T-cell-based Therapies for Malignancy and Infection in Childhood




One major advance in T-cell–based immunotherapy in the last 20 years has been the molecular definition of numerous viral and tumor antigens. Adoptive T-cell transfer has shown definite clinical benefit in the prophylaxis and treatment of viral infections that develop in pediatric patients after allogeneic transplant and in posttransplant lymphoproliferative disease associated with the Epstein-Barr virus. Developing adoptive T-cell therapies for other malignancies presents additional challenges. This article describes the recent advances in T-cell–based therapies for malignancy and infection in childhood and strategies to enhance the effector functions of T cells and optimize the cellular product, including gene modification and modulation of the host environment.


Tumor vaccines


One major advance in T-cell-based immunotherapy in the last 20 years has been the molecular definition of numerous viral and tumor antigens. Immunodominant epitopes have been defined for major viral pathogens, including Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus, human papilloma virus (HPV) and hepatitis B virus (HBV), that can be used to target infections in immunocompromised hosts or tumors that express viral antigens. Many tumor antigens have also been identified in adult cancers, and some of these are expressed in pediatric tumors ( Table 1 ). Current concepts in tumor immunology suggest that tumor antigens comprise unique tumor-specific molecules or tumor-associated molecules that are rare on normal tissues but highly expressed on tumors. Unlike viral antigens, which generally induce vigorous immune responses in healthy hosts, tumor antigens are not naturally immunogenic as a result of a combination of factors including the immuno-evasive nature of cancer, which diminishes the presentation of antigens to the immune system, and co-expression of tumor-associated antigens on normal tissues, which leads to immune tolerance. The basis of tumor vaccine therapies is that administration of tumor-specific or tumor-associated antigens in the context of immune costimulation induces tumor-specific immunity, resulting in antitumor effects.



Table 1

Antigens expressed on pediatric tumors








































































Antigen Pediatric Tumors References
MAGE-1, -2, -3 Gliomas, medulloblastoma, neuroblastoma, osteosarcoma
GAGE Gliomas, medulloblastoma, neuroblastoma, ESFT
BAGE AML
XAGE ESFT, alveolar rhabdomyosarcoma
NY-ESO-1 Synovial sarcoma, osteosarcoma, neuroblastoma
PRAME AML, Wilms tumor, neuroblastoma
N-Myc Neuroblastoma
Proteinase-3 CML, AML, MDS
WT1 AML, ALL, rhabdomyosarcoma
Survivin Universal
Telomerase (hTERT) Universal
Translocation breakpoints Synovial sarcoma t(X;18); CML t(9;22), ALL t(12;21), DSRCT t(11;22), alveolar rhabdomyosarcoma t(2;13)
Mutant p53 Variable across histologies
HBV and HCV Hepatocellular carcinoma
EBV EBNA2, 3 EBV lymphoproliferative disorder
EBV EBNA1, LMP1, -2 Hodgkin disease, nasopharyngeal carcinoma, EBV lymphoproliferative disorder, Burkitt lymphoma

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; DSRCT, dermoplastic small round cell tumor; ESFT, Ewing sarcoma family tumor; MDS, myelodysplastic syndrome.


Thus far, ample data are available from studies on adult cancer to conclude that tumor vaccines administered as single agents do not reliably induce regression of established tumors. However, tumor burden is 1 critical factor that influences the effectiveness of immunotherapy for cancer. Essentially all animal models of cancer show that minimal residual tumor burdens are more readily treated by the immune system than bulk tumors. In human studies, this is clearly shown in the context of donor leukocyte infusions for chronic myelogenous leukemia (CML), which show an 85% response rate when tumor burdens are low and a <20% response rate in accelerated phase. Therefore, randomized studies are needed to determine whether vaccines administered in the adjuvant setting can prevent tumor recurrence. Indeed, recent results using an antigen-loaded dendritic cell vaccine approach in men with advanced prostate cancer has shown benefit over placebo in a large phase 3 trial (31.7% vs 23% 3-year survival and 25.8 months vs 21.7 months median survival), increasing the prospect that this may be the first cancer vaccine approved for general use by the US Food and Drug Administration. Studies of tumor vaccines in pediatric oncology have largely followed these principles. Several types of vaccines have been administered, all primarily aimed at delivering a tumor antigen (or antigens) in a manner that induces robust immune responses. Many different approaches to tumor-based vaccination are currently used and even within each approach, the choice of appropriate adjuvant, antigen, timing of vaccine, route, and so forth remains under study ( Table 2 ). Although essentially all studies of tumor vaccines in pediatric oncology have demonstrated safety, only a few instances of shrinkage of established tumors were observed. Thus, as with adult tumors, current efforts in pediatrics are focused on administering tumor vaccines in the setting of minimal residual disease (MRD) or combining vaccines with other cell-based therapies for patients with established disease. This is discussed in the following sections.



Table 2

Current approaches to tumor vaccination














































Approach Antigen Restrictions Pros Cons
Peptide vaccines 9–20 amino acids HLA allele specific (eg, HLA-A2) Nontoxic, cheap to produce Restricted to patients with 1 specific HLA allele, targets only 1 epitope, requires adjuvant
Protein Whole antigens None No restrictions based on HLA type Expensive to produce, unclear how best to administer
Pox viruses Whole antigens Some concern in immunosuppressed hosts Can also administer costimulatory molecules Antipox immune response limits repetitive administration
DNA Whole antigens None Relatively simple to produce May be better at boosting existing immune responses than inducing primary responses
Dendritic cells Peptides or protein antigens or whole tumor cells Requires cell harvest, not off the shelf Individualized therapy, can deliver multiple antigens Labor intensive, unclear how best to prepare dendritic cells
Genetically engineered tumor cell banks All antigens expressed by the tumor will be presented None Presentation of multiple antigens, can specifically modulate costimulation Individual cell banks difficult to produce, allogeneic cells banks may or may not be as effective




Adoptive cell therapies


Adoptive T-Cell Therapy for Infections


Infections are a major cause of morbidity and mortality in pediatric patients who are immunosuppressed following hematopoietic stem cell transplantation (HSCT). As risk is clearly correlated with impaired virus-specific immunity in the early posttransplant period, there is considerable interest in developing means to adoptively transfer a protective T-cell response to more rapidly reconstitute immunity without transferring alloreactive T cells. Initial studies to evaluate such strategies targeted viruses such as CMV and EBV for which the immune response is well defined and the approach has now been extended to other pathogens such as adenovirus, BK virus and aspergillus. The methodology used in these studies has been to generate cytotoxic T cells ex vivo from the transplant donor by repeated stimulation of donor-derived peripheral blood mononuclear cells (PBMCs) with antigen-presenting cells expressing viral antigens ( Fig. 1 ). These cells are subsequently administered to the recipient either preemptively to prevent viral infection or to treat documented infections. To identify suitable viral antigens for such immunotherapeutic strategies, it is necessary to know which antigens are required for viral persistence. There must also be a source of the identified viral antigen suitable for clinical use and an appropriate antigen-presenting cell that will effectively present viral antigen and produce the costimulation required to activate an effective T-cell response.




Fig. 1


Generating cytotoxic T lymphocytes (CTLs) by repeated ex vivo stimulation. Low-frequency virus-specific CTLs in a PBMC population are expanded by primary and secondary stimulation with antigen expressed on APCs followed by expansion with IL2. The resulting population is enriched for T cells specific for the viral antigen.


Although the overall incidence of EBV-associated posttransplant lymphoproliferative disease (PTLD) following HSCT is less than 1%, the risk may be much higher in recipients with congenital immune deficiencies or in those who receive highly immunosuppressive conditioning regimens and T-cell–depleted grafts (which are becoming more commonly used as discussed elsewhere in this issue). EBV-PTLD is almost always derived from donor B cells, which express all EBV latency proteins and would normally be eliminated by an EBV-specific immune response. The proliferating cells have the same phenotype and pattern of EBV gene expression as EBV transformed B lymphoblastoid cell lines (LCLs), and these can be readily prepared from any donor by infecting PBMCs with a laboratory strain of EBV. LCLs are excellent antigen-presenting cells (APC) because they present EBV antigens efficiently on the cell surface with robust expression of costimulatory molecules. LCLs have been used after irradiation as effective stimulator cells to generate EBV-specific T cell lines from transplant donors. When EBV-specific cytotoxic T lymphocytes (CTLs) have been administered as prophylaxis or therapy for EBV lymphoma in high-risk HSCT recipients, immunity to EBV has been expanded and reconstituted. In addition, CTLs have been effective in preventing EBV-LPD in high-risk recipients and in treating patients who received CTL for established EBV-LPD with sustained response rates of more than 85%.


The immune response to CMV is also well defined and several studies have transferred donor-derived T cells specific for the immunodominant CMV pp65 protein to HSCT patients and shown that the transferred cells can prevent reactivation and treat CMV reactivation and disease in humans. In these studies, several sources of antigen were used including purified CMV antigen, CMV-infected cell lysates, and peptides. APCs have included dendritic cells, fibroblasts, and PBMCs. The first studies performed by the Seattle group infused CD8+ CMV-specific T-cell clones, reactive against CMV virion proteins, and showed protection against CMV viremia and disease but long-term persistence only in patients who recovered CD4+ CMV-specific responses. Peggs and colleagues produced CMV-specific CTLs using dendritic cells pulsed with CMV antigens as stimulator cells and after infusion saw rapid expansion and long-term persistence of CMV immunity. Several other groups have confirmed that adoptive transfer of donor-derived CMV-specific T cells reconstitutes immunity to CMV and not only prevents transplant patients from developing CMV infection but also treats active disease.


These approaches target only 1 virus in a patient population that is at risk of infection with many viruses following transplant. Leen and colleagues therefore developed an approach to generate CTLs specific for 3 of the viruses that cause morbidity and mortality post transplant: CMV, EBV, and adenovirus. To achieve this they used mononuclear cells transduced with a recombinant adenoviral vector encoding the CMV antigen pp65 for the initial stimulation followed by stimulation with EBV-LCLs transduced with the same vector. Responding T cells were therefore exposed to all 3 antigens. In 2 sequential studies with CMV seropositive and seronegative donors they showed that trivirus (CMV-, EBV- and adenovirus-specific) CTLs could expand in response to viral challenge and clear all 3 viruses in more than 90% of patients with active viral disease. In preclinical studies this approach is being extended to target BK virus.


These methodologies for generating virus-specific CTLs are complex and require considerable time. More rapid selection techniques are therefore being evaluated to provide virus-specific T cells for transplant recipients in a timely manner when they have active infections. Two methodologies to select virus reactive T cells from donor blood have been evaluated in clinical trials. In the first, T cells specific for the CMV-derived antigens were selected from apheresis products obtained from donors by incubating cells with HLA-peptide tetramers (4 joined major histocompatibility complex class I complexes that bind directly to T-cell receptors of a particular specificity) specific for the viral peptides followed by selection with magnetic beads. After infusion, the cells were able to expand by several logs and reconstitute immunity to CMV. A limitation of this approach is that the product has limited specificity for 1 epitope and is only available for some HLA types. A second rapid selection technique is γ-interferon capture assay where donor blood cells are briefly stimulated with antigen and cells are selected that respond to antigenic stimulus based on γ-interferon secretion. Adenovirus-specific donor T cells isolated by this technique were infused into 9 children with systemic adenovirus infection post transplant and responses were seen in 5 of 6 evaluable patients. An alternative to rapid selection is to develop banks of virus-specific cells lines so that the most closely matched product can be accessed rapidly if a patient develops an infection. A recent phase 2 study using banked EBV-specific CTLs to treat PTLD showed a response rate of 64% with no adverse events related to alloreactivity reported. These studies have all targeted viral antigens. However, the T cell immune response may also be important for the clearance of other infections. The Perugia group has generated donor T-cell clones specific for Aspergillus and shown it is possible to transfer high-frequency T-cell responses associated with control of Aspergillus infections.


Adoptive T-Cell Therapy for Pediatric Malignancies


Development of adoptive T-cell therapies for malignancy presents additional challenges. Although adoptively transferred T cells can in theory be redirected toward antigens that are relatively or absolutely restricted to the cancer cells, as discussed earlier, tumor-specific antigens are not as well defined or as immunogenic as viral antigens. In addition to tumor antigens defined in autologous hosts (see Table 1 ), alloantigens selectively expressed on hematopoietic cells in the context of allogeneic HSCT are also a potential target and several groups are developing methods for selection of such T cells based on the γ-interferon capture assay. As described earlier, EBV-PTLD has served as a prototype disease for successful targeting of viral antigens in cancer. Other EBV-associated malignancies, such as Hodgkin lymphoma, some types of non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, have also been targeted using this strategy, but show lower response rates compared with EBV-CTL immunotherapy. These tumors, which develop in previously immunocompetent individuals, express a more restricted array of EBV-encoded antigens than EBV-PTLD with only the weakly immunogenic EBV antigens (EBNA1, LMP1, and LMP2) being expressed. They also possess a myriad of immune evasion mechanisms that are active in the tumor microenvironment. To overcome these obstacles, investigators have developed ways to tailor CTL specificity to the subdominant tumor antigens expressed in EBV-associated lymphomas by stimulating T cells with latent membrane protein (LMP) antigens transferred to APCs (dendritic cells or LCLs) using adenoviral vectors. The resulting CTLs are enriched for T cells specific for LMP antigens and show increased activity compared with EBV-CTLs when administered to patients with EBV+ Hodgkin disease or NHL, either post transplant or in the setting of relapsed disease.


Although T cells specific for tumor antigens can be identified, most are present at a low frequency, may have receptors with low avidity for the tumor antigens, and are commonly anergic. One strategy to overcome these limitations is to activate T cells ex vivo to circumvent these limitations and to overcome suppressive factors present in vivo thus augmenting the antitumor activity. In 1 study, leukemia reactive T cells derived from an allogeneic donor were selected based on their ability to inhibit in vitro growth of CML progenitor cells, and subsequently expanded to generate CTL lines. When transferred to the recipient they were able to induce remission in a patient with recurrent CML. However, this labor-intensive process is not widely applicable. In a simpler approach, donor lymphocytes activated ex vivo were expanded nonspecifically by incubation with CD3- and CD28-coated beads and administered to 18 patients with relapsed lymphoreticular malignancies after HSCT. Objective responses were seen in 8 patients, 4 of whom had a sustained response at a median 23 months of follow-up. However, such products may also contain alloreactive cells that can induce graft-versus-host disease (GVHD) and thus may be problematic when administered in the context of allogeneic HSCT.


An alternative approach to target tumor antigens is to genetically modify T cells with artificial antigen receptors to redirect their potent effector functions toward tumor cells. This has been achieved by expression of either αβ T-cell receptor (TCR) heterodimer pairs or tumor antigen-specific chimeric antigen receptors (CAR). High-avidity αβ TCR heterodimer pairs are either generated by immunizing HLA-A2 transgenic mice with tumor antigen or cloned from human autologous CTL cultures. This approach, albeit attractive, is limited to individuals with a particular HLA type, mostly HLA-A2. Moreover, although αβ TCR T cells mediate antitumor activities in vitro, their in vivo effector functions may be limited by the inadvertent pairing between the native TCR and the transduced αβ chains. Such limitations may be overcome by using CARs, which are artificial molecules custom made by fusing an extracellular variable domain derived from a high-affinity monoclonal antibody specific for a tumor-restricted antigen of interest to an intracellular signaling domain usually derived from the ζ-signaling chain of the TCR. On encountering the specific antigen by the extracellular antibody-derived domain, the T-cell–derived signaling domain initiates an intracellular signal that results in T-cell activation. To promote cell activation and survival, investigators have incorporated additional signaling domains from costimulatory molecules to the intracellular portion of the CAR. CARs recognize antigens in an HLA-independent manner (like an antibody), and thus overcoming a major limitation of the αβ TCR. In addition, the CAR approach circumvents HLA molecule down-regulation, an important mechanism of tumor evasion, and allows for recognition of unprocessed tumor antigens on the surface of the cell. Such artificial molecules can theoretically be designed to target any tumor-restricted or tumor-associated cell surface antigen of interest including those carbohydrate and glycolipid moieties such as the disialoganglioside GD2 in neuroblastoma. Genetically modified T cells have shown promising preclinical effector functions and CARs targeting CD20 and GD2 have already been evaluated in clinical trials in patients with lymphoma and neuroblastoma, respectively. Clinical responses were seen in some patients in both studies although the persistence of the transferred T cells was suboptimal. Several trials are currently underway evaluating whether T cells genetically modified with a CAR targeting CD19 have activity in patients with relapsed CD19+ malignancies post transplant.


Among the many hurdles that must be crossed for adoptive T-cell immunotherapy to be successful is the necessity for infused T cells to access the long-term memory pool. There are concerns that excessive ex vivo stimulation can render T cells senescent, and unable to sustain long-term proliferation required of memory T cells. A recent study showed that it may be possible to take advantage of the longevity of virus-specific CTLs and genetically incorporate antitumor specificities onto these cells. Two distinguishable GD2-specific CARs were transferred to EBV-CTL or primary T cells activated with OKT3 and IL2 administered to neuroblastoma patients in a phase 1/2 clinical trial and the EBV-specific CTLs did survive longer than T cells perhaps because of the costimulation received through their native receptor.




Adoptive cell therapies


Adoptive T-Cell Therapy for Infections


Infections are a major cause of morbidity and mortality in pediatric patients who are immunosuppressed following hematopoietic stem cell transplantation (HSCT). As risk is clearly correlated with impaired virus-specific immunity in the early posttransplant period, there is considerable interest in developing means to adoptively transfer a protective T-cell response to more rapidly reconstitute immunity without transferring alloreactive T cells. Initial studies to evaluate such strategies targeted viruses such as CMV and EBV for which the immune response is well defined and the approach has now been extended to other pathogens such as adenovirus, BK virus and aspergillus. The methodology used in these studies has been to generate cytotoxic T cells ex vivo from the transplant donor by repeated stimulation of donor-derived peripheral blood mononuclear cells (PBMCs) with antigen-presenting cells expressing viral antigens ( Fig. 1 ). These cells are subsequently administered to the recipient either preemptively to prevent viral infection or to treat documented infections. To identify suitable viral antigens for such immunotherapeutic strategies, it is necessary to know which antigens are required for viral persistence. There must also be a source of the identified viral antigen suitable for clinical use and an appropriate antigen-presenting cell that will effectively present viral antigen and produce the costimulation required to activate an effective T-cell response.


Oct 3, 2017 | Posted by in PEDIATRICS | Comments Off on T-cell-based Therapies for Malignancy and Infection in Childhood

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