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Treatment of Hematologic Malignancies With Genetically Modified T Cells
Conventional modalities for treating cancer remain unsatisfactory. Despite the introduction of small molecules that target specific molecular lesions or pathways within the cancer cells, cure rates for many common tumors remain low, while adverse events are still distressingly high. Cancer immunotherapy represents a promising extension of highly targeted cAncer therapy with potentially favorable toxicity and pharmacoeconomic profiles. Until recently, most attention has been on the development of conventional monoclonal antibodies that target specific tumor-expressed antigens. Over the past few years, the focus of monoclonal antibody therapy has shifted to agents that recruit innate or adaptive immune responses against the tumor, either by blocking immune regulation by tumors or by simultaneously engaging tumor cells and effector lymphocytes (bispecific antibodies). More recently, however, strikingly beneficial results with direct (adoptive) transfer of immune system cells are now being reported. Although to date these have primarily been obtained in patients with leukemia, lymphoma, multiple myeloma, melanoma, or neuroblastoma, methodologies are being developed to allow us to extend the tumor range.
Many human tumors express tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) that can be recognized by the host immune system and induce antitumor cell-mediated and humoral immune responses. Although these responses may be transient and are not always associated with clinical responses, they provide evidence for the existence of tumor-directed immunity in humans that may also have antitumor activity. Several barriers block the development of more effective antitumor immunity in people with cancer. First, many human tumors express few major histocompatibility complex (MHC) molecules or have poor processing of their potential tumor antigens. Even when TAA/TSA are processed and presented, most tumors lack the costimulatory molecules necessary to implement long-lived and effective immune responses. In addition to these passive defenses against immunity, many tumors can “edit” the immune system to their advantage, secreting cytokines such as transforming growth factor beta (TGFβ) or by expressing molecules such as programmed death-ligand 1 (PD-L1) that act as inhibitory or check point signals to cytotoxic effector T-cell growth, function, and survival, or that favor expansion of Th2/regulatory T cells rather than effector T cells. Finally, intensive chemotherapy and radiotherapy can themselves severely reduce immune function by destroying antigen presenting cells and dividing T lymphocytes.
As our understanding of the molecular basis of tumor immune escape has increased, it has been possible to derive countermeasures that may allow us to induce more potent antitumor immune responses, and that will soon allow us to extend effective therapies to a broad range of common tumors.
Types of Cellular Immunotherapy
Cellular immunotherapy may use cell-based vaccines derived from tumor cells or antigen-presenting cells expressing TAA/TSA from proteins/peptides, or may depend on the direct adoptive transfer of viable immune cells. The former approach relies on the intact afferent and efferent immune system of the host responding to the stimulus with an effective antitumor response, while the latter is the cellular equivalent of antibody serotherapy, in which the transferred immune cells are expected to attack the tumor cells directly, albeit with a phase of in vivo expansion, and to subsequently establish a pool of memory cells to provide long-term protection against resurgent disease. Several cell subsets are currently being studied in adoptive transfer protocols, including activated T lymphocytes (ATL), tumor infiltrating T lymphocytes, antigen-specific cytotoxic T lymphocytes (CTL), natural killer (NK) cells, γβ T cells, and natural killer T (NKT) cells. In this chapter, we discuss adoptive transfer of genetically modified ATL and CTL.
Adoptive Cell Therapy With T Lymphocytes
In principle, lymphocytes have the ability to traffic through multiple tissue planes and to be self-renewing. These assets, coupled with their ability to destroy tumor or viral infected target cells through a range of mechanisms makes them an appealing resource for adoptive transfer, and a multiplicity of clinical studies using this approach have now been described. Adoptive lymphocyte therapies may use allogeneic or autologous cells, which may be of tightly defined specificity (e.g., T-cell clones) or broad phenotype and activity (e.g., tumor infiltrating lymphocytes). As we have learned more about the molecular basis of immune recognition and immune regulation, it has become possible to genetically modify the infused lymphocytes to alter their specificity or behavior. In this section, we describe examples of each type of T-cell adoptive transfer and discuss the relative merits and limitations of each.
Allogeneic Donor Lymphocyte Infusion
It has long been apparent that the curative effects of allogeneic hematopoietic stem cell transplants (HSCTs) for many hematologic malignancies can be attributed to a graft-versus-leukemia (GVL) effect largely mediated by the incoming T cells within the donor graft. Thus, patients with chronic graft-versus-host disease (GVHD) were well recognized as having a lower probability of relapse than individuals without this unpleasant complication. Similarly, recipients of syngeneic grafts have the lowest rate of GVHD and the highest risk of relapse. In 1990, Kolb and colleagues took advantage of this observation and deliberately infused donor lymphocytes in an attempt to eliminate recurrent disease in patients with chronic myeloid leukemia (CML). Their positive results have been confirmed in multiple studies worldwide, and remission can be induced in more than 50% of CML patients who relapse after transplantation by stopping immunosuppressive treatment or infusing donor lymphocytes. Unfortunately, donor lymphocyte infusion (DLI) is much less effective at treating other types of relapsed leukemias after transplantation, with a 29% remission rate for acute myeloid leukemia (AML) and only 5% for acute lymphoblastic leukemia (ALL). It is not clear why these differences occur, since all these leukemias present the minor histocompatibility antigens (mHags) that are likely the targets of this GVL effect, although many mHags have yet to be defined. DLI therapy may also produce severe adverse effects, since the frequency of broadly alloreactive effector cells is usually much higher than the frequency of lymphocytes targeted exclusively to the relapsed malignancy. As a consequence, patients receiving DLI often develop GVHD. This complication of DLI usually increases in frequency and severity if the donor and recipient are either unrelated or human leukocyte antigen (HLA) haploidentical. Strategies aimed at retaining the benefits of GVL while preventing GVHD have included the depletion of alloreactive T cells in the donor lymphocyte product and the incorporation of suicide genes into the infused donor T cells so that they may be killed if the GVHD activity exceeds the benefits from GVL. Manipulation of the stem cell graft to deplete only the αβ T-cell receptor (TCR)+ T lymphocytes while retaining the γδTCR+ T-cell compartment may reduce GVHD without compromising stem cell engraftment and may retain some protection against opportunistic infections. Ultimately, investigators may wish to identify tumor-restricted target antigens on the malignant cells and infuse antigen-specific T cells directed to them. As discussed in Chapter 25 the potential efficacy of this approach has been demonstrated in preliminary studies, although scalability remains a challenge. Furthermore, genetic evolution and neoantigen depletion on tumor cells has been demonstrated as a mechanism of resistance to the GVL effect following allogeneic hematopoietic stem cell transplantation and could similarly limit the efficacy of infusing autologous T cells that target tumor-restricted antigens.
Infusion of Activated T Lymphocytes
When T cells are polyclonally stimulated, for example by simultaneously cross-linking their CD3 and CD28 receptors by CD3/CD28 monoclonal antibodies on beads, they proliferate, secrete tumoricidal cytokines such as tumor necrosis factor alpha (TNFα), and can mediate MHC-unrestricted cytotoxicity toward a range of tumor target cells. Efforts have been made to harness these effects by producing large numbers of CD3/CD28-activated T cells for cancer patients and infusing them. Although infusion of CD3/CD28-ATL after autologous stem cell transplant may improve patients’ T-cell reconstitution, there is not yet evidence to suggest improved antitumor activity. ATL that are additionally primed with interferon gamma (IFN-γ) and interleukin (IL)-2 (so called cytokine-induced killer [CIK] cells) may have superior clinical potential for hematologic malignancies and early phase clinical trials have shown clinical benefits.
Genetic Modification of T Cells
Early clinical studies using genetic modification only attempted to “mark” the T-cell infused to follow their fate in the peripheral blood or other tissues. More recently, efforts have been devoted to “redirecting” the antigen-specificity of T lymphocytes and thus providing them with robust antitumor activity. To overcome the low affinity of tumor-specific CTLs detected in vivo, investigators have cloned T-cell receptor α and β chains (αβTCR) of high affinity. Alternatively, tumor-specificity has been generated by the construction of chimeric antigen receptors (CARs), which are most commonly composed of the binding domains of a monoclonal antibody and the ξ signaling domain of the CD3αβ TCR as well as components of costimulatory molecules to ensure signaling and T-cell activation once the CAR has been engaged ( Fig. 26.1 ). Finally, interest in genetic modification of T cells has also arisen as a means of incorporating countermeasures to the multiplicity of immune evasion strategies used by potentially immunogenic tumor cells or to enhance the “survival' of T cells in vivo (see Fig. 26.1 ). Because T lymphocytes can be long-lived cells and may proliferate extensively in vivo, most gene transfer studies have used integrating vectors such as gamma-retroviral vectors, lentiviral vectors, or transposon/transposase integrating plasmids to ensure long-term expression of the therapeutic transgene.
Artificial αβT-Cell Receptors
The large-scale culture of T lymphocytes to enrich the scanty precursors specific for weak TAAs is often unsuccessful and always tedious. This process can be bypassed by introducing additional TCR genes with predetermined specificity and high affinity for the weak tumor antigen into a polyclonal population of T cells. Technical improvements in retroviral transduction mean that greater than 30% of polyclonal T lymphocytes can now be induced to express a transgenic TCR with high affinity for TAAs including melanoma-associated antigen recognized by T cells (MART)-1, melanoma antigen (MAGE)-3, mouse double minute 2 homolog (MDM2), Wilms’ tumor 1 (WT1), New York esophageal squamous cell carcinoma 1 (NY-ESO-1), survivin, and for mHags such as HA1 and infectious agents such as human immunodeficiency virus (HIV)-1 and Epstein-Barr virus (EBV).
Early studies, primarily among solid tumor patients, have been hampered by “on target, off tumor” toxicities in normal tissues that physiologically express the target antigen at low level. Melanoma patients treated with polyclonal T cells expressing transgenic MART-1 specific αβTCRs developed toxicity in normal tissues containing melanocytes (e.g., skin and uvea). Patients with metastatic colon carcinoma developed colitis after treatment with T cells expressing a TCR directed to the carcinoembryonic antigen that is also expressed at low level in normal gut epithelia cells.
Nonetheless, promising results have been reported among patients with AML and multiple myeloma. In a trial including 12 AML patients who were required to be HLA-A*0201 (HLA-A2)-positive, EBV-specific CD8 + T cells (to minimize GVHD and enhance T-cell survival) were engineered to express an HLA-A2-restricted WT1-targeted TCR and were infused following allogeneic HSCT. No on-target, off-tissue toxicities were identified and, at a median follow up of 44 months, no patient had relapsed. Another study among MM patients evaluated NY-ESO-1–specific TCR-engineered T cells, which did not appear to cause any significant toxicities and were associated with a 19-month progression-free survival when infused following autologous HSCT.
The major problem of TCR gene transfer in polyclonal T lymphocytes harboring their own native αβTCR was hypothesized to be the “cross-pairing” between transgenic α or β receptor chains and the reciprocal endogenous TCR α- and β-chains, that could create loss of function or—and potentially worse—gain-of-function receptors that may produce autoimmune disease, an adverse effect clearly demonstrable in mouse models. Although these events have not been reported in clinical trials so far, the issue of potential “cross-pairing” has been addressed in one recent study via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated knockout of the endogenous TCR. In prior studies, other unexpected toxicities have unfortunately been observed. For example, patients with myeloma or melanoma were given T cells modified to express a TCR specific for MAGE-A3 that had been synthetically affinity-enhanced. Two of the recipients rapidly developed lethal cardiotoxicity caused by an unanticipated cross-reactivity of the transgenic TCR against peptide epitopes derived from Titin, which were expressed only by cardiac myocytes. These results strongly indicate that T cells engineered with high-affinity TCR can be effective, but can also reveal unexpected and lethal cross-reactivity with other peptide epitopes that would not be recognized by TCR with more physiologic binding affinity. While such unanticipated toxicities may be avoided by ever more extensive preclinical evaluation, reliance on a “superaffinity” TCR may be intrinsically hazardous.
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