells. However relapse and GVHD continue to present significant challenges with DLI, particularly in acute leukemias and non-Hodgkin's lymphoma (NHL).

The GVT response post-DLI is observed most clearly in CML where DLI +/- the TK inhibitor Imatinib after allo-HSCT is particularly effective in treating relapse^34,71. A retrospective analysis of 500 CML patients treated with DLI after post-allo-HSCT relapse identified some of the factors associated with increased overall survival (OS) without secondary GVHD⁴³. In patients with cytogenetic, molecular and hematological relapse the probability of greater OS without GVHD was higher in patients who i) received DLIs early in CML relapse (at molecular or cytogenetic, rather than hematological diagnosis); ii) did not have chronic GVHD prior to infusion; and iii) received DLIs more than 1 year after allo-HSCT. For patients with hematological relapse the dose of CTLs, donor-recipient sex mismatch and chronic GVHD prior to DLI were independently associated with greater probability of reduced OS and secondary GVHD. While they occur at lower levels, GVT effects in the absence of clinical GVHD were also identified in this study, and have been reported elsewhere⁴⁴. Thus GVHD is not a prerequisite for a successful GVT effect, though GVHD often accompanies an effective GVT response.

Whereas DLI is extremely effective in CML, this approach has more limited utility in acute leukemias. The efficacy of DLI in acute leukemias as a pre-emptive measure post-allo-HSCT (i.e. administering DLI to patients with minimal residual disease (MRD) or mixed chimerism), has been systematically investigated by the Acute Leukemia Working party of the European Society for Blood and Marrow Transplantation (EBMT)⁴⁵. The results showed that DLI given preemptively was effective in 69% of patients. Reduction in MRD was achieved in 71% (15/21) of patients and increased donor chimerism in 68% (110/163). While encouraging, the authors note that further refinement is required to identify which patients may benefit from preemptive DLI as well as discussing the potential role for prophylactic DLI (i.e. DLI administered to high risk patients when in complete response [CR]). It was also noted that GVHD-related mortality was as high as 5% when administered as a pre-emptive strategy. Other groups have consolidated the knowledge that MRD and GVHD status are prognostic for patient relapse, and identify that multiple consolidation therapy and DLI can drive patients into MRD negative status^31,46,47,56. Specifically, there is a robust association between cGVHD and achievement of a successful anti-tumor response to DLI suggesting that clinically manageable cGVHD may be an acceptable goal for the majority of patients with high-risk hematologic cancers post-allo-HSCT. However, evidence of GVT responses in the absence of GVHD confirms that we need to identify how to uncouple the GVT and GVHD responses, ameliorating both unnecessary patient suffering and the ongoing treatment expense of immune suppression agents.

Selective Depletion and Allodepleted DLI

Investigators have tried various approaches to enhance the GVT effect of DLI including: chemotherapy plus DLI⁵⁶ (NCT02046122; NCT01390311; NCT00003145; NCT01369368); total body irradiation plus DLI (NCT00003196); GM-CSF mobilization pre-donor harvesting (NCT01158118); or accompanying peptide administration⁷² (NCT00270452); and administration of interferon or interleukin-2 (NCT02331706; NCT00005802) to stimulate an antitumor immune response. Several groups have however evaluated “selective depletion” DLI strategies to enhance the GVT response while limiting GHVD.

Investigators at the National Heart Lung and Blood Institute (NHLBI), National Intitutes of Health used anti-CD25 immunotoxin to selectively deplete host-reactive donor T cells from HLA-identical sibling peripheral blood stem cells. The results from that trial in elderly patients with advanced hematological malignancies suggested that elimination of alloreactive donor cells can enhance lymphocyte recovery and reduce the incidence of severe GVHD⁷³. Those conclusions were subsequently supported by another trial from Baylor College of Medicine where they evaluated immune reconstitution after adding back two different doses of allo-depleted donor T cells in the haploidentical setting. That study similarly demonstrated low acute and chronic GVHD and enhanced T cell reconstitution following anti-CD25 immunotoxin allodepleted T cell infusion⁷⁴. The group at the NHLBI also evaluated allodepletion using a novel photodepletion approach. Allo-reactive T cells that retained the photosensitizer 4, 5-dibromorhodamine 123 (TH9402) were eliminated by exposure to visible light. Results from this study in HLA-matched sibling donors likewise suggested that depletion of host-reactive T cells reduced the severity of GVHD, however, unlike the two previous studies, delayed immune recovery was observed in this cohort and clinical outomes were not improved⁷⁵. A more recent study using photo-mediated allo-depletion in haploidentical transplants reduced transplant-related mortality (TRM) to 17% compared to 34% for HSCT alone⁷⁶. One-year overall survival was increased to 67% in the allo-depleted group compared with 20% in the HSCT-only arm. The reduced TRM was attributed to the retention of immune protection in the group receiving allo-depleted T cells, as infections were the primary cause of TRM in the control cohort. Another promising pre-clinical study has demonstrated adenosine-mediated selective depletion of allo-reactive T cells in HLA-mismatch mixed lymphocyte reactions⁵². Virus- and leukemia-specific non-alloreactive naïve, central, and effector memory T cells were retained and T cells recognizing leukemia-associated antigens were efficiently generated in vitro from the adenosine-treated cell product. However these pre-clinical studies are yet to be tested in clinical trials.

A group at Dana-Farber more recently hypothesized that selective depletion of regulatory CD4+CD25+ cells (T_REGs) from DLI populations prior to infusion could boost the anti-tumor effect, by removing their suppression of the GVT response⁵¹. Their Phase I trial compared a cohort of patients who received unmodified DLI with a cohort that received CD4+CD25+-depleted DLI. CD25+ cells were removed by antibody conjugated magnetic bead selection. Rates of severe GVHD were 19% and 33% at 8wks and 1 year respectively. These rates were similar to rates observed in other studies of unmodified DLI and, importantly, anti-tumor responses were not dependent upon GVHD. Improved outcomes were achieved in the CD4+CD25+-depleted DLI group (60% response rate at 8-12 weeks versus a 14% response rate in patients receiving unmodified DLI). Moreover, in patients receiving unmodified DLI, the 1 year event-free survival (EFS) was 0%, compared with 27% in the T_REG-depleted group. Increased proportions of naïve (T_N) and T central memory (T_CM) cells, and reductions in T effector memory (T_EM) and terminal effectors were observed⁴⁹. Overall, the results from this small trial suggest that CD25-depleted DLI may be somewhat more effective than unmodified DLI for the management of relapsed acute leukemia post-allo-HSCT.

Finally, following the observation in mice that naïve T cells cause more severe GVHD than memory T cells, a group in Seattle evaluated naïve T cell depletion as a strategy to prevent GVHD while preserving GVL⁷⁷. Positive selection of CD34+ progenitor cells was followed by depletion of CD45RA+ cells from the CD34-negative fraction by antibody-conjugated magnetic beads. In this trial, incidence of acute GVHD was not reduced but an impressive reduction in chronic GVHD was achieved (9% compared with historical rates of approximately 50%). Furthermore, T cell tolerance was dramatically faster in the T_N-depleted arm: time to 60% completion of corticosteroids was around 150 days in the T_N-depleted cohort compared with approximately 1400 days in the control arm. Figure 1 provides a visual illustration of the main selective depletion approaches that have been evaluated in clinical trials to date.

Figure 1. Non-gene-modified selective depletion-based T cell immunotherapeutics for hematological malignancies.

Donor peripheral blood stem cell (PBSC) populations can be enriched for cell subsets of interest or depleted of undesirable subsets such as regulatory T cells (T_REGs), naïve T cells (T_N) and allo-reactive T cells. A) Monoclonal antibodies (mAB) linked to metal spheres bind to cellular targets and, when passed through a magnetic column, cells attached to the spheres are retained within the column. This positive fraction, or the negative fraction, can be collected for downstream application, such as T_N (CD34-CD3+CD45RO-), T_REG (CD3+CD4+FoxP3+CD25+) or allo-reactive T cell (CD69+) selective depletion. B)The interleukin-2 (IL-2) receptor α chain CD25, a T cell activation marker, can also be targeted by pharmaceutical approaches. CD25 immunotoxin is a murine anti-CD25 mAb linked to deglycosylated ricin α chain. Co-culture of recipient lymphoblastoid cell lines (LCL) with donor peripheral blood mononuclear cells (PBMC) results in allo-reactivity of donor T cells. Allo-reactive cells can be removed by overnight treatment with CD25 immunotoxin, or by 72 hour culture with 2 mmol/L adenosine. Following haploidentical stem cell transplant (HSCT) the allo-depleted donor cells are also transfused. C) Allo-depletion can also be achieved by incubating mixed lymphocyte reactions (MLR) in the presence of the photo sensitizer 4,5-dibromorhodamine 123 (TH9402). Activated cells retain TH9402 and are eliminated upon exposure to visible light. Patients received infusions of autologous photo-depleted (PD) effector T cells along with CD34-selected stem cells from HLA-matched siblings.

Antigen-specific T cells

Targeting leukemia-associated antigens

In contrast to selective depletion or suicide gene DLI strategies, another approach to enhance the GVL effect while minimizing GVHD is to selectively expand the tumor antigen-specific T cells of interest. GVT responses have been attributed to CD8+, CD4+ and NK cells. Consequently tumor-specific CTLs and, to a lesser extent, NK cells targeting leukemia antigens or minor histocompatibility antigens (mHag), in relapsed patients have attracted strong attention by investigators^{59,61–63,78–80}. (NCT02203903; NCT02074657; NCT00052598; NCT00052520; NCT00107354; NCT02895412; NCT00620633; NCT00460629). While the focus of this review is on T cells it is appropriate to mention that other lymphocytes can contribute to the anti-leukemia/lymphoma response. NK cells and cytokine-induced killer cells are also relatively prevalent in the field of immunotherapy for hematologic malignancies and are regarded as a feasible approach. Data has shown that they can provide benefit in the context of acute leukemia, particularly for AML, with little or no GVHD⁴¹.

Targeting Minor Histocompatibility Antigens

Minor histocompatibility antigens that cause graft rejection in transplant recipients and play a role in GVHD, may yet be harnessed to drive a GVT effect in acute leukemia⁸¹. mHAgs are peptides presented on the cell surface by HLA molecules. These peptides differ between individuals by just a few amino acids and can induce anti-mHAg T cell responses from mHAg-negative donors. The best-characterized human mHAg is hematopoietic tissue-restricted HA-1, which is presented by HLA-A2. HA-1's ability to drive a GVL response has been reported, however T cells specific for HA-1 could potentially induce collateral damage to surrounding tissue and thereby contribute to GVHD in the early post-HSCT period when DCs of host origin remain in recipient tissues⁷⁹. In one trial Warren and Riddell targeted mHAgs that were identified on the basis of T cell clonal response, but not molecularly characterized. Five of seven patients achieved complete, though transient, remissions⁸² (NCT00107354). Grade 3 or 4 pulmonary toxicity occurred in three patients within 2 hours of CTL infusions (>1 × 10⁹ cells), and was attributed to the T cell therapy. The fact that expression of some of the targeted mHAgs was not restricted to hematopoietic cells was revealed during post-trial analysis of these 3 patients when the genes encoding a number of the targeted minor H antigens were identified and tissue expression examined. To minimize damage to non-leukemic tissue it is possible to isolate mHAg-specific CTLs that do not recognize the same mHAg on surrounding epithelial, GI tract or liver tissues⁸³. However the sophisticated nature of these time-heavy, expensive assays currently limits their availability.

Targeting Tumor-Associated Antigens

Various approaches for targeting specific leukemia-associated antigens (LAA) have been attempted. Subcutaneous administration of the peptides Wilms Tumor protein 1 (WT1) and pathogenesis-related protein 1 (PR1) in Montanide adjuvant along with GM-CSF-mediated immune stimulation resulted in detection of CD8+WT1+PR1+ CTLs in 8/8 myeloid leukemia patients and coincided with reduction of WT1 mRNA transcripts⁶⁹ (NCT00270452). However, this anti-leukemia response was not durable and further boosts of peptides+Montanide failed to elicit further benefit^84,85. The significant decline in vaccine-specific CD8+ T cells with further peptide+Montanide boosts was also observed by another group who cautioned against the use of Montanide and/or CpG as vaccine adjuvants in the leukemia setting⁸⁶. To improve on these vaccine strategies, several groups have evaluated the use of leukemia-specific T cells. One of the first reports of ex vivo expanded leukemia-specific T cells for the treatment of patients in accelerated phase CML showed not only complete eradication of CML after the third dose, but interestingly treatment success was not accompanied by GVHD⁷⁸. These results were particularly exciting as they demonstrated that separation of the GVHD and GVL responses was possible. This early trial selected leukemia-specific T cells, not by their ability to recognize known leukemia antigens, but by their ability to inhibit CML progenitor cell growth in vitro. In this simple but elegant approach three T cell products were generated that lysed CML cells but not donor hematopoietic progenitor cells.

Leukemia-associated antigen-specific T cells

Studies have attempted to enhance leukemia-specific T cell specificity by expanding T cells specific for known leukemia-associated antigens such as cancer/testis antigens (MAGE-A3, A4, PRAME; NCT02203903), transcription factors (WT1⁸⁷; NCT02203903; NCT00052520; NCT02895412; NCT00620633; NCT00460629), pro-survival/anti-apoptosis proteins (survivin; NCT02203903), serine proteases (PR3; NCT00052598; PR1; NCT00460629⁶¹) and oncogene products (BCR-ABL; NCT00460629) (Table 4). These approaches are achieved by priming T cells with specific LAA epitopes or mixtures of overlapping peptide sequences that span known or predicted antigenic regions. The first report of adoptively transferred WT1-specific CTLs for high risk leukemia patients post-HSCT described administration of allogeneic CD8+ T cells specific for the 9mer HLA-A*0201-restricted WT1 epitope, WT1_126-134 (RMFPNAPYL)⁶³. Seven patients that received IL-2, IL-7, IL-15-expanded WT1 CTLs relapsed, whereas 4/4 patients who received IL-2, IL-7, IL-15, IL-21-expanded WT1 CTLs were alive at 18-30 months post-infusion. The addition of IL-21 to the expansion medium appears to have improved function and persistence of the infused CTLs. A confounding variable in this study is the fact that 5 of the 7 IL-2 only patients had detectable disease at the time of transplant compared with only one of the group that received the WT1 CTLs. Thus it is unclear whether the superior persistence of the IL-21-expanded CTLs was due to the less suppressive in vivo leukemic milieu in these patients. However the impressive result in the one patient with detectable disease encouraged further study. Another Phase I trial of allogeneic WT1-specific CTLs utilized pooled peptides encompassing the full WT1 protein to expand a broader range of WT1-epitope-specific clones, that were not HLA-A*0201-restricted. These WT1-specific CTLs were also able to mediate transient reduction or elimination of WT1-expressing cells in the absence of toxicities or GVHD⁸⁷. Further refinement of this approach has seen studies utilizing CTLS specific for multiple LAA. Fourteen CML patients received prophylactic PR1, WT1 and/or BCR-ABL-specific CTLs that had been expanded in the presence of IL-2 and IL-7⁶¹. No toxicity or GVHD was reported and 50% obtained molecular remission (median follow-up 45 months).

Table 4. Non-gene modified T cells for leukemia.

NCT # (ACRONYM)	Disease Target	Cell Type	Age	Inclusion Criteria	Exclusion Criteria	Phase	Sponsor
NCT01627275	Hematological malignancies	Naive T cell depleted DLI	≥ 18	HLA-matched allo-transplant; ≥ 60d post-transplant; KPS 50-100%; Donor engraftement ≥ 40%; Acute GvHD ≤ grade II; No extensive cGvHD; Corticosteroids ≤ 20 mg; Mycophenolate mofetil ≤2000mg/d Cyclosporine/tacrolimus ≤ therapeutic levels; No change in immunosuppressants 2 wks before DLI.	Pregnant or lactating; Major medical or psychiatric illnesses.	I	Duke University
NCT00675831	Hematological malignancies	CD25+ T cell-depleted DLI	≥ 18	Relapse after HLA-A, -B, -C, and-DRBI matched allo HSCT; ≥ 2 months post-HSCT; Off immunosuppressants for GVHD 2 wks prior to DLI; Donor chimerism ≥ 20% 6 wks prior to DLI; ≤ 50% BM involvement (% cellularity) and ≤ LNs 6 wks prior to DLI; ECOG score 0-2; Prior stem cell donor medically fit to undergo leukapheresis.	Chronic phase relapsed CML; Prior DLI or other immunotherapy treatment ≤ 8 wks prior to enrollment; Chemotherapy ≤ 4 wks prior to enrollment; Clinically significant and active autoimmune disease in donor or patient; Acute or cGVHD; Active infection.	I	Dana-Farber Cancer Institute
NCT02203903 (RESOLVE)	Relapsed/Refractor y Hematopoietic Malignancies	Autologous or allogeneic WT1, NE, PR3, PRAME, MAGE-A3, MAGE-A4, NY-ESO, and survivin-specific CTLs	6 m-80 y	Allo-HSCT pts with high risk for relapse or residual/recurrent dz OR relapsed/refractory dz (> 2 regimens with > M1 marrow or persistent HD) with anticipated allo- HSCT pre- and/or post-HSCT; ALL, AML, Ambiguous lineage leukemia or lymphoma, CML, MDS by flow cytometry, morphology, or cytogenetic evaluation within BM or extramedullary sites; HD by morphology, PET/CT uptake in site of previous dz in absence of other etiologies; KPS/LPS > 50; ANC > 500/μL;Bilirubin < 2.5 mg/dL; AST/ALT <5× ULN; Serum creatinine < 1.0 or 2× ULN (whichever is higher); pulse oximetry > 90%; On contraception during study; LVEF > 50% or LVSF > 27% if history of TBI (within 6 mths); Steroids ≤ 0.5 mg/kg/day;	Uncontrolled infection; HIV infection; GVHD > grade 2 or bronchiolitis obliterans syndrome, sclerotic GVHD, or serositis; Pregnant or lactating; Patients on ATG, or Campath or other immunosuppressive T cell mABs within 28 days of screening for enrollment	I	Children's Research Institute, Johns Hopkins University
NCT00052598	Accelerated Phase CML, AML With Multilineage Dysplasia Following MDS, Adult AML With 11q23 (MLL) Abnormalities/Del(5q)/Inv(16)(p13;q22)/t(15;17)(q22;q12)/t(16;16)(p13;q22)/t(8;21)(q22;q22), Blastic Phase CML, Childhood CML, Childhood MDS, Recurrent Adult AML, Recurrent Childhood AML, Relapsing CML, Secondary AML,	Autologous and allogeneic proteinase 3 (Myeloblastin)-specific CTLs + IL-2	Child Adult Snr	Pts undergoing alloHSCT for CML in accelerated/blast phase, AML beyond first remission, 1° refractory AML, tx-related AML any stage, acute leukemia any stage, antecedent dx of MDS/MPS (incl CML, polycythemia vera, essential thrombocytosis, and agnogenic myeloid metaplasia with my elofibrosis); Pts and donors HLA-A2+; Able to provide blood & BM; Eligibility for prophylactic treatment with CD8+ CTL after Transplant (Highest Risk Subgroup): PR3-specific CD8+ CTL generated & QC tested; > 5% morphologic blasts in BM or PB just prior to or at time of transplant; Posttransplant ANC > 500/mm̂3 7 d prior to CTL infusions; Pts on immsupp tx for GVHD eligible if not on corticosteroids or if dose can be tapered to ≤ 0.5 mg/kg/day prednisone; pt's symptoms must remain stable and unlikely to increase to stage III or IV aGVHD; or cGVHD unlikely to progress following change in immsupp tx, after monitoring period, as deemed by the pts' physician & the PI; Morphologic/flow cytometric/cytogenic/molecular evidence of recurrent/progressive dz posttransplant	Pts for whom PR3-specific CD8+ CTL clones have not been generated before dz relapse/progression post-transplant Pts whose malignant cells do not overexpress PR3, based on BM sample with > 50% blasts or on leukemia cells; KPS/LPS ≤ 30; Stage III or IV GVHD unresponsive to tx or requiring tx with anti-CD3 mAb, prednisone > 0.5 mg/kg/day (or equivalent), or other txs that ablate or inactivate T cells (eg anti-T cell mABs); Concurrent use of cyclosporine, FK506, or my cophenolate mofetil (MMF) not strictly an exclusion criterion, but attempts should be made to discontinue; Concurrent therapy with hydroxyurea or other agents that may interfere with the function or survival of infused CTL clones; Nonhematopoietic organ toxicity; graft rejection or failure	I/II	Fred Hutchinson Cancer Research Center, NCI
NCT00052520	Advanced MDS, CML, AML or ALL	Autologous and allogeneic CD8+ WT1-specific CTLs + IL-2	Child Adult Snr	Eligibility for Enrollment: a.i) Pre-transplant: Pts undergoing alloHSCT for RAEB, RAEB-t, CML beyond chronic phase, AML beyond 1st remission, BCR-ABL+ ALL any stage, any ALL beyond 1st remission, 1° refractory AML or ALL, tx-related AML any stage, or acute leukemia any stage in pt with antecedent dx of MDS/MPS; ii) Post-transplant: Pts relapsed after transplant (morphologic, flow cytometric, cytogenetic and molecular) may undergo tx if dz control possible while awaiting generation of study tx b. Pts and donors must both express an HLA- allele from which it is possible to generate WT1-specific clones c. Pts must be able to provide blood and BM Eligibility for Prophylactic Tx with CD8+ CTL After Transplant (Highest Risk Subgroup): a. > 5% blasts in BM or PB just prior to/at time of transplant b. post-transplant ANC > 500/mm̂3) at least 7d prior to CTL infusions c. Pts on immsupp tx for GVHD eligible if not on corticosteroids or if dose can be tapered to ≤ 0.5 mg/kg/day prednisone; pt's symptoms must remain stable and unlikely to increase to stage III or IV aGVHD; or cGVHD unlikely to progress following change in immsupp tx, after monitoring period, as deemed by the pts' physician & the PI; Eligibility for Treatment with CD8+ CTL at Time of Relapse after Transplant (All Others): a. Morphologic/flow cytometric/cytogenetic/molecular evidence of recurrent dz post- transplant b. Pts on immsupp tx for GVHD eligible if not on corticosteroids or if dose can be tapered to ≤ 0.5 mg/kg/day prednisone; pt's symptoms must remain stable and unlikely to increase to stage III or IV aGVHD; or cGVHD unlikely to progress following change in immsupp tx, after monitoring period, as deemed by the pts' physician & the PI; DONOR: undergo separate leukapheresis for PBMC ≥ 2 weeks before/after stem cell mobilization and harvest if possible; if separate leukapheresis not possible, portion of PBMC from PB stem cells may be used to generate WT1-specific CTL clones; ≥18 yrs	Pts for whom CD8+ WT1-specific CTL clones have not been generated in time for planned infusion; Pts whose malignant cells do not over-express WT-1; KPS/LPS =< 30% Current stage III or IV GVHD unresponsive to tx or requiring tx with anti-CD3 mAb, prednisone > 0.5 mg/kg/day (or equivalent), or other txs resulting in ablation/inactivation of T cells (eg anti-T cell mABs); concurrent use of cyclosporine, FK506, or MMF not strictly an exclusion criteria, butattempts should be made to discontinue if possible; concurrent tx with hydroxyurea or other agents that interfere with function or survival of infused CTL clones; preexisting nonhematopoietic organ toxicity deemed by PI to place pt at unacceptable risk; graft rejection or failure DONOR: Medical conditions precluding either leukapheresis or blood donation: Inadequate age or weight Active infection, with or without antibiotic treatment Recent hepatitis exposure, hepatitis A or B antigenemia, or hep C antibody + Pregnancy or nursing; HIV or human T-lymphotropic virus (HTLV) infection Severe cardiovascular disease (e.g., uncontrolled hypertension, recent myocardial infarction [MI], or unstable angina)	I/II	Fred Hutchinson Cancer Research Center, NCI
NCT00107354	Leukemia, MDS	Autologous and allogeneic minor histocompatibility antigen-Specific-CTLs	≥ 14 yrs child adult Snr	AlloHSCT from MHC-identical donor for 1° refractory AML or ALL, AML or ALL beyond 1st remission, Tx-related AML any stage, BCR- ABL+ ALL, RAEB, RAEBt Morphologic, flow cytometric/cytogenetic/molecular evidence of recurrent dz pos-t transplant No grade III or IV aGVHD at post-transplant relapse; No extensive cGVHD at post-transplant relapse KPS 60-100% at post-transplant relapse; No preexisting major nonhematopoietic organ toxicity ≥ grade 3 Concurrent immsupp steroid tx for GVHD allowed provided: Able to taper steroid dose to < 0.5 mg/kg/day No increase of > 1 grade in aGVHD OR progression of cGVHD within 14 d after dose change		I	Fred Hutchinson Cancer Research Center, NCI
NCT02895412 (INTACT-WT1)	Leukemia, Myelocytic, Acute	Allogeneic WT1 and multiple pathogen specific-CTLs (cytomegalovirus (CMV), Adenovirus (Adv), Epstein Barr virus (EBV), Varicella-Zoster virus (VZV), Influenza, BK virus (BKV), and fungal infections)	Child Adult Snr	my eloablative or non-myeloablative allo transplant from HLA (A, B and DR) identical or 1-3 antigen mismatched donor; Transplant performed for AML; Leukemia blasts WT1+ by ELISA (> 50 copies/104ABL in BM or >50 copies/104ABL in PB); PB HSCT recipients; Adequate hepatic & renal function (< 3 × ULN AST, ALT, < 2 × ULN total bilirubin, serum creatinine); Estimated life expectancy ≥ 12 m	Anti-lymphocyte globulin (ALG, ATG, Campath or other broad spectrum lymphocyte antibody) in 4 wks immediately prior to infusion or planned within 4 wks after infusion; ≥ Grade II GVHD within 1 wk prior to infusion; Prednisone/methyl-prednisone > 1 mg/kg (or equivalent) within 72 hours prior to cell infusion; Prior allo HSCT; Privately insured in or outpatients	I	University of Sydney
NCT02074657 (LANK-2)	Relapsed/Refractor y Paediatric Acute Leukaemia	Allogeneic activated and expanded natural killer cells	Child Adult ≤23y	AML in second relapse, post-transplant relapse or refractory, or AML relapsed or refractory; Lansky index > 60%; Mild (<2) functional organ alteration (hepatic, renal, respiratory) per (NCICTCAE v 4; LVEF > 39%; compatible haploidentical donor (father or mother or sibling)	History of poor therapeutic compliance; Patients not valid after psycho-social evaluation; HIV+ serology	II	Hospital Universitario La Paz
NCT00620633	Leukemia, MDS	Allogeneic WT1-specific CTLs	Child Adult Snr All ages	WT1+ leukemic or MDS blasts (IRS 4-12 considered positive); Leukemia relapse/persistent MRD following allo-HSCT; Pts with expected of risk of relapse post-transplant of >30% once MRD confirmed or relapse occurs; ALL, AML or MDS refractory to 1° induction therapy; ALL or AML any stage later than 1° relapse; CML ≥ 2° chronic phase after chemotx; CML in persistent accelerated phase or blast crisis; High risk MDS (RAEB and RAEB+) that failed to respond or recurred following induction chemotx; Donor must consent to 2 vol leukapheresis or whole blood donations obtained at one phlebotomy, approx 250 ml from which WT-1-specific T cells for adoptive transfer will be generated; KPS or LPS ≥ 40; Adequate BM, renal and hepatic function at time of recieving WT1-specific T cells; ANC ≥ 1000/mm3 +/- G-CSF (may be waived if pt relapsed or counts not yet recovered from chemotx); Platelets ≥20,000/mm3 (may be waived if pt relapsed or counts not yet recovered from chemotx); Creatinine ≤2.0mg/dL; ALT, AST <3.0 × institutional ULN; bilirubin < 2.5 × institutional ULN; Pts with CNS relapse may be treated however T cells should not be administered until ≥ 24 h after intrathecal chemotx	Active (grade 2-4) aGVHD, cGVHD or overt autoimmune dz (e.g. hemolytic anemia) requiring high dose glucocorticosteroid (>0.5 mg/kg/day prednisone or equivalent); other conditions not related to leukemic relapse (e.g. v eno-occlusive disease or uncontrolled bacterial, viral or invasive fungal infection) which are also life-threatening and which would preclude evaluation of the effects of a T cell infusion; pregnancy; DONOR ELIGIBILITY: WT1-specific T cell donor = HSCT donor; EBV+ T cells will also be generated from EBV+ donors and infused along with WT1 cells and growth and persistence compared; Initial donation of 25ml blood prior to HSCT donation, to generate EBV-APCs; Leukapheresis/whole blood for WT1-specific T cells 2 wks post transplant donation; Consent to use blood for virus-specific T cells for recipient if required Donor exclusion: HIV+ Other uncontrolled infection; Anemia (Hgb ≤10 gm/dl) persisting since original transplant donation; MI or stroke since HSCT donation;	I	Memorial Sloan Kettering Cancer Center
NCT00460629	CML	Allogeneic BCR-ABL, PR1, and WT1-specific CTLs	Adult 18-60y	Ph+ CML HLA A0201, 0301, 1101, B0801 BCR-ABL b3a2+ No significant comorbidities Resistance to or intolerance of imatinib	HIV+ Blast crisis	I/II	University Hospital Carl Gustav Carus

Allo: allogeneic; ALL: Acute lymphoblastic Leukemia; AML: Acute Myeloid Leukemia; CML: Chronic Myeloid leukemia; MDS: Myeloid Dysplastic Syndrome; MPS: Myeloproliferative Syndrome; CTL: Cytotoxic T Lymphocyte; NCI: National Cancer Institute; KPS: Karnofsky Performance Status; LPS: Lansky Play Score; BM: bone marrow; LN: lymph nodes; ECOG: ECOG (Eastern Cooperative Oncology Group) Performance Status Score; aGVHD: acute GVHD; cGVHD: chronic GVHD; ANC: absolute neutrophil count; mABs: monoclonal antibodies; Snr: Senior; Pts: patients; dz: disease; dx: diagnosis; tx: treatment or therapy; PB: peripheral blood; immsupp: immunosuppressive; RAEB: refractory anemia with excess blasts; RAEB-t: refractory anemia with excess blasts in transformation; HLA: human leukocyte antigen; ELSA: European LeukaemiaNet standardised assay; UL: Upper limit; NCI CTCAE v4: National Cancer Institute Common Terminology Criteria for Adverse Events; LVEF: Left ventricular ejection fraction; IRS: German Immunoreactive Score; ULN: upper limit of normal; MI: myocardial infarction; EBVBLCL: EBV+ B cell line; 1°: primary; APCs: antigen-presenting cells.

Targeting multiple TAA

To circumvent the more limited applicability of HLA-restricted approaches, strategies to expand cell products with specificity for multiple TAAs, expressed by a range of hematological malignancies have been developed. Both MHC-I and MHC-II-restricted epitopes are included in the composite peptide pools, thus both CD8+ and CD4+ T cells can be primed. Trial NCT02203903 is an important step toward multi-specific T cells for post-allo-HSCT leukemia patients, an approach that could ultimately be used in a variety of hematological malignancies without the need to first identify specific tumor antigens in the patient. Targeting multiple antigens also minimizes the possibility of tumor escape since selection pressure is not applied to a single antigen.

Lymphoma antigen-specific T cells

Clinical trials for T cell-mediated lymphoma have revealed that potent immune responses can be generated against lymphoma-associated viral antigens, but that poorly immunogenic tumor antigens constitute more difficult targets. T cells specific for PRAME, SSX2, MAGE-A4, NY-ESO1 and survivin can be generated ex vivo from HL and NHL patients and their effectiveness is being assessed in two clinical trials (Table 5): (NCT01333046 (TACTAL); and NCT02203903 (RESOLVE). Table 5 summarizes lymphoma trial data.

Table 5. T cells for lymphoma.

NCT # (ACRONYM)	Disease Target	Cell Type	Age	Inclusion Criteria	Exclusion Criteria	Phase	Sponsor
NCT00002663	Lymphoma, leukemia, other adult and pediatric solid tumors	Allogeneic EBV-specific CTLs	Child Adult Snr	EBV+ LPD, lymphoma, or other EBV-associated malignancy OR Severely immunocompromised pts at high risk for EBV LPD(> 500 copies/ml EBV DNA in PB) following allo BMT; or allo organ transplant; or HIV-induced immunodeficiency; or congenital immune deficit; or anti-neoplastic or immune suppression tx; or non-immune-suppressed pts with EBV+ lymphoma, EBV+ HD, EBV+ NHL, EBV+ nasopharyngeal carcinoma, EBV+ hemophagocytic lymphohistiocytosis, or EBV+ leiomyosarcoma; Pregnant women	Moribund pts with heart, kidney, liver, lung, or neurologic dysfunction not related to lymphoma, who are unlikely to survive the 6-8 weeks required for in vitro generation and expansion of EBV-specific T cells to be infused and the subsequent 3 weeks required assess effects of infusions	I/II	Memorial Sloan Kettering Cancer Centre and NCI
NCT01948180 (CITADEL)	EBV+ extranodal NK/T cell Lymphoma	Autologous EBV-specific CTLs (CMD-003)	≥ 18y Adult Snr	SCREENING PHASE 1. extranodal NK/T lymphoma, EBV+ by EBER or LMP1 immunostaining 2. Active dz: relapse/progression,1st/2nd relapse following at least one cycle ABC OR initial dz or 1st/2nd relapse and unable to tolerate one full cycle ABC OR High-risk dz (stage III/IV, KPI grps 3-4 or IPI intermediate-high) prior to second CR regardless of previous chemotx. Weigh ≥ 35 kg; ECOG performance score 0-2, inclusively; Negative β-hCG women of childbearing potential; TREATMENT PHASE Relapse/progression following at least one prior cycle of ABC; Active dz: based on any one of the following at baseline or within 2 wks prior to baseline: Imaging; clinical sign(s): lymphomatous skin lesions, organ dysfunction or organomegaly not attributable to other causes; or other clinical sign(s); PB/plasma ENV DNA; Chemotx completed ≥ 2 wks prior to 1st study dose; Recovery from acute hematological, hepatic and renal chemotherapy-related toxicities as defined by ≤ Grade 1 according to NCI CTCAE v4.0; Life expectancy ≥ 8 weeks.	SCREENING PHASE CNS lymphoma; NK cell leukemia; Hemophagocytic lymphohistiocytosis; HIV+, hep B+, hep C+, syphilis+, HTLV+; Systemic corticosteroids >0.5 mg/kg/day within 10 d prior to obtaining 200 mL PB starting material; Pregnant or lactating; Active second malignancy; Prior alloHSCT or solid organ transplant; ARD:Progression during initial ABC & up to 3 mth after end of initial ABC; OR failure to achieve at least PR with initial ABC. ALC <400/μL; Any previous autologous EBV-specific T cell treatment; Any uncontrolled systemic infection; Third or greater relapse; TREATMENT PHASE Use of any investigational agents within prior 4 wks; Radiotherapy within prior 3 wks; Major surgery within prior 2 wks; Systemic corticosteroids within 24 h prior to study drug administration; Hepatic dysfunction: serum bilirubin >3 × ULN or ALT >5 times ULN or AST >5 times ULN	II	Cell Medica Ltd
NCT00779337	Lymphoma	Autologous AdE1-Latent Membrane Protein-specific CTLs	≥ 18y Adult Snr	EBV+ lymphoma by in situ hybridization or equivalent (excluding Burkitts Lymphoma). ECOG performance status 1, 2 or 3 Life expectancy ≥ 6 mths; Measurable disease: relapsing/PR/refractory/PD dz by clinical examination or radiography (incl CT scans/functional imaging), or plasma EBV viral load; No chemotx/radiotx &/or antibody tx ≥ 2 wks prior to anticipated first infusion.	EBV negative tumour; Malignant cells in PB by flow cytometry or morphology; infection, positive serology for HIV I&II, H TLV1 or syphilis; Negative serology for EBV; Psychiatric, addictive or any condition which may compromise trial participation Serious infection within 28 days that has not adequately responded to tx; Pregnancy/not using contraception; Serology (within 3 mths of CTL release date) indicating active HBV or HCV	I	Queensland Institute of Medical Research, The Atlantic Philanthropies, Australian Department of Industry, Tourism and Resources, British Society Health and Medical Research Council, Australia for Haematology, National
NCT00005606	Lymphoma, Leukemia, Multiple Myeloma Plasma Cell Neoplasm	Allogeneic EBV-specific CTLs	Child Adult Snr (≤ 65 yrs)	Pts who have received or will receive a solid organ transplant or T cell depleted BMT EBV DNA+and seroneg OR EBV sero+ Matched or 1 HLA mismatched sib donor HIV neg Hep B surface antigen neg Hep C antibody neg No prior 1° malignancy within past 5 yrs in donor except previously resected skin cancer	None specified	II	Northwestern University, NCI
NCT01636388	HL	Allogeneic LMP-specific CTLs	Child Adult (≤ 45 yrs)	Off other investigational tx for 1 mth prior to entry in this study Adequate renal function: Serum creatinine <2.0 × ULN, or Creatinine clearance or radioisotope GFR > 40 ml/min/m2 or >60 ml/min/1.73 m2 or equivalent GFR; Adequate liver function: bilirubin <2.0 × ULN; and SGOT (AST) or SGPT (ALT) <5.0 × ULN; Adequate cardiac function: Shortening fraction of >27% by ECG; HL with: 1° induction failure (failure to achieve initial CR) and/or 1°refractory dz. 1st relapse. Early relapse (within 12 mth off tx) (excl those who received no tx or radiation tx only for initial tx); Late relapse (> 12 mths off tx). Only pts with recurrent Stage III or IV dz &/or B symptoms at relapse (all other late relapses excluded); 2nd relapse; 3rd relapse. History of prior ablative auto HSCT or ineligible for ablative auto HSCT or ≥25% RD after at least two reinduction chemo cycles. EBV sero+ IgG HLA matched family/MUD HLA matched family donor (6/6 or 5/6) MUD (7/8 or 8/8). Ideally have tissue from original dx specimen &/or relapse reviewed centrally for confirmation of HL. If no specimen avail, pathology documenting EBV+ status. Immunophenotyping to confirm dx will be performed. In situ hybridization for EBV (LMP1, and/or EBER) will be performed. All central morphologic, IHC/insitu hybridization performed in Sherrie Perkins & Rodney Miles lab at U of Utah.	HD with ≥ 4th CR, PR, and/or SD; Rapid PD unresponsive to reinduction chemo, radio, or immunotx; EBV neg HL; Patients with no eligible donor; Pregnancy	II	New York Medical College, Children's Research Institute Baylor College of Medicine M.D. Anderson Cancer Center City of Hope Medical Center Johns Hopkins University Ohio State University University of Utah University of Michigan
NCT01956084	HL, NHL, SCAEBV Lymphoepithelioma, Leiomyosarcoma	Allogeneic LMP1/2-specific CTLs	Child Adult Snr	EBV+ HL/NHL or EBV-associated-T/NK-LPD or lymphoepithelioma/leiomyosarcoma or severe Chronic EBV and in remission (group A) or with detectable dz (group B) after allo SCT Life expectancy > 6 weeks Tumor tissue EBV+; KPS/LPS > 50; Donor HIV neg; ≥ 50% donor chimerism in either PB or BM; Bilirubin <2× ULN, AST <5× ULN, and Hgb >8.0; Creatinine <2× ULN; Off other investigational tx 1 mth prior to entry in this study;	HIV+ GVHD > Grade II Pregnancy	I	Children's Research Institute
NCT02057445	HL, NHL, Lymphoproliferative disorder,	Allogeneic LMP-specific CTLs	Child Adult Snr (≥ 1yr)	Off other investigational tx 1 mth prior to entry in this study; Adequate renal function: Serum creatinine <2.0 × Must meet criteria as per 21 CFR 1271; ULN, or Creatinine clearance or radioisotope GFR > 40 ml/min/m2 or >60 ml/min/1.73 m2 or equivalent GFR; Adequate liver function: Total bilirubin <2.0 × ULN; and SGOT (AST) or SGPT (ALT) <5.0 × ULN; Adequate pulmonary function: Pulse oximetry >94%. LPS (< 16yr) or KPS (> 16 yrs) ≥ 50%; Life expenctancy ≥ 6 weeks; Negative urine pregnancy test; Clinical status at enrollment allows tapering steroids to < 0.5mg/kg/day prednisone at time of tx. HL/NHL LPD, SCAEBV: > 4000 genomes per μg PBMC DNA, &/or biopsy tissue EBV+ At dx unable to receive conventional chemotx or in 1st relapse AND not a candidate for HSCT PR after conventional tx. Refractory to conventional tx. In 2nd or subsequent relapse. RD after auto, syngeneic or allo HSCT; Ideally have tissue from original dx specimen &/or relapse reviewed centrally for confirmation of HL. If no specimen avail, pathology documenting EBV+ status. Immunophenotyping to confirm dx will be performed. In situ hybridization for EBV (LMP1, and/or EBER) will be performed. All central morphologic, IHC/insitu hybridization performed in Sherrie Perkins & Rodney Miles lab at U of Utah. DONOR ELIGIBILITY for LMP-CTL 3rd Party Banking: Adequate hematopoietic function: ANC > 1000/mm3, hemoglobin > 10 g/dl, platelet count >50,000/mm3 and EBV IgG sero+; ≥ 12 kg or 24 pounds; <18 years: max 3cc/kg blood in 8 wk period; min 60 cc PB × 2; For stem cell collection, PB for LMP specific CTLs collected prior to stem cell collection, no specific day specification; Must meet criteria as per 21 CFR 1271;	Currently receiving any investigational agents or have received any tumor vaccines within previous 4 wks; Active acute grade III-IV GVHD; Severe refractory intercurrent infection other than EBV; Received alemtuzumab or other anti-Tcell antibody within 28 days. HIV sero+; Pregnancy or lactation; PTLD post solid organ transplantation eligible for the COG PTLD LMP/CTL protocol	I	New York Medical College, Children's Research Institute, Baylor College of Medicine, M.D. Anderson Cancer Center, University of Michigan, University of Utah, City of Hope Medical Center, Ohio University, Johns Hopkins University
NCT01498484	NHL, EBV infection	Allogeneic EBV-specific CTLs	Child Adult Snr	EBV+ LPD, lymphoma or other EBV-assocd malignancy; OR Current or prior PB EBV DNA > 500 copies/ml by qRT PCR; OR EBV DNA > 500 copies/ml in pts treated for EBV-LPD with chemotx &/or rituximab with no clinically or radiologically evaluable dz but high risk of recurrence; Availability of EBV-specific T cells for adoptive immune cell therapy from donor; 3rd party EBV-CTLs will be: 1) matched for ≥ 2 HLA antigens & 2) restricted by an allele shared with the EBV+ malignancy (if known), or with the donor in HSCT recipients, or patient in organ transplant or immunodeficient patients; KPS/LPS ≥ 20; Life expectancy ≥ 6 weeks; Adequate BM, heart, lung, liver & kidney function at tx initiation with EBV-specificT cells, including: ANC ≥ 1,000/μL +/- GCSF support; Platelets ≥ 20,000/μL; Creatinine ≤ 2.0mg/dl; ALT, AST < 3.0× ULN; Bilirubin < 2.5× ULN; Stable BP & circulation not requiring pressor support; Adequate cardiac function (EKG &/or ECG within 30 days prior to treatment); Abnormalities of specific organs not grounds for exclusion if arising from EBV+ malignancy or its tx (e.g. renal allograft recipient with EBV LPD on dialysis due allograft rejection after immune suppression stopped); EBV+ lymphoma or LPD following allo HSCT; or allo organ transplant, or due to HIV-induced profound acquired immunodeficiency; or other EBV-assocd malignancy due congenital immune deficit or sequela of antineoplastic or immunosuppressive tx; EBV-assocd malignancies without pre-existing immune deficiency, including: EBV+ HD & NHL, EBV+ nasopharyngeal carcinoma, EBV+ hemophagocytic lymphohistiocytosis, or EBV+ leiomyosarcoma.	Active (grade 2-4) aGVHD, cGVHD or overt autoimmune disease (e.g. hemolytic anemia) requiring glucocorticosteroid (>0.5 mg/kg/day prednisone or equiv); Pregnancy; Severe comorbidities, unrelated to EBV-associated malignancy, that would be expected to preclude their survival for the 6 wks required to assess response of T cell therapy Patients eligible for MSK protocol #16-803 (EBV-CTL-201)	II	Atara Biotherapeutics, Memorial Sloan Kettering Cancer Centre
NCT01447056	HL, NHL, Lymphoproliferative disease, Nasopharyngeal Carcinoma, Leiomyosarcoma, SCAEBV	Allogeneic LMP-specific CTLs	Child Adult Snr	SCREENING EBV+ HL, NHL, LPD, Nasopharyngeal carcinoma, Leiomyosarcoma, SCAEBV, EBV load in plasma or PBMC > 4000 genomes per ug PBMC DNA &/or biopsy tissue EBV+; KPS/LPS ≥ 50%; TREATMENT EBV+ HL, NHL, LPD, Nasopharyngeal carcinoma, Leiomyosarcoma, SCAEBV, EBV load in plasma/PBMC > 4000 genomes per ug PBMC DNA &/or biopsy tissue EBV+; At dx or in 1st relapse AND unable to receive chemotx OR in 2nd or subsequent relapse OR with RD after auto, syngeneic or allo HSCT; Life expectancy ≥ 6 wks; Tumor tissue EBV+; KPS/LPS score ≥ 50%; Bilirubin < 3 × ULN; AST < 5 × ULN; Hgb > 8.0 g/dL; Serum creatinine < 3 less × ULN; Pulse oximetry > 90%; If post allo HSCT, patient ≥50% donor chimerism in either PB or BM; Clinical status at enrollment allows tapering steroids to < 0.5 mg/kg/day prednisone at tx; Willing to utilize birth control during study and 3 mths after study conclusion.	SCREENING HIV+; TREATMENT Currently receiving any investigational agents or have received any tumor vaccines within previous 4 wks; Active acute grade III-IV GVHD; Severe intercurrent infection; Alemtuzumab or other anti-T-cell antibody within 28 days; HIV seropositivity; Pregnancy or lactation; Tumor in location where enlargement could cause airway obstruction	I	Baylor College of Medicine
NCT01555892 (GRALE)	HL, NHL, T/NK lymphoproliferative Disease Lymphoma	Autologous or syngeneic donor LMP1/2, EBNA-1 and BARF-specific CTLs	Child Adult Snr	EBV+ HL/NHL or EBV-assocd T/NK-LPD or SCAEBV ≥ 12 kg In 2nd or subsequent relapse (or 1st relapse or with active dz if immunosuppressive chemotx contraindicated; or multiply relapsed patients in remission who have a high risk of relapse); OR any patient with 1° dz or in 1st remission if immunosuppressive chemotx contraindicated, e.g. HD after solid organ transplant or if Lymphoma is a 2nd malignancy e.g. Richter's transformation of CLL. (Group A) OR In remission or MRD after auto or syngeneic SCT (Group B); Life expectancy ≥ 6 wks; Bilirubin ≤ 3× ULN; AST ≤ 5× ULN; Hgb > 8.0; creatinine ≤2× ULN; Pulse oximetry > 90%; Off other investigational therapy 4 wks prior to entry in this study. KPS/LPS ≥ 50; Willing to utilize birth control during study and 3 mths after study conclusion.	Active infection with HIV, HTLV, HBV, HCV; Pregnant or lactating; Severe intercurrent infection; Current systemic corticosteroids > 0.5 mg/kg/day	I	Baylor College of Medicine, NCI
NCT00062868	HL, NHL, Lymphoepithelioma, Leiomyosarcoma	Autologous or allogeneic LMP1/2-specific CTLs	Child Adult Snr	In 2nd or subsequent relapse (or 1st relapse or with active dz if immunosuppressive chemotx contraindicated; or multiply relapsed patients in remission who have a high risk of relapse); OR any patient with 1° dz or in 1st remission if immunosuppressive chemotx contraindicated, e.g. HD after solid organ transplant or if Lymphoma is a 2nd malignancy e.g. Richter's transformation of CLL. (Group A) OR In remission or MRD after autol or syngeneic SCT. (Group B); OR detectable dz after alloSCT (Group C); Life expectancy ≥ 6 wks; KPS/LPS ≥ 50; ≥ 50% donor chimerism in either PB or BM if post alloSCT; Bilirubin ≤ 3× ULN; AST ≤ 5× ULN; Hgb > 8.0; creatinine ≤2× ULN; Off other investigational tx 1 mth prior to entry into study	Severe intercurrent infection; HIV+ donors or HIV+ pts if autologous product to be used > Grade II GVHD Pregnancy	I	Baylor College of Medicine
NCT02287311 (MABEL)	HL, NHL, SCAEBV, T/NK-lymphoproliferative Disease	LMP, BARF-1 and EBNA1- specific	Child Adult Snr	EBV+ HL; EBV+ NHL; EBV-assocd T/NK-LPD, or SCAEBV (>4000 EBV genomes per ug PBMC DNA) &/or biopsy tissue EBV+ AND in 1st or subsequent relapse (Group A); with active dz persisting despite tx (Group B); with active dz if immsupp chemotx contraindicated e.g. pts who develop HD after solid organ transplant or if lymphoma = 2nd malignancy e.g.Richter's transformation of CLL (Group C); ≥ 12kg;	Pregnant or lactating; Severe intercurrent infection; Current systemic corticosteroids > 0.5 mg/kg/day; Receiving ATG, Campath, or other immunosuppressive T cell monoclonal antibodies within 30 days.	I	Baylor College of Medicine
NCT00002663	Lymphoma, Leukemia, Unspecified Adult Solid Tumor, Protocol Specific Unspecified Childhood Solid Tumor, Protocol Specific	Allogeneic EBV-specific CTLs	Child Adult Snr	EBV+ LPD, lymphoma, or other EBV-assocd malignancy OR Severely immunocompromised pts with PB levels EBV DNA > 500 copies/ml at high risk for EBV LPD; Incl pts with/at risk for EBV lymphomas or LPDpost allo BMT or allo organ transplant; AIDS pts w/EBV lymphomas/LPD due HIV-induced acquired immunodeficiency; EBV lymphomas/LPD due congenital imunodeficiencies or acquired as asequela of anti-neoplastic or immsupp tx; EBV+ HD & NHD, EBV+ nasopharyngeal carcinoma, EBV+ hemophagocytic lymphohistiocytosis, or EBV+ leiomyosarcoma in immune compentent pts; Pregnancy not contraindicated for infusions of EBV-specific T cells	Moribund patients who due heart, kidney, liver, lung, or neurologic dysfunction unrelated to lymphoma, are unlikely to survive the 6-8 wks required for in vitro generation and expansion of EBV-specific T cells to be used for tx & the subsequent 3 wks required to assess effects of infusions of EBV-specific T cells.	I/II	Atara Biotherapeutics, Memorial Sloan Kettering Cancer Centre, NCI
NCT02973113 (PREVALE)	HL, NHL, Lymphoproliferative Disorders, EBV-related Lymphoma, EBV-Related PTLD, EBV-Related NHL EBV-Related HL	Autologous EBV-specific CTLs+nivolumab (anti-PD-1 mAB)	Child Adult Snr	PROCUREMENT EBV+ HL or NHL or EBV-assoc T/NK/B cell LPD. 1st 3 pts will be adults. Pts <18 years of age eligible if 1st 3 pts don't experience DLT considered to be primarily related to EB-VST or Nivolumab; Relapsed or refractory lymphoma who failed or are ineligible for autoHSCT; ≥12kg; Life expectancy ≥ 6 weeks. TREATMENT As above AND HL in 2nd relapse or 1st relapse & refractory to at least 2 lines salvage chemotx incl Brentuximab Vedotin or 1° refractory dz after at least 2 lines tx OR NHL in 1st relapse and/or refractory to at least one salvage chemotx or with 1° refractory disease after at least 2 lines of tx or in 2nd or subsequent relapse OR T/NK- or B LPD in 1st relapse &/or refractory to at least one salvage chemotx or with 1° refractory dz after at least 2 lines of tx or in 2nd or subsequent relapse; Life expectancy > 6 weeks; Bilirubin ≤ 3× ULN; AST ≤ 5× ULN; Hgb > 8.0; creatinine ≤2× ULN; pulse oximetry > 90%; Off other investigational therapy 4 wks prior to entry in this study; KPS/LPS > 60; Recovered from acute toxic effects of chemotx at ≥ 1 week before entering study; Willing to utilize birth control during study and 6 mths after study conclusion.	PROCUREMENT Active HIV, HTLV, HBV, HCV infection; Hx of solid organ transplant; TREATMENT Pregnant or lactating; Severe active intercurrent infection; Current systemic corticosteroids >0.5 mg/kg/day; Current investigational agents or radiotx within 4 wks prior to entering study; CNS involvement; Hx of allergic rx n attributed to nivolumab or other checkpoint inhibitors; Uncontrolled autoimmune dz needing systemic steroids or steroid sparing agents except for hypothyroidism or type I diabetes. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhy thmia, or psychiatric illness/social situations that would limit compliance with study requirements. Lipase > 70U/ml	I	Baylor College of Medicine
NCT01333046 (TACTAL)	HL, NHL	5-azacytadine+ autologous NY-ESO-1, MAGEA4, PRAME, Survivin and SSX-specific CTLs	Adult Snr ≥ 18y	PROCUREMENT HL or NHL Life expectancy ≥ 6 weeks; Hgb > 8.0 TREATMENT: Group A: ≥ 18 years old with active dz: in 2nd or subsequent relapse; in 1st relapse for indolent lymphoma after first-line tx for relapse; or 1st relapse if immsupp chemotx contraindicated; 1° refractory dz or persistent dz after 1st-line tx of relapse; multiply relapsed patients in remission at high risk of relapse; lymphoma is a 2nd malignancy e.g. Richters transformation of CLL after failing front line tx. Group B: ≥ 18 years old after auto or syngeneic SCT (as adjuvant tx) Group C: 5-azacytidine plus multiTAA-T cells. ≥18 years old with active dz in: 2nd or subsequent relapse; 1st relapse for indolent lymphoma after 1st line tx for relapse; 1st relapse if immsupp chemotx contraindicated; with 1° refractory dz or PD after 1st line tx of relapse; or lymphoma as 2nd malignancy e.g. Richters transformation of CLL after failing front line therapy GROUP D: ≤18 yrs old with active disease in: 2nd or subsequent relapse; 1st relapse for indolent lymphoma after 1st line tx for relapse; 1st relapse if immsupp chemotx contraindicated; with 1°refractory dz or persistent dz after 1st line tx of relapse; lymphoma as 2nd malignancy e.g. Richters transformation of CLL after failing front line tx; Life expectancy ≥ 6 weeks; Pulse oximetry ≥95 percent having previously received radiation tx; KPS/LPS > 50; Bilirubin ≤ 2× ULN; AST ≤ 3× ULN; Hgb > 8.0; platelets > 25,000 (Group C only); Creatinine ≤ 2× ULN; Off other investigational tx for 1 mth prior to entry in this study; Off conventional tx ≥ 1 wk prior to entry in this study, including rituximab; Females of child-bearing potential: at least 2 forms contraception unless hx of hysterectomy or tubal ligation;	PROCUREMENT: Severe intercurrent infection; Active HIV infection; Systemic corticosteroids. TREATMENT: Severe intercurrent infection; Systemic corticosteroids; Pregnant or breastfeeding; Abnormal coagulation parameters (PT > 15 s, PTT > 40 s &/or INR > 1.5) Significant active cardiac dz within previous 6 mths incl: NYHA class 4 CHF; Unstable angina; MI; Active HIV or hepatitis B or C infection defined PB reactive for Hep B, C and/or HIV confirmed with qPCR; Known or suspected hypersensitivity to azacitidine or mannitol; Advanced malignant hepatic tumors	I	Baylor College of Medicine, NCI
NCT02203903 (RESOLVE)	Relapsed/Refractor y Hematopoietic Malignancies	Autologous or allogeneic WT1, NE, PR3, PRAME, MAGE-A3, MAGE-A4, NY-ESO, and survivin-specific CTLs	Child Adult 6mo-65y	PROCUREMENT Allo-HSCT with high risk for relapse or residual/recurrent dz OR relapsed/refractory dz (> 2 regimens with > M1 marrow or persistent HD) with anticipated allo-HSCT pre- and/or post-HSCT; ALL, AML, Ambiguous lineage leukemia or lymphoma, CML, MDS: Evidence of active leukemia or lymphoma by flow cytometry/morphology/cytogenetic evaluation in BM or extramedullary sites; HD by morphology, PET/CT uptake in site of previous dz in absence of other etiologies; KPS/LPS > 50; ANC > 500/μL +/- GCSF; Bilirubin < 2.5 mg/dL, AST/ALT <5× ULN, Serum creatinine < 1.0 or 2× ULN (whichever is higher); Pulse oximetry > 90%; LVEF > 50% or LVSF > 27% if hx of TBI; Agree to contraception during study protocol participation (age appropriate) TREATMENT Steroids < 0.5 mg/kg/day prednisone or equivalent KPS/LPS > 50 Pulse oximetry > 90%	PROCUREMENT Uncontrolled infections; HIV infection; Current GVHD > grade 2 or bronchiolitis obliterans syndrome, sclerotic GVHD, or serositis; Pregnancy or lactating TREATMENT Receiving ATG, or Campath or other immsupp T cell mABs within 28 days of screening for enrollment. Uncontrolled infections Acute GVHD > grade 2 or cGVHD manifestations: bronchiolitis obliterans syndrome, sclerotic GVHD, or serositis.	I	Children's Research Institute, Johns Hopkins University

HD: Hodgkin Disease; HL: Hodgkin lymphoma; NHL: Non-Hodgkins Lymphoma; SCAEBV: severe Chronic Active EBV Infection Syndrome; CTL: cytotoxic T lymphocyte; NCI: National Cancer Institute; DZ: disease; HTLV: human T Cell leukemia virus; ARD: Asparaginase refractory disease; ABC: asparaginase-based chemotherapy; ALC: Absolute lymphocyte count; EBV: Epstein Barr virus; EBER: EBV encoded RNA; ECOG: Eastern Cooperative Oncology Group; NCI CTCAE v4.0: National Cancer Institute Common Terminology Criteria for Adverse Events; PR: partially responsive/partial response; PD: progressive disease; incl: including/includes; sib: sibling; ECG: echocardiogram; neg: negative; 1°: primary; 2°: secondary; RD: residual dz; MUD: or matched unrelated adult donor; SCAEBV: Severe chronic active EBV infection syndrome;qRT-PCR: quantitative real time PCR; Immsupp: immunosuppressive; DLT: dose limiting toxicity; Hx: history; rxn: reaction; MI: Myocardial infarction; TBI: total body irradiation; LVEF: left ventricular ejection fraction; LVSF: left ventricular shortening fraction; mAB: monoclonal antibodies;

Targeting viral antigens on lymphomas

The “right” antigens for lymphoma are currently unknown. Epstein Barr virus (EBV) remains dormant in around 1% of B cells following infection resolution and is highly associated with approximately 40% of cases in immune-competent HL and NHL. EBV is also >90% associated with post-transplant lymphoproliferative disorder (PTLD), thus it has been a valuable target for T cell therapy of EBV+ malignancies. In contrast, for patients with EBV-negative lymphomas, identification of lymphoma-specific tumor antigens has been vital for generating lymphoma-specific therapies. Both EBV-specific and LAA-specific T cells are under current investigation by multiple institutions to identify which are able to most effectively clear lymphoma.

EBV-specific T cells for PTLD

EBV-mediated lymphomas that occur in patients post-HSCT have not been exposed to the influence of immune-selection and as such are exceptionally sensitive to immune cell-mediated therapy. Rituximab, a monoclonal antibody (mAB) targeting CD20 is prescribed for EBV-mediated lymphoproliferative disease in immune-compromised transplant patients. In a proportion of patients Rituximab alone can constrain B cell proliferation throughout the post-transplant immune cell regeneration stage. Thereafter the patient's immune system can resume regulation of EBV-infected B cells, quelling their proliferation. T cells specific for EBV antigens have proven highly beneficial for regaining control of EBV-driven proliferation in patients with PTLD for whom Rituximab is not successful. This approach elucidated the mechanism of action of non-specific DLI, which was first demonstrated to reestablish EBV immunity and suppress lymphoproliferative disease in the mid-1990s - although this was often accompanied by severe GVHD⁸⁸. The approach was later refined with the generation of EBV-specific T cells designed to eliminate EBV+ tumors and reduce the risk of GVHD^89–91. In a trial of 114 HSCT patients, from 3 study sites, none of the patients who received prophylactic infusion of third party, EBV-primed CTLs developed lymphoproliferative disease⁹¹ establishing the robustness of this approach.

EBV-specific T cells for HL and NHL

EBV+ Hodgkin's lymphoma (HL) and NHL arise in an immune-competent setting. These tumors have been exposed to immune editing and display minimal expression of EBV antigens. Consequently these tumors are less immunogenic and more resistant to immune control. Nevertheless a 20% response rate, and persistence up to a year, has been observed following treatment with autologous EBV-CTLs in these patients⁶⁵. Further studies with antigen presenting cells (APC) genetically modified to express LMP1/2 proteins for expanding both autologous and allogeneic CTLs demonstrated safety and efficacy of these EBV-CTLs⁶⁴.

The limits of success for adoptive T cell therapy

Transferred T cells are also subject to endogenous immune suppression in the host tumor microenvironment (TME). Clinicians can evaluate the effectiveness of cell-based therapeutics by monitoring adoptively transferred cells' capacity to home to the tumor site(s), engage with the target(s) in vivo, expand and survive. Unfortunately, in the absence of gene “marking” definitive determination of whether such responses are related to the adoptive T cell transfer can be difficult to achieve. However, more complete knowledge of where and when inhibition of infused cells occurs will enable design of sound tactics to thwart immune-restricting factors in vivo. Lymphodepletion pre-T cell infusion is one approach employed to overcome the tumor-induced immune suppressive microenvironment and to promote homeostatic lymphoproliferation. Proliferation of donor T cells in the host is also fostered by administering exogenous cytokines, such as interleukin-2 (IL-2) and IL-15, that promote T cell survival and expansion. Ex vivo expansion of T cells outside of the inhibitory elements of the malignant microenvironment in vivo can produce highly activated, antigen-specific, cytotoxic T cells. Nevertheless following adoptive transfer these cells remain susceptible to the actions of steroids administered to control GVHD post-HSCT, which can inhibit the function of the infused cells and stimulate apoptosis.

Tumors can avoid direct CD8+ T cell-mediated lysis by downregulating their HLA Class I molecules. Thus treatments that induce tumors to upregulate MHC-I can reestablish antigenicity, and the efficacy of some chemotherapies lies in their capacity to stimulate MHC-I expression⁹². Further inhibitory mechanisms include ownregulation of costimulatory molecules thus limiting CTL activation, and secretion of immune-modulating factors. TGF-β, for example, promotes regulatory functions in dendritic cells and macrophages and directly inhibits T cell function⁹³. Restoration of anti-tumor immunity via engineering antigen-specific T cells that are resistant to TGF-β is one hypothesis that is currently being explored (NCT00368082)⁹⁴. Tumor-secreted factors also contribute to the accumulation of myeloid-derived suppressor cells (MDSC) that in turn inhibit T cell function and drive CD4+ T cells down the regulatory pathway into T_REGs. Strategies for overcoming the pro-tumor effects of these immature myeloid cells include targeted depletion, deactivation, induction of maturation and blocking their development^95,96.

The existence of tumor stem cells that are ignored by the immune system may provide a reservoir of MRD cells that remain undetected after an apparently successful treatment and cause disease relapse. The bone marrow, which is a source of hematopoietic progenitors, may provide this leukemia-favoring niche. Hence, strategies designed to eliminate leukemia stem cells are focused on survival and self-renewal pathways (NF-κB, PI3K/Akt/mTOR, Wnt/β-catenin, Hedgehog); cell surface antigens (CD123, CLL-1, CD25, CD47, CD33); and the bone marrow microenvironment (adhesion and homing [CXCR4/SDF-1, VLA-4, CD44, MUC1-C] and hypoxia [HIF-1a, VEGF, hypoxia-activated prodrugs])⁹⁷.

Finally, targeting immune checkpoints has seen impressive results in the treatment of patients with some solid tumors and hematological malignancies. CTLA4 is upregulated on activated T cells to regulate contraction of the expanded clonal population. CTLA4 binding to CD80/86 on antigen presenting cells or tumor cells triggers ‘off’ signaling pathways in the T cell, hence anti-CTLA4 mABs are utilized in cancer patients to block CTLA4 binding with its ligand and thus maintain T cell activity⁹⁸. Likewise the PD-1–PD-L1 interaction between T cells and tumor cells inhibits T cell proliferation, and anti-PD-1/PD-L1 mABs are able to rescue this inhibition, particularly in HL⁹⁹.

However not all patients respond to PD-1/PD-L1 blockade and the degree to which T cell exhaustion can be reversed is unknown. Nevertheless, exhaustion reversal has been observed after DLI and is associated with disease responses in patients with relapsed CML⁵⁰. Therefore, the consensus is that single immune or cell-based therapeutics probably will not work in isolation and combination approaches (checkpoint inhibition with cell-based therapies) may be the best way forward for a potent anti-tumoral response in vivo (NCT02973113 (PREVALE); NCT02846376; NCT02733042).

Conclusion

T cell therapeutics for hematological malignancies are making steady progress towards more efficacious treatments but are currently restrained by certain limitations: Donor Lymphocyte Infusions have achieved very positive results in CML but lymphoma and acute leukemia patients remain less responsive to this approach; and the means to disengage the GVT effect from GVHD is one “Moon Shot” that still awaits discovery. Researchers are elucidating various means by which heightened cell-mediated anti-tumor effects can be achieved, including selective depletion of certain T cell subsets, and ex vivo production of T cells that target tumor- or viral-associated antigens. More finely-tuned T cell-based immunotherapies, combined with: (i) rational immune checkpoint blockade, (ii) genetically engineered TCRs or chimeric antigen receptors, and/or (iii) highly precise pharmacological molecular targeting, will likely lead to effective, durable and less toxic treatments for patients.

Practice Points.

• Bone Marrow Transplant represents the original form of successful cell therapy for hematological malignancy.

• Donor lymphocyte infusions administered to patients after BMT can achieve sustained remissions especially in patients with CML

• Selective depletion strategies can permit the infusion of large numbers of leukemia-reactive donor T cells without GVHD, or the need for immunosuppression to prevent GVHD, leading to superior GVL effects in patients after BMT

• Selective expansion of tumor antigen-specific T cells has shown efficacy in patients with leukemias and lymphomas without the toxicity profile of gene engineered T cells

Research Agenda.

• Collaborative studies are required to extend the tumor antigen-specific T cell field beyond boutique centers

• Combining antigen-specific T cells with other immunotherapeutic modalities such as checkpoint inhibitors may be required to enhance efficacy