Augmentation of Anti-tumor Immunity by Adoptive T-cell Transfer After Allogeneic Hematopoietic Stem Cell Transplantation

Marie Bleakley; Cameron J Turtle; Stanley R Riddell

Disclosures

Expert Rev Hematol. 2012;5(4):409-425. 

In This Article

MHC-dependent Tumor-associated Antigens

MHC-dependent antigens are peptides anchored to the cleft between the a-1 and a-2 domains of MHC class I molecules or the a-1 an b-1 domains of MHC class II molecules. MHC molecules are polymorphic; therefore, a given MHC-dependent antigen can only be presented by the subpopulation of allogeneic HCT recipients who express the corresponding MHC allele.

Self-antigens Overexpressed in Hematologic Malignancies

Several nonpolymorphic self-proteins that are expressed at high levels in hematologic malignancies and relatively low levels in normal cells can be targets for T-cell immunotherapy in both the allogeneic HCT and non-HCT settings (Table 1).[8–25] An important advantage of overexpressed self antigens for T-cell immunotherapy is that they may be expressed in a wide range of hematopoietic malignancies and solid tumors and therefore have broad applicability. However, the presentation of self antigens on normal tissues poses a risk of toxicity, and self-tolerance mechanisms restrict the repertoire of reactive T cells present in vivo to those with low-affinity T-cell receptors (TCRs). The isolation of high-avidity T cells for tumor-associated self antigens can be even more challenging in patients with malignancy because the tumor may further promote their deletion or result in functional impairment.[26] As discussed later in this review, the problem of low affinity can be overcome by transferring genes into T cells that encode TCRs selected or modified to have higher affinity for the antigen. This approach can reveal unanticipated problems. Survivin is highly expressed in many malignancies and was once viewed as an attractive candidate antigen; however, this protein is expressed in activated T cells, and T cells endowed with a high-affinity survivin-specific TCR commit fratricide and cannot be propagated in vitro.[27]

Despite the disadvantages of many self antigens as targets for T-cell therapy, a few have emerged as promising targets for differential recognition of normal and malignant cells and are advancing to clinical trials. Recent studies in a HLA-transgenic murine model demonstrate that high-avidity T-cells specific for Wilm's tumor 1 (WT1) can be generated in the thymus and acquire a memory phenotype and persist in the periphery without inducing autoimmunity.[28] Moreover, by optimizing in vitro culture conditions for human T cells, it has been possible to isolate T cells from normal human donors with sufficient avidity for self antigens such as WT1 and proteinase 3 in order to recognize leukemia.[8,29–31] WT1 has been ranked as the top priority cancer antigen for translational immunotherapy research and is currently being investigated in clinical trials of T-cell immunotherapy following allogeneic HCT, either using T cells derived directly from the donor or engineered by gene transfer to express a WT1-specific TCR (Box 1).[25,32]

Tumor-specific Antigens

Novel proteins that arise as a consequence of chromosome translocations and other mutations can provide unique peptide sequences that can be presented by MHC molecules and are potential targets for T-cell immunotherapy (Table 1).[33–38] Mutated proteins may also contribute to the malignant phenotype, making them particularly attractive targets for immunotherapy, since antigen loss would be expected to decrease. Whole exome sequencing of primary tumor samples is providing new information on the mutational landscape in leukemias and lymphomas and will likely identify novel targets for both pharmaceutical and immunologic approaches to therapy. Because mutant proteins are truly tumor-specific, high-avidity T cells specific for novel peptides derived from these proteins should not have been deleted in normal HCT donors, and T-cell immunotherapy targeting these antigens should have little risk of on-target toxicity to normal cells in the recipient. However, many mutations are unique to the tumors in individual patients or shared by only a subset of patients, which, combined with the necessity that the mutated epitope be appropriately processed and conform to the peptide-binding requirements for the MHC molecules expressed by that patient, will limit the broad applicability of T-cell therapy for many mutation-associated antigens.

B-cell malignancies can express immunogenic determinants derived from the variable regions of the heavy and light chains of the immunoglobulin idiotype (Id) that is uniquely expressed by tumor cells. Id has been validated as a tumor-specific target by vaccination studies in patients with indolent lymphoma that elicited Id-specific T cells, and in some studies improved survival.[39,40] In principle, an Id vaccine could also be used to immunize HCT donors before stem cell collection to elicit tumor-specific T cells that would be transferred with the graft or that could be isolated and expanded for adoptive T-cell transfer.[41] However, exome sequencing has shown that many diffuse large cell lymphomas have mutations in b-2 microglobulin and are class I MHC negative, suggesting that efforts to target this particular tumor with MHC-restricted T cells may be difficult.[42] Moreover, Id is patient specific, thus its applicability as a target for adoptive T-cell therapy is in the realm of personalized medicine.[41] T-cell responses to peptides derived from framework regions within the variable regions of the immunoglobulin that are shared among patients have also been observed, but T-cell immunotherapy trials targeting the immunoglobulin framework peptides have not been reported and could potentially be complicated by toxicity against normal B cells.[43]

Minor Histocompatibility Antigens

Minor histocompatibility (H) antigens, which are HLA-binding peptides derived from endogenous proteins in cells of the HCT recipient that differ from those of the donor due to genetic polymorphisms, represent a unique class of antigens that can only be targeted after allogeneic HCT to promote a GVL/T effect. Recipient cells that are homozygous or heterozygous for a polymorphic minor H antigen allele may be recognized by T cells from an HLA-matched individual who is homozygous for the alternative 'negative' allele. Because minor H antigens are not 'self' to the donor immune system, they can elicit high-avidity CD8+ and CD4+ T-cell responses. Thus, minor H antigens that are expressed on hematopoietic cells including leukemic stem cells represent an important class of tumor-associated antigens for GVL/T responses after allogeneic HCT.[44–46] Unfortunately, many minor H antigens are not rigorously restricted in their expression to hematopoietic cells and specific T cells may recognize nonhematopoietic cells that express the antigen and contribute to GVHD.[47–50]

The challenge of separating the GVL/T effect from GVHD after allogeneic HCT may be addressed by only transferring donor T cells that specifically target minor H antigens expressed predominantly or exclusively on hematopoietic cells, preferably following a HCT that is designed to minimize broad alloreactivity such as by complete or selective T-cell depletion of the graft. A Phase I clinical trial of adoptive immunotherapy was performed by our group in which T-cell clones that were specific for minor H antigens that exhibited preferential expression on hematopoietic cells by in vitro cytotoxicity assays against representative target cells were adoptively transferred to patients with leukemia relapse after allogeneic HCT.[51] This study demonstrated that generating minor H antigen-specific T cells in sufficient quantities for therapy was feasible in most patients, and that the transferred T cells infiltrated the bone marrow and mediated antileukemic activity in vivo. However, some patients experienced reversible pulmonary toxicity following the infusion of high doses of T cells that was subsequently shown in one patient to reflect the unexpected expression of the targeted minor H antigen in pulmonary epithelial cells. This on-target toxicity emphasized the need in future trials to focus on only targeting molecularly characterized minor H antigens that can be assayed for transcript expression exclusive to hematopoietic cells.[51] The amino acid sequence, HLA-restriction, gene and chromosomal location of more than 40 human minor H antigens have been determined by the authors and others, and this list is now expanding rapidly as the methodology for gene identification improves.[46,52] Examples of hematopoietic-restricted minor H antigens are provided in Table 1 & Box 2. It is anticipated that a sufficient number of hematopoietic-restricted minor H antigens presented by common HLA alleles will soon be identified and make it feasible to begin T-cell immunotherapy trials targeting antigens with a defined and desirable tissue distribution. Clinical trials of post-HCT vaccination to elicit T cells against hematopoietic-restricted minor H antigens have also been initiated but are not yet reported.

Viral Antigens Expressed by Hematopoietic Tumors

Epstein–Barr virus (EBV)-associated post-HCT B-cell lymphoproliferative disease (PTLD) can develop after allogeneic HCT, nearly exclusively in patients who receive a T-cell-depleted HCT or T-cell-depleting monoclonal antibodies. PTLD tumors express the EBV antigens, EBNA3A, 3B and 3C which, unlike the EBV antigens expressed by EBV-associated malignancies that develop in immunocompetent hosts, are highly immunogenic and can be effectively targeted by EBV-specific T cells. Polyclonal EBV-specific T cells derived from HCT donors were effective in both preventing and treating established EBV-associated PTLD after allogeneic HCT.[53,54] The potential to rapidly treat PTLD with stored, partially HLA-matched, 'third-party', EBV-specific T cells may increase feasibility and broaden the applicability of this approach; however, additional studies are needed to determine if these cells will persist sufficiently long enough to provide durable tumor regression.[55] Other EBV-associated tumors such as Hodgkin's lymphoma have been treated with EBV-specific T cells;[56,57] however, these tumors are less responsive than PTLD, in part due to the lower immunogenicity of the expressed EBV antigens, and incorporation of T-cell therapy after allogeneic HCT for these diseases has not been reported.

Targeting MHC-restricted Antigens by TCR α & β Gene Transfer

One of the major limitations of targeting MHC-dependent tumor antigens after allogeneic HCT is that tumor-reactive T cells are present at an extraordinarily low frequency in normal HCT donors, and their isolation often requires repetitive antigen stimulation and a long duration of culture to generate sufficient numbers of T cells for therapy. The long culture process may drive T cells toward an exhausted effector phenotype and compromise therapeutic efficacy. One promising approach to rapidly derive tumor-reactive T cells is to use gene transfer technology to introduce genes that encode the TCR a- and b-chains from previously isolated tumor-specific T-cell clones. There are several potential advantages of TCR gene transfer for immunotherapy following allogeneic HCT, including improved feasibility and short culture duration due to the availability of TCRs as 'off the shelf' reagents; the potential to select TCRs with sufficiently high avidity to confer lysis of malignant hematopoietic cells; and the ability to direct the transfer of the TCR into T cells selected for particular functional characteristics to improve T-cell persistence in vivo and for defined antigen specificity of the endogenous TCR to minimize the risk of GVHD.

TCR gene transfer has been successful for generating T cells that are effective in murine models of adoptive immunotherapy,[58,59] and the first human trials of TCR gene-modified T cells to treat metastatic melanoma have been performed with some success.[60,61] The most attractive antigens to target with TCR gene transfer for initial translation in the allogeneic HCT setting are WT1 and the hematopoietic-restricted HA-1 minor H antigen.[62–68] Clinical trials of adoptive immunotherapy using allogeneic HCT donor T cells engineered to express WT1-specific TCRs have been developed by our group and others and will open in the near future.[67,68] Similarly, progress has been made in efforts to transduce allogeneic donor T cells with TCRs specific for HA-1 and clinical trials are expected to open shortly.[66]

There are limitations of TCR gene transfer including mispairing of the introduced and native TCR chains leading to low expression of the desired complex, or mispaired complexes that are potentially autoreactive; competition for the CD3 coreceptor between the gene-transferred TCR with the native TCR or mixed TCR dimers leading to reduced function of the introduced antigen-specific TCR; and inefficient gene transfer and unstable transgene expression. Potentially harmful neo-reactivity, including HLA class I and II alloreactivity and autoreactivity, has been observed in human T cells in vitro as a consequence of mispairing of the gene-transferred minor H antigen or tumor-specific TCR a- or b-chains with native TCR chains.[69] Of greater concern was the observation in murine models that a lethal GVHD-like syndrome developed as a result of TCR mispairing.[70] TCR mispairing can be reduced by modifying the introduced a and b chains to increase their pairing with each other,[69,71–74] or by knockdown of the endogenous TCR b-chain through the introduction of a TCR b-chain-specific zinc-finger nuclease or siRNA.[59,75,76] Alternatively, TCR a- and b-genes can be introduced into g/d T cells that lack endogenous ab chains.[64,77]

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