Cancer Chemotherapy & Biotherapy: Principles & Practices, 4th Edition

Adoptive Cellular Therapies

Carl H. June

INTRODUCTION

Adoptive immunotherapy is the isolation, ex vivo activation, and infusion of antigen-specific or antigen-nonspecific lymphocytes. Adoptive cellular therapy can be considered a strategy aimed at tumor elimination through direct antineoplastic effects or through indirect effects such as immune-mediated antiangiogenic effects. Adoptive cellular therapy may also have a role in replacing, repairing, or enhancing the immune function damaged as a consequence of cytotoxic therapy by means of autologous or allogeneic cell infusions. Genetic engineering can be used to enhance the function, engraftment, or persistence of the adoptively transferred cells. The analysis of the presently available clinical results suggests that, despite some disappointments, there is room for optimism that both adoptive immunotherapy and active immunotherapy (vaccination) may eventually become part of the therapeutic arsenal and help prevent or combat cancer in a more efficient way. Although adoptive immunotherapy has thus far added little to the routine treatment of most human cancer, it can now be considered “front-line” therapy for patients with chronic myeloid leukemia in relapse after allogeneic stem cell or marrow transplantation and for certain Epstein-Barr virus (EBV)–related tumors. This chapter describes the history of using adoptive cellular therapies for the treatment of cancer, the rationale for such use, and current clinical and experimental approaches.

HISTORY

The seminal discovery made by William B. Coley in the 1890s—that patients with certain malignancies responded to the intratumoral inoculation of live bacterial organisms or bacterial toxins—became the impetus for the development of immunotherapy for cancers.1 In a series of experiments addressing mechanisms of skin allograft rejection, Bellingham and coworkers first coined the term “adoptive immunity” to describe the transfer of lymphocytes to mediate an effector function.2 Based on these studies, immunologists have categorized immunotherapies as either active or passive. Active immunizations require an intact host immune system and are typically delivered as prophylactic or therapeutic vaccines. In contrast, passive or adoptive immunotherapies transfer serum, antibodies, or lymphocytes to the host and do not require an intact host immune system to generate the response. One characteristic of an adoptively transferred immune response is that the host has never experienced the primary immune response. This is particularly attractive for patients with late-stage tumors who may not have the time or capability to mount a primary immune response. However, as noted later, with the advent of dendritic cell transfer therapies, in a practical sense the distinction between active and passive (or adoptive) cellular therapy is blurring.

The concept of adoptive cellular therapy for tumor allografts was first reported for rodents over 45 years ago by Mitchison.3 The cloning of T-cell growth factors made possible the first ex vivo expansion of tumor-specific T cells for adoptive immunotherapy in mouse syngeneic tumor models.4 There are several excellent reviews of the rationale and experimental basis for adoptive T-cell therapy of tumors.5, 6, 7, 8 In early clinical trials, patients were given adoptive transfers of autologous, allogeneic, and xenogeneic lymphocytes for a variety of tumors. The results of these early trials were not promising, and this is not surprising, since they were carried out before the principles of T-cell biology and tumor antigens were understood. The field of adoptive cellular therapy during its first 25 years was reviewed by Rosenberg and Terry.9

TUMOR IMMUNOLOGY AND CELL BIOLOGY

Rational use of adoptive cellular therapy is predicated upon an understanding of the relevant principles of cellular and molecular immunology and cancer cell biology. The reasons for the shortcomings of many previous forms of adoptive cellular therapy are now clear, based on current advances in the basic sciences.

Immunosurveillance and Immunoediting

Sir MacFarlane Burnet proposed a theory of immunological surveillance.10 The concept of immunosurveillance remains controversial; the basic idea is that a function of the immune system is to control the outgrowth of cancer cells by eliminating cells bearing malignant mutations. Thus, immunocompromised humans do have a propensity to develop tumors, and the tumors are often found in immunologically privileged sites such as the brain. However, there is evidence that goes against this concept. For example, athymic nude mice do not develop tumors with greater frequency than normal mice.11 However, more immunosuppressed mice that lack interferon-γ, interferon-γ receptors, or perforin, as well as mice deficient in the recombinase-activating gene 2, have an increased incidence of spontaneous or methylcholanthrene-induced tumors.12, 13 In humans, patients with melanoma frequently have tumor-specific T-cell immunity that developed spontaneously.14 The most direct evidence to support immunosurveillance in human tumors is from studies that provide a clear demonstration that patients with advanced ovarian carcinoma can expect to have much longer overall and progression-free survival if the tumor is infiltrated by T cells than if it lacks infiltrating T cells.15 Furthermore, as discussed later, the occurrence of certain immunologically mediated paraneoplastic syndromes also provides strong support for tumor immunosurveillance in some circumstances. In retrospect, the theory of immunosurveillance appears to have been largely correct; however, sophisticated mechanisms used by tumors in many instances probably thwart the natural immune response.

Immune Escape Mechanisms

Current immunologic dogma is that tumor cells are antigenic but not immunogenic. The fact that tumors frequently survive and prosper in the face of measurable immune responses underscores the importance of regarding living tumors as complex entities rather than clonal aggregates of transformed cells. There are multiple means that tumors use to escape or prevent immune-mediated elimination.16, 17 These are broadly classified as (a) mechanisms leading to decreased immunogenicity and (b) mechanisms leading to tolerance or immunosuppression. Tumor cells often have low expression of MHC class I molecules, and absent or low density of peptide/MHC complexes may cause lack of recognition, a phenomenon termed immune ignorance. Tumor cells themselves are poor antigen-presenting cells (APCs), as the lack of cell surface costimulatory molecules such as B7 may induce T-cell anergy. Ineffective presentation of tumor antigens by the tumor itself as well as by APCs leads to a failure to provide the necessary costimulatory signals required to activate antigen-specific T cells and then to a subsequent functional clonal inactivation of tumor-specific T cells.

Multiple tumor immunosuppressive events may also blunt or eliminate a tumor-specific immune response. For example, tumor cells often secrete suppressive cytokines such as TGF-β or IL-10, which can down-regulate T-cell responses. Tumor cells may also express an enzyme termed indoleamine 2,3-dioxygenase, leading to the catabolism of tryptophan and the subsequent inhibition of T-cell proliferation.18 Finally, to sustain an ongoing T-cell response, a continued production of IL-2 is required; other cytokines such as IL-15 may also play a role.19 In the absence of cytokines, cytotoxic T lymphocytes (CTLs) undergo a few cell divisions and die. CTLs require helper T cells to supply these cytokines, and the tumor microenvironment is generally deficient in helper T cells.

Tumor Antigens

Adoptive cellular therapy is based on the premise that tumor cells possess intracellular or surface antigens that are qualitatively or quantitatively distinct from those present on normal cells. Furthermore, the adoptively transferred T cells must be able to recognize the tumor antigens. The initial successes of adoptive T cell therapy in the 1950s were discredited when it was realized that the tumors were allogeneic and that tumor rejection was simply a form of allograft rejection. However, Klein and Hellström provided strong evidence for specific tumor immunity by demonstrating that after resection of a methylcholanthrene-induced tumor, the host could subsequently reject challenge with its own resected tumor.20 Indeed, tumor immunology was not considered a respectable field until the last decade, when a clear molecular understanding of the nature of antigenic targets presented by tumors became available. The present consensus is that most, if not all, human tumors express tumor antigens.21

Tumor antigens can be classified according to the type of immune response they elicit: humoral, cellular, CD4+, or CD8+, CTL responses. Humoral antigens must be expressed on the surface of the tumor cells for it to be a therapeutic target, whereas T-cell antigens may be derived from cytosolic as well as membrane proteins. A classification of human tumor rejection antigens was proposed.22 Tumor antigens include the following:

§  Nonself tumor antigens such as transforming proteins of viral origin for tumors caused by viruses. For example, a number of human cancers are caused by transforming viruses such as EBV and human papillomavirus.

§  Mutated “self” antigens occur because tumor cells have genetic instability and may accumulate mutations or chromosomal translocations.

§  Overexpressed mutated oncogene products, of which the best examples are p53 and Her-2/neu.

§  In addition, tumor antigens may be normal self-tumor antigens such as reexpressed embryonic antigens, which were not expressed during the development of the immune system; differentiation antigens that are transiently expressed in tissue development and may be reexpressed in tumor cells; and reexpressed retroviral gene products encoded in the mammalian genome (these have been identified for mouse tumors but not yet for human tumors); as well as tissue-specific antigens that may be from immunologically privileged sites or from so-called dispensable tissue.

§  Self-antigens modified chemically by carcinogens such as methylcholanthrene in the mouse.

§  “Silent” genes, which are cellular genes that are not normally expressed but become transcriptionally active in tumor cells, such as cytochrome P450 subfamily 1 (CYP1B1).23

There is currently an explosive growth in the understanding of human tumor antigens and the immune response to tumors. This is based on the development of technologies to identify human tumor rejection antigens.24, 25 A second major advance in the understanding of the human tumor immune response has been the development of tetramers and other immunoglobulin-based reagents that display antigen-specific binding to T-cell receptors. These reagents permit for the first time the tracking and quantification of tumor-specific T cells. Initial studies with tetramers of the MHC class I restricted CD8 T-cell response in patients with melanoma indicate that tumor-specific or tissue-specific T cells are present in most patients with ovarian cancer and melanoma.

Dispensable Tissues: A New Paradigm for Tumor Immunotherapy?

For decades the goal of most cancer immunotherapy centered on the induction of immune responses against tumor-specific “neoantigens.” However, the coalescence of results from many laboratories now indicates that the generation of tissue-specific autoimmune responses represents an alternate approach to cancer immunotherapy that is gaining momentum.26, 27 This is because it is now clear that for most tumors there is no special set of tumor proteins targets; tumor antigens are by and large normal self proteins. Furthermore, for many and perhaps most tumor proteins, active immunologic tolerance has not been induced, as these proteins have simply been ignored previously by the immune system because they have not been presented in an immunogenic form.28

For common tumors derived from “dispensable tissues” such as prostate, pancreas, breast, ovary, and skin, an acceptable toxicity could include the immune-mediated damage or destruction of normal as well as neoplastic tissue. Given that many common cancers such as melanoma, prostate cancer, pancreatic cancer, and breast cancer are derived from dispensable tissues, the induction of immune responses against tissue-specific antigens shared by these tumors might represent an immunotherapy approach whose autoimmune side effects would represent acceptable “collateral damage.” Thus, a new hypothesis in cancer therapy is that the ability to induce tissue-specific autoimmunity could permit the treatment of many important cancers.

The rationale for the induction of tissue-specific rather than tumor-specific responses is derived from several observations. First, in the 1980s immunologists learned that, for many peripheral tissues, T-cell tolerance to self antigens is not maintained purely by clonal deletion of autoreactive T cells in the thymus but also by peripheral mechanisms that result in the functional silencing of the T cells or ignorance to the peripheral tissue. Autoimmunity is the disruption of this mechanism and leads to the loss of self-tolerance to tissues. Second, in the 1990s it was discovered that patients who experienced immune-based rejection of tumors often had responses that were directed against normal self antigens. For example, Brichard and colleagues29 discovered that the target for a melanoma-specific CD8+ T-cell clone isolated from a melanoma patient was wild-type tyrosinase, a melanosomal enzyme selectively expressed in melanocytes and responsible for one of the steps in melanin biosynthesis. Additional evidence for the relevance of tissue-specific responses in melanoma immunotherapy came from the finding that patients whose tumors responded to IL-2–based immunotherapy occasionally developed vitiligo, an autoimmune depigmentation of patches of skin, whereas vitiligo was essentially never seen among melanoma patients who failed to respond to immunotherapy.30 Third, recent therapeutic vaccine trials and adoptive transfer studies in melanoma patients indicate that it is now routinely possible to induce vitiligo.31 Thus, the ability to break self-tolerance does not appear to require rare MHC backgrounds or other unusual polymorphisms.

The ability of adoptive cellular therapies to “break” tolerance to tissue-specific antigens for cancer therapy will probably depend on the antigenic target as well as other host-specific genetic elements that set immune trigger sensitivity such as the patient's MHC background and CTLA-4 alleles.32 It is likely that both the specific antigen as well as the form of immunologic adjuvant will be critical in defining the qualitative and quantitative nature of immune responses generated. Under physiologic circumstances, the level of endogenous immunity against an antigen is below a critical threshold necessary for clinical autoimmunity or antitumor immunity. A successful antitumor response would require the elevation of immunity against a particular antigen above a critical threshold. The stringency of tolerance against a particular antigen will dictate how potent the vaccination strategy will have to be to raise the immune response above the threshold level. For antigens that are difficult to elicit responses against, approaches to interfere with the normal down-regulation of the immune responses, such as blocking CTLA-4 or PD-1 interactions, might be required for the induction of clinically evident antitumor immunity.33, 34, 35

Paraneoplastic Syndromes

One premise for adoptive immunotherapy, especially when using an autologous source of T cells for infusion is that T-cell immunity occurs in response to tumors and that clinically evident tumors have developed the means to escape or overwhelm the cellular response. Unfortunately, it has been extraordinarily difficult to demonstrate naturally occurring T-cell immunity to most human tumors. Other than recent tetramer data, paraneoplastic syndromes provide perhaps the clearest examples of naturally occurring tumor immunity in humans. A recent series of studies in a rare subset of patients with occult carcinomas indicates that spontaneous and potent T-cell immunity in fact occurs in some human tumors. Studies indicate that patients with paraneoplastic neurologic disorders often harbor systemic tumors that express proteins whose normal expression is restricted to the central nervous system. Thus, the expression of these antigens by tumors outside of the normal immunologically privileged site of expression allows for their recognition by the immune system and, consequently, the fortuitous antitumor response. A Purkinje neuronal protein termed “cdr2” has been identified as a target that is responsible for paraneoplastic cerebellar degeneration (PCD) in patients with ovarian and breast tumors.36 Cdr2 mRNA is expressed in almost all tissues, whereas the protein is expressed only in the brain and testis.37 Previous studies had shown that although tumor immunity and autoimmune neuronal degeneration in PCD correlates with a specific antibody response to the tumor and brain antigen cdr2, this humoral response has not been shown to be pathogenic. Darnell and coworkers have detected expanded populations of MHC class I–restricted cdr2-specific CTLs in the blood of PCD patients.38 Thus, it is likely that tumor-induced peripheral activation of cdr2-specific CTLs contributes to the subsequent development of the autoimmune neuronal degeneration in the central nervous system. These studies raise the hope that therapeutically induced immunity to this antigen and perhaps other similar “self” antigens might be an effective immune-based therapy for a variety of carcinomas. They also raise the possibility that effective immunotherapy against some carcinomas could be subject to CNS toxicity, as they may share immunodominant antigens with CNS tissue.

Principles of T-Cell Growth

Adoptive cellular therapy depends on the ability to optimally select or genetically produce cells with the desired antigenic specificity and then induce cellular proliferation while preserving the effector function, engraftment, and homing abilities of the lymphocytes. Unfortunately, many previous clinical trials were carried out with adoptively transferred cells that were propagated in what are now understood to be suboptimal conditions that impair the essential functions of the adoptively transferred cells. Our understanding of T-cell activation through cell surface receptors and proteins now indicates that this is a complex multistaged process of recognition, adhesion, and stimulation. In vivo, the generation of antigen-specific T cells requires the interaction of dendritic cells and naïve T cells in a secondary lymphoid organ, usually a lymph node.39

For over a half a century, immunologists have sought to understand how self-tolerance is induced and maintained. Bretscher and Cohn first proposed a two-signal model of B lymphocyte activation that was later modified by Lafferty and Cunningham for T-cell activation and allograft rejection.40 The essential features of these models were that activation of lymphocytes requires an antigen-specific signal 1 as well as a second antigen-nonspecific event termed “signal 2.” Moreover, these theories and later modifications proposed that signal 1 in the absence of the costimulatory signal 2 led to tolerance or apoptosis. Indeed, in some instances, the binding of tumor antigen presented to the T-cell receptor in the absence of costimulation not only fails to activate the cell but also leads to functional inactivation.41 Antigenic stimulation of T cells leads to at least three distinct outcomes: (a) activation, clonal expansion, and differentiation to produce cells that secrete distinct subsets of cytokines or to express lytic machinery; (b) induction of an unresponsive state termed “anergy,” and (c) induction of apoptosis.42, 43

The most appropriate methods of ex vivo T-cell activation and propagation mimic the physiologic processes whereby dendritic cells generate a constellation of antigen-specific and costimulatory signals in the T cells. Polyclonal T-cell proliferation can be induced by mimicking the antigen signal by anti–T-cell receptor antibodies or anti-CD3 antibodies.44, 45 However, anti-CD3 stimulation without the addition of IL-2 or another costimulus is not sufficient for full activation of T cells and long-term growth.46 Enhanced polyclonal T-cell activation and proliferation results when cells are stimulated via the T-cell receptor as well as the CD28 receptor. 47 This culture system has been adapted for clinical use, and starting with an initial apheresis product, it is possible to generate the total number of mature T cells found in adults within 2 weeks of ex vivo culture.48, 49 Antigen-specific T-cell proliferation can be induced by the addition of autologous dendritic cells that have been loaded with the desired antigen or by the use of tetramers to activate the T cells with the desired specificity.50, 51 Dendritic cells are most efficient for the activation of naïve T cells; however, other forms of APC may suffice for previously primed T cells. Schultze and coworkers have shown that CD40-stimulated B cells are an efficient means to propagate antigen-specific T cells.52

In addition, cell lines and beads can be engineered to create artificial APC in order to generate antigen-specific T cells and avoid the need to use autologous APC for patient-specific cultures.53 General approaches have been to produce artificial APC, either by coating beads with peptide:MHC “tetramer” complexes or by transfecting MHC-negative cells with MHC molecules and costimulatory molecules. Magnetic beads were coated with MHC class I molecules loaded with specific peptide, the beads were used as a substrate for T-cell capture, and, following isolation and expansion, the recovered cells specifically killed target cells in vitro and displayed antiviral therapeutic effects in vivo in a rodent model.54 Others have used nonmagnetic microspheres coated with complexes of recombinant MHC molecules to successfully generate CTLs ex vivo from naïve precursor cells.55 Peptide-MHC tetramers specific for the melanoma proteins MART-1 and gp100 have been used to isolate high-avidity tumor-reactive CD8+ T cells from a heterogeneous population by flow cytometry. The tetramer reactive cells could be cloned, and they retained their functional activity on reexpansion.56, 57 Sadelain and colleagues engineered APC that could be used to stimulate the T cells of any patient with a given HLA type.58 Mouse fibroblasts were retrovirally transduced with a single HLA class I molecular complex along with the human accessory molecules CD80 (B7.1), CD54 (ICAM-1), and CD58 (LFA-3). These artificial APCs consistently elicited and expanded CTLs specific for the melanoma tumor antigens gp100 and MART-1. Our approach has been to create artificial APCs based on expression of the 4-1BB ligand 59; such APCs efficiently expand human central memory CD8 T cells.60

Other T cell culture techniques have been developed to selectively activate and/or clonally expand tumor-specific T cells, with the goal of retaining in vivo antitumor reactivity, trafficking, and engraftment potential. The identification of tumor antigens has permitted the expansion of antigen-specific CTLs that can specifically lyse tumor cells. Theoretically, oligoclonal T-cell lines are preferred to CTL clones for treatment, because a more broadly directed response is likely to minimize the emergence of tumor cells that fail to express the targeted epitope. They are also less costly to develop. However, CTL clones represent a powerful tool for identifying the precise sequence of tumor or viral epitopes, and by virtue of their specificity, they are the least likely cell preparations to trigger adverse effects such as autoimmunity. Initial studies have used dendritic cells or other APC loaded with antigen to activate and expand T-cell clones or lines. The limited general availability of autologous tumor cells to serve as a source of antigen for repeated in vitro stimulation of T cells is a practical limitation for clinical therapy. This approach is also not desirable because many tumor cells secrete immunosuppressive cytokines such as TGF-β or IL-10. When tumor cells themselves are used as APCs, only MHC class I reactive T cells are usually obtained. Obtaining sufficient autologous dendritic cells for repeated pulsing of the T cells is also a practical limitation. However, this limitation may be circumvented with the advent of flt3 ligand–mediated dendritic cell mobilization protocols. APCs should be autologous or MHC-matched for propagation of tumor-specific T cells. The most widely used method currently employed for the generation of CTLs in vitro is to pulse dendritic cells with tumor-specific peptides that are presented by the appropriate MHC-restricting allele. It is critical that the correct concentration of peptide be used to pulse the dendritic cells. Berzofsky and colleagues showed that if high peptide concentrations are used in vitro, only low-avidity T cells are propagated because the high-avidity T cells die by apoptosis,61 suggesting that a submaximal concentration of peptides may be required for in vitro induction of tumor-reactive CTLs. Furthermore, CTLs generated with peptide-pulsed APCs are often peptide-reactive but not reactive with tumors that express the gene of interest due to low-level expression or impaired antigen processing by the tumor cells. To circumvent this, Greenberg and colleagues have used recombinant vaccinia virus encoding the tyrosinase gene to infect autologous APCs and have generated tyrosinase-specific and melanoma-reactive CTL cells from the peripheral blood of five out of eight patients with melanoma.62 Tyrosinase-specific CD4+ T-cell clones were isolated from six of the eight patients by stimulation with autologous APCs infected with recombinant vaccinia virus, and all of these clones were capable of recognizing autologous tumor cells.

An obstacle to adoptive T-cell therapy with antigen-specific T cells is that each T-cell culture is patient-specific and tumor-specific. Thus, if universal tumor antigens could be identified that are presented by most MHC types, are expressed and presented in most tumors, have limited expression in normal tissues, and are directly involved in the malignant phenotype of the tumor, a more widely applicable form of cellular therapy could be developed. Several candidates for universal tumor antigens have been identified, including the catalytic subunit of telomerase (hTERT), cytochrome P450 isoform 1B1, survivin, WT-1, and MDM2.

Limitations to Adoptive Cellular Therapy

The major rationale for the use of T cells is that these cells have the capacity to specifically kill tumor cells, proliferate, and persist after transfer, and therefore they could completely eliminate all residual tumor cells or newly emerging tumor cells. Mathematical modeling suggests that adoptive transfer of CTLs should augment antitumor immunity in a variety of scenarios.63 T-cell survival and replication in the host is essential for efficacy, as irradiation of adoptively transferred T cells before their transfer abrogates therapeutic efficacy in most animal models.64, 65 Factors leading to failure or suboptimal efficacy of adoptive cellular therapies can be classified as those due to intrinsic limitations of the infused cells and as immunosuppressive conditions in the tumor-bearing host (Table 33.1). Large tumor burdens present qualitative and quantitative problems for immunotherapy, and as with all therapies, cell transfer therapy has most promise when conducted in the setting of minimal residual disease. Immunologists have long observed a process termed “tumor sneaking through,” by which is meant that small tumors grow progressively, medium-sized tumors are rejected, and large ones break through again. De Boer and colleagues developed mathematical models studying tumor kinetics in the setting of adoptively transferred T cells.66 In De Boer's model, the magnitude of the cytotoxic effector cell response depends on the time at which helper T cells become activated: early helper activity steeply increases the magnitude of the immune response. Thus, tumor rejection is most favored if the tumor-specific CD4 helper T cells are induced early, as this helps to magnify the induction of CTLs. Recent studies have shown DeBoer's work to be remarkably prescient, as there is an emerging consensus that one of the primary limitations in the immune response to tumors is the failure to develop antigen-specific CD4+ helper T cells.67 Given the accumulating evidence that CD4+ T cells are critical participants in effective antitumor immune responses, a number of potential roles have been suggested. While it had long been known that CD4+ T cells provide help for the priming of CTLs,68 more recent evidence has indicated that CD4 cells are required to maintain the optimal effector function of CD8 cells throughout their lifespans.69

Another host-specific factor that can prevent therapeutic efficacy of adoptively transferred T cells is the inactivation of the T cells in the immunosuppressive environment of the host. The priming of tumor antigen–specific T cells is critical for the initiation of successful antitumor immune responses, yet the fate of such cells during tumor progression is unknown. In a lymphoma model in mice, when naive CD4+ T cells specific for an antigen expressed by tumor cells were transferred into tumor-bearing mice, transient clonal expansion occurred early after transfer. The adoptively transferred cells then developed a diminished tumor-specific response, suggesting that tolerance to tumor antigens may impose a significant barrier to therapeutic vaccination, at least in the case of tumors that express MHC class II antigens.70 T cells from patients with Hodgkin's disease have defects in activation that are reversible in vitro by stimulation with anti-CD3 and anti-CD28.71 Numerous strategies are being tested in animal models to overcome limitations to adoptive immunotherapy.72, 73, 74, 75 With the exception of homeostatic proliferation, these strategies have yet to be tested clinically.

TABLE 33.1 POTENTIAL EXPLANATIONS FOR LACK OF EFFICACY OF ADOPTIVE T-CELL THERAPY

Limitations of the infused cells
Ex vivo expanded T cell population approaches or reaches replicative senescence or loss of CD28 expression
Tumor antigen–specific T cells have been deleted or tolerized in the donor by previous chemotherapy or by the tumor itself
Ex vivo expansion of T cell population introduces failure to home to tumor or lymph nodes
Culture process renders transferred T cells immunogenic, leading to failure of sustained engraftment
Limitations in the host
Unrealistic effector to target ratio: host tumor burden exceeds killing capacity of adoptively transferred cells
Poor engraftment of adoptively transferred CTLs: lack of CD4 T cell help, cytokine “addiction”
Infertile soil: previous chemotherapy or radiotherapy has ablated stroma and lymph nodes, leading to impaired survival signals and lack of “niche”
Regulatory cells in the host kill or inactivate adoptively transferred T cells
Tumor cells kill or inactivate adoptively transferred T cells
Transferred T cells do not recognize tumor-associated antigens due to loss of tumor antigen expression
Lack of tumor costimulation induces anergy or apoptosis of adoptively transferred cells

 

ADOPTIVE CELLULAR THERAPY FOR HUMAN TUMORS

Natural Killer (NK) and Lymphokine-Activated Killer (LAK) Cell Adoptive Therapy

Unlike T cells or B cells, which recognize antigen using clonally restricted receptors generated by gene rearrangement, natural killer (NK) cells appear to use a variety of different, non-rearranging receptors to initiate cytolytic activity and cytokine production. NK cells are cytolytic for targets even in the absence of MHC class I expression, and inhibitory receptors expressing immunoreceptor tyrosine-based inhibition (ITIM) motifs prevent NK cells from harming tissues expressing normal levels of MHC class I. This latter characteristic, the ability to cause non-MHC–restricted lysis, is a major feature of NK cells that distinguishes them from T cells. Many receptors have been implicated in NK cell activation, including NKG2D, CD94/NKG2C, NKR-P1, CD2, and CD16.76 NK cells, unlike T cells, do not have an extensive replicative potential, perhaps due to the fact that telomerase is expressed at much lower levels in NK cells than in T cells.77 Tumors may shed soluble ligands for NK receptors as a means of promoting immune evasion.78

Human lymphocytes that mediate non-MHC–restricted cytotoxicity can be divided into multiple subpopulations. The physiologic basis for this is the differential expression of multiple killer receptors at the single cell level. For example, T cells develop NK-like cytotoxicity after activation with anti-CD3 or cytokines, and the most likely explanation for this phenomenon is the de novo expression of one or more NK receptors. The ability of NK and LAK cells to kill a variety of tumor cell targets in vitro made them attractive candidates for adoptive cellular therapy. This T-cell receptor-independent form of cellular cytotoxicity was originally reported to spare normal tissues; however, autologous human lymphocytes and cultured normal human kidney cells can be killed by LAK cells.79, 80Another major drawback of NK and LAK cells is their relative inability to traffic to the tumor.

The availability of recombinant IL-2 permitted the first clinical trials of adoptively transferred autologous NK cells.81 An extensive series of trials has demonstrated clinical responses in a minority of patients, particularly those with melanoma and renal cell carcinoma.82, 83 Randomized studies at the National Cancer Institute with LAK cells have failed to show clinical efficacy.84 Subsequent analysis has shown that the IL-2 that was administered concomitant with the LAK cells accounted for the majority of the clinical responses that were observed. Human MHC-unrestricted cytotoxic NK cell leukemia lines have been shown to display antitumor effects and can be grown ex vivo for clinical use.85 For example, the xenogeneic adoptive transfer of human TALL-104 killer cells into a dog with metastatic mammary adenocarcinoma resulted in a 50% reduction of the largest lung metastasis and stabilization of the other lesions for 10 weeks, accompanied by the development of tumor-specific immune responses.86 A phase I trial with TALL-104 cells in patients with metastatic breast carcinoma demonstrated safety and showed some indications of antitumor efficacy.87

At present, adoptive therapy with NK cells has been largely abandoned. However, it is likely that variations of this form of therapy will be explored again, given that adoptively transferred T cells can not kill MHC class I negative tumors and also given that T-cell therapy will likely select for tumor cell loss variants of this phenotype. Furthermore, NK cells express both activating and inhibitory receptors. The same NK cell can express both inhibitory and activating receptors. Because of the expression of inhibitory receptors (KIRs) for certain major histocompatibility complex (MHC) class I allotypes, a person's NK cells will not recognize and will therefore kill cells from individuals lacking their own KIR epitopes. Recently “alloreactive” NK cells were shown to mediate antileukemic effects against acute myeloid leukemia after mismatched transplantation when KIR ligand incompatibility existed in the direction of graft versus host disease (GVHD) (i.e., MHC class I KIR ligand that is absent in the recipient but present in the donor).88 This beneficial antitumor effect does not appear to occur against acute lymphoblastic leukemia. Alloreactive NK cells are cytotoxic for melanoma and renal cell cancer cells in vitro,89 suggesting that HLA-mismatched hematopoietic stem cell transplantation may be a setting to exploit NK cell adoptive therapies for patients with solid tumors. Once the complexity of the NK system is better understood, it is likely that clinical trials using combinations of tumor-specific T cells and NK cells will be done.90, 91

Adoptive T-Cell Therapy

The principles of adoptive immunotherapy established in animal models have formed the basis for the testing of therapeutic strategies for human tumors. The primary attraction of the use of T cells for adoptive therapy is their ability to specifically target tumor cells that express small peptides, even if the intact target protein itself is not expressed on the cell surface. A second attraction is the potentially long clonal lifespan of T cells. Finally, unlike NK and LAK cells, adoptively transferred human T cells have been shown to traffic to tumor.92

Autologous T-Cell Therapy

In mice, nearly all successful immunotherapies have required the use of large numbers of T cells derived from multiple immunized syngeneic animals. In humans, it is not possible to use this approach, and therefore a central issue for the development of clinical adoptive immunotherapy strategies has been the development of culture systems that produce adequate numbers of effector T cells. Two basic approaches are being tested (Fig. 33.1). In one case, polyclonal ex vivo activation of the T cells is done, based on results from mouse syngeneic tumor models.44, 93 This approach is based on the assumptions that tumor-specific T cells are present in the patient but that they have not been primed in the patient and/or that the in vivo function of the cells in the patient is impaired. The cells are activated polyclonally by various means in vitro and are then reinfused to the patient in the hope that they will now respond directly to tumor or to tumor antigen presented by APCs in the patient. The second approach is to isolate and activate antigen-specific T cells or tumor-infiltrating lymphocytes (TILs) in vitro and then clonally expand antigen-specific cells in vitro by various approaches.

Figure 33.1 General approaches for ex vivo T cell expansion. The initial T cells are obtained from peripheral blood, TIL, or draining lymph nodes. The starting T-cell repertoire can be expanded by polyclonal stimulation via CD3/CD28 stimulation or other methods to generate cells with enhanced effector function or to maintain the TCR repertoire of the initial population (left). Antigen-specific CTLs can be generated without prior selection or enrichment by repeated stimulation with antigen-pulsed antigen-presenting cells (APC) or tumor cells. This process usually requires several rounds of stimulation (middle). Selection of CTLs via tetramers can improve the efficiency of antigen-specific T-cell generation, and several methods can be used for ex vivo expansion of these antigen-specific cells (right). At least 27 cell divisions are required from a single precursor T cell in order to generate 1 billion clonal T cells.

As to the first approach, one of the first human trials of activated autologous polyclonal T cell transfers was done by Mazumder and colleagues at the National Cancer Institute.94 They and others had shown that the in vitro activation of T cells from cancer patients with the lectin phytohemagglutinin (PHA) generated cells that were lytic for fresh autologous tumor. In a phase I clinical protocol, 10 patients with late-stage cancers were given repeated infusions of up to 1011autologous T cells after in vitro culture in PHA for 2 days. Ten patients were treated, and the toxicities encountered included fever and chills in 10 of 10 patients, headaches in 5 of 10, and nausea and vomiting in 3 and 10. No tumor regressions were seen.

Investigators have used mouse tumor models to show that antibodies that bind to the CD3 complex can mimic antigen, and even though all T cells are activated ex vivo nonspecifically, it has been demonstrated that subsequent specific antitumor responses can be enhanced. For example, tumor-specific T cells from the spleens of mice immunized with the FBL-3 leukemic cell line could be expanded in number in vitro by culture with anti-CD3 and IL-2.44 In a related approach, Osband and colleagues developed a technique termed “autolymphocyte therapy for activating human T cells ex vivo.” Peripheral blood mononuclear cells are activated ex vivo for 5 days by low doses of the mitogenic monoclonal antibody OKT3 in conditioned medium, a mixture of previously prepared culture supernatant that contains autologous cytokines in the presence of cimetidine and indomethacin.95

In a phase I trial in patients with advanced cancer, Curti and coworkers tested autologous adoptive transfers of T cells activated with anti-CD3 ex vivo for 4 days.96 They showed that the anti-CD3–activated CD4+ T cells could traffic to tumor sites in vivo and mediate antitumor effects. Of four lymphoma patients in this study, three had tumor regressions, one of which was a complete response. Patients in this trial were given IL-2 infusions following the T-cell infusion, so that it is not possible to distinguish the relative contributions of the adoptively transferred T cells and the IL-2 to the clinical responses. Repeated ex vivo stimulation with anti-CD3 may cause cell death in vitro and preferential expansion of CD8+ T cells.97 Using a related approach, based on the idea that anti-CD3 and anti-CD28 can more efficiently activate T cells ex vivo46, 47 and that tumor-draining lymph node cells cultured with anti-CD3 and anti-CD28 can mediate antitumor effects in mice,98 encouraging results have been observed in several phase I trials in which the adoptive transfer of anti-CD3–activated and anti-CD28–activated autologous T cells was performed in patients with refractory lymphoma, chronic lymphocytic leukemia, myeloma, and renal cell cancer.99,100, 101 This T-cell culture process has been scaled up for clinical testing48 and commercial development.102

In the second general approach—activation and expansion of tumor-specific T cells ex vivo—infusions of TILs have been tested most extensively. This approach is based on the hypothesis that tumor-specific T cells will be preferentially present in the resected tumor specimens.103 TIL reactivity to tumor is enhanced compared to unselected peripheral blood T cells, and the antitumor response is generally MHC class I restricted. Furthermore, the TILs are relatively more specific to the tumor of origin. TILs are generated by in vitro culture of dissociated tumor cell preparations in the presence of high concentrations of IL-2. In mice, TILs are 50- to 100-fold more potent than NK cells in several tumor models.104TILs have proven to be very useful for the identification of tumor antigens.105 The failure to consistently generate TILs from tumor specimens has limited widespread clinical application of the approach.106 Furthermore, clinical studies with TILs have shown poor engraftment efficiencies in patients.92 Gene-marked TILs have been infused in patients with melanoma and renal cell carcinoma, and selective trafficking of the TILs to the tumor site could not be demonstrated.107, 108 It is likely that the poor clinical results initially obtained with TILs were in part due to the prolonged 4- to 6-week in vitro culture time, which leads to replicative senescence and loss of homing abilities in the TILs. It is also possible that TIL trials have been disappointing because the populations of TIL cells infused also contain regulatory T cells that inhibit the antitumor response.109, 110 Finally, it is possible that the TIL populations that were tested have only contained effector cells and that the response is limited by a lack of tumor-specific T helper and central memory cells.111

Shu and Chang have developed an approach that should circumvent many of the limitations posed by TIL therapy. They and others have shown in mice that tumor-draining lymph nodes harbor T cells that are not capable of mediating tumor rejection in adoptive transfer experiments. In contrast, if the draining lymph node cells are activated in vitro with anti-CD3 and IL-2, the cells are now capable of mediating tumor rejection after adoptive transfer.8 They have shown that for mouse T cells optimal generation of effector T cells occurs when anti-CD3 is added to the culture for the first 2 days and IL-2 is added subsequently on days 3 to 5 of culture.93 In further studies, T cells were isolated from vaccine-primed lymph nodes obtained from patients with melanoma, renal cell, and head and neck cancer. In the absence of antigen-presenting cells, activation with anti-CD3 and anti-CD28 greatly enhanced subsequent T-cell expansion in IL-2 (>100-fold), compared with anti-CD3 alone.112 Thus, these results define conditions in which tumor-draining lymph node cells could be stimulated in the absence of tumor antigen to develop into specific therapeutic effector cells during an abbreviated period of cell culture. Based on these preclinical studies, Chang and coworkers carried out a phase I trial in patients with late-stage melanoma and renal cell carcinoma.113 Patients were given intradermal vaccination with irradiated autologous tumor cells and bacille Calmette-Guérin (BCG) as an adjuvant. The draining lymph nodes were harvested 7 to 10 days later, and the vaccine-primed T cells cultured with anti-CD3 and IL-2. Among the 11 melanoma patients, 1 had a partial tumor response, and there were two complete and two partial responses among the 12 patients with renal cell carcinoma. Thus there may be some clinical activity with this approach in patients with metastatic renal cell carcinoma. One potential limitation of this approach is that the infused T cells may be immunogenic with this particular culture process, as they will contain the murine anti-CD3 OKT3 antibody that is bound to the T cells.

The clinical utility of tumor-specific CTLs has not yet been extensively evaluated. In one study, CTLs were induced in vitro by repeated stimulation with inactivated autologous tumor cells, and the CTL lines were administered intravenously to 11 patients with advanced cancers once every 2 weeks for 10 weeks. The cell infusions were not toxic, and tumor reduction or decreased tumor markers were observed in 4 patients.114 In another study, autologous CTLs were generated against primary-cultured malignant gliomas from peripheral blood mononuclear cells in vitro in 4 patients.115 The CTLs specifically recognized the corresponding autologous glioma in vitro. The CTLs were injected 3 times into the primary tumor–resected cavity via an Ommaya tube, and reduction of the tumor volume was observed in 3 of the 4 patients. These results suggest that adoptive immunotherapy with autologous CTLs may be a promising approach for malignant gliomas; however, further testing will be required to establish whether there is clinical benefit.

Tumor-specific CTLs generated ex vivo with the rapid expansion method116 appear to have substantial activity in melanoma, as 8 of 10 patients with refractory, metastatic melanoma had minor, mixed, or stable responses.117 However, recent studies at the NCI indicate that host conditioning can increase the response to adoptive immunotherapy with TILs. When 13 patients with progressive metastatic melanoma were given cyclophosphamide and fludarabine, a regimen that is immunosuppressive but does not have antimelanoma efficacy, 6 patients had partial responses as judged by RECIST criteria, and 4 others had mixed responses.31 Significantly, the patients had prolonged engraftment with the adoptively transferred TILS, and the levels of engraftment correlated with the clinical responses.31, 118 In contrast, 34% of patients with melanoma who were treated with TIL administration and high-dose IL-2 therapy and who received no prior conditioning therapy to induce lymphodepletion achieved objective clinical responses119; most of the responses were transient, and the patients had limited persistence of the transferred cells.92 Adverse effects included opportunistic infections and the frequent induction of vitiligo and uveitis, presumably due to autoimmunity. If confirmed, these results indicate that induction of immunosuppression in the host is essential to improve the antitumor efficacy of adoptive immunotherapy.

Virally Induced Lymphomas

Virally induced lymphomas that retain some expression of the inciting viral genome are likely to present a good target for adoptive cellular therapy. Unlike in the case of most spontaneous tumors, the repertoire of T-cell receptors contains receptors with a high affinity for the viral protein as a consequence of the lack of deletion of these T cells in the thymus. In patients recovering from allogeneic bone marrow transplantation, a severe defect in the cellular immune system exists, and this often results in the death of the patients from reactivation of systemic CMV or EBV infection. Donor-derived, CMV antigen–specific CD8+ CTLs have been administered to the patients, and an extremely promising restoration of immune function has been noted.120, 121 EBV often reactivates after bone marrow or organ transplantation, resulting in aggressive lymphoma. Infusions of allogeneic peripheral blood T cells (DLI) have proved effective, as have infusions of EBV-specific T-cell lines. The latter approach has the benefit of reduced risk of inducing or exacerbating GVHD, since the T cells with allospecificity are eliminated or greatly reduced before infusion. In vitro cultured T-cell lines or clones that recognize viral antigens can be effective in suppressing EBV-associated lymphoproliferative disorders. Even relatively modest doses of T cells (1 × 106 cells/kg) are an effective treatment or prophylaxis for EBV-associated lymphoma, with complete remissions recorded in most patients.122, 123 Pneumonitis and tumor swelling with respiratory obstruction have been reported as adverse events following CTL infusion for lymphoma.123 EBV-specific CTLs are safe and have significant antitumor activity in Hodgkin's disease and nasopharyngeal carcinoma associated with EBV infection.124, 125

A single patient with an aggressive EBV-associated lymphoma treated with adoptive immunotherapy using autologous LAK cells was reported.126 The patient had leukapheresis, autologous peripheral blood mononuclear cells were cultured in IL-2 for 10 days, and the IL-2–activated LAK cells were returned to the patient in the absence of systemic IL-2 therapy. The patient experienced a complete response.

Donor Lymphocyte Infusions and Allogeneic T-Cell Therapy

The first form of human adoptive T-cell therapy was given “inadvertently” as passenger T cells contained in stem cell infusions from bone marrow harvests. The bone marrow infusions were given to patients receiving allogeneic marrow grafts using myeloablative regimens as therapy for leukemia. At the time, the T cells were regarded as “contaminants” of the stem cell grafts. Weiden and coworkers performed a retrospective analysis of a series of patients treated with total body irradiation and high-dose cyclophosphamide,127 and to their surprise they discovered that the probability of tumor recurrence was significantly lower in patients receiving allografts than in those who had syngeneic (twin) grafts. Later studies showed that the probability of tumor recurrence was inversely related to the occurrence of GVHD. Resting donor peripheral blood T cells are now given routinely to patients with chronic myeloid leukemia who relapse following a marrow allograft, and this procedure results in the induction of molecular complete remissions in a high proportion of cases.128, 129, 130, 131This form of therapy is now termed “donor lymphocyte infusion” (DLI).

The mechanisms of DLI-mediated antitumor effects are not yet well understood. It is likely that T cells and/or dendritic cells are involved and that the antigenic targets are minor histocompatibility antigens or leukemia-specific antigens. It is noteworthy that the kinetics of the clinical response are delayed, as clinical response following DLI takes weeks to months, and often 6 to 8 months are required for maximal antileukemic effects; these kinetics are typical of an acquired immune response that is mediated by T cells. Recently, Falkenburg and coworkers used T-cell leukemia-reactive CTL lines generated from a patient's HLA-identical donor to induce a complete remission in a patient with chronic myelogenous leukemia (CML).132 The CTLs did not react with normal lymphocytes from the donor or recipient and did not affect donor hematopoietic progenitor cells. The CTL lines were infused at 5-week intervals at a cumulative dose of 3.2 × 109 CTLs, and following the third infusion, a complete eradication of the leukemic cells was observed. The interpretation of the clinical benefit in this experiment is difficult, since the patient had been given previous DLI that was terminated due to failure to induce a response and the onset of GVHD. If the clinical response that was eventually observed was in fact due to the infused CTLs, then it is likely that the CTLs engrafted and proliferated in vitro. This is due to the fact that the initial effector to target ratio was only ~1:1,000 in vivo immediately after the CTL infusion, given the patient's estimated leukemic burden of 1 × 1012 to 3 × 1012 cells.

One critical issue with DLI is that it often results in the induction of chronic GVHD. Under some experimental conditions in transgenic mice, high-avidity T cells reactive for tumor antigens as well as self antigens are deleted, and the remaining low-avidity T cells appear to be sufficient to provide protection against subsequent tumor challenge but do not suffice to provoke autoimmunity.133 This may explain why clinically evident GVHD does not appear necessary for clinically evident antitumor effects. However, clinical trials are evaluating whether depletion of CD8+ T cells from the adoptively transferred cell population might reduce GVHD while retaining the antileukemic effects.134, 135 Given the early results of these trials, it is likely that CD8+ T lymphocytes are important as effectors of GVHD but may not be essential for the DLI-mediated antitumor effect. Another limitation of DLI is that, though it is very effective for CML, it is much less effective for other forms of leukemia, and it is not yet known if DLI will have a role in treating solid tumors. Limited but impressive data indicate that potent allogeneic antitumor effects can be observed in selected patients with renal cell carcinoma, ovarian cancer, and breast cancer.136, 137, 138, 139

It is possible that the antitumor effects of donor leukocytes can be used for therapeutic benefit outside the setting of allogeneic transplantation. Xenogeneic and allogeneic adoptive T-cell therapy has the obvious advantage that the in vivo antitumor effect is not dependent on the condition that all tumor cells express tumor-specific antigens, since alloantigens or minor histocompatibility antigens can serve as targets. Furthermore, the donor repertoire has not been contracted by previous chemotherapy, and therefore it is more likely to contain naïve T cells reactive with tumor antigens. Xenogeneic adoptive lymphocyte transfers have been done in humans by Symes and coworkers.140 Pigs were immunized with transitional cell carcinoma fragments, and later the draining porcine lymph node cells were harvested and injected into patients with bladder carcinoma. Two of seven patients had objective responses, and no significant toxicity was observed, in part because of the rapid rejection of the porcine allograft that likely occurred.

Allogeneic MHC-mismatched human lymphocytes have also been transferred to patients in a variety of settings without the myeloablative or immunosuppressive conditioning required to permit engraftment. As can be seen in Figure 33.2, the general design of some of these early trials was remarkably similar to current approaches. In spite of the limitations of these early trials due to the failure to obtain engraftment of the adoptively transferred cells, objective tumor regressions were observed when patients were given infusions of allogeneic MHC-mismatched lymphocytes from donors immunized with the recipient's tumor.141, 142 However, in the absence of MHC matching, it is unlikely that allogeneic or xenogeneic T cells can mediate tumor-specific responses, and it is possible that the responses observed in these early trials were due to NK cells.

Terasaki and coworkers performed the first adoptive transfers of HLA-matched allogeneic lymphocytes and observed objective responses in two of six patients with advanced cancers.143 Haploidentical lymphocytes have been given in conjunction with cyclophosphamide to a small group of patients with various tumors as primary therapy without inducing GVHD.144 Six patients received infusions of lymphocytes after alloactivation and expansion in vitro, and this resulted in a complete response in one patient with lymphoma. More recently, MHC-matched allogeneic DLI has been given to patients without minimal immunosuppressive conditioning145, 146; remission has been induced in a significant fraction of patients with refractory hematologic malignancies. Slavin and coworkers have obtained similar results in patients with breast cancer given allogeneic lymphocyte infusions following autologous stem cell transplants.147 The main limitations of these approaches are the generally short-term engraftment, the suboptimal response rate, and the unpredictable onset of GVHD in a subset of patients.

In order for the full antitumor potential of allogeneic T cells to be realized, it is necessary to achieve long-term donor engraftment. Previously, this was only possible in young patients who could survive the rigorous myeloablative protocols. This fact largely precluded the use of allografting for patients older than 55 years or for younger patients with certain kinds of preexisting organ damage. A major step toward decreasing the rigors of allogeneic stem cell transplants occurred with the development of nonmyeloablative stem cell transplantation (NMSCT), a procedure where the preparative regimen is designed only to provide sufficient immunosuppression to achieve engraftment of an allogeneic stem cell graft.148, 149 The nonmyeloablative regimen does not by itself completely eliminate residual host hematopoietic cells, but rather the allogeneic T cells over the period of weeks to months may either maintain partial hematopoietic chimerism or eliminate all residual host elements and achieve full donor chimerism. Barrett and coworkers have studied the kinetics of engraftment in patients receiving NMSCT consisting an allogeneic peripheral blood stem cell transplant from an HLA-matched donor after a preparative regimen of cyclophosphamide and fludarabine.150 Donor myeloid chimerism gradually supplanted recipient hematopoiesis, and the myeloid compartment became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism occurring in some patients by day 30 and in other patients by day 200 after cyclosporine withdrawal and DLI. Ten of 14 patients surviving more than 30 days had delayed tumor regression, consistent with a T cell–mediated tumor rejection.

Figure 33.2 A clinical adoptive transfer approach used by Nadler and Moore in the 1960s.141 MHC unrelated patients with cancer were immunized with viable tumors by subcutaneous injection. The allogeneic donor was then subjected to leukapheresis, and the buffy coat given as a form of donor leukocyte infusion (DLI) to another cancer-bearing patient. (Reproduced with permission from Nadler SH, Moore GE. Immunotherapy of malignant disease. Arch Surg 1969;99:376.) This approach is not endorsed by the author or by the editors, and this experiment is illustrated simply to indicate the general similarity to currently ongoing clinical trials.

An unanticipated benefit of NMSCT is that the incidence of acute GVHD is markedly decreased, permitting allogeneic marrow grafts to be done in much older patients. This is likely due to the absence of chemotherapy-induced mucositis, which leads to the secretion of cytokines, the generation of other danger signals, and the subsequent activation of allogeneic donor T cells. Another attractive feature of NMSCT is that adoptive cell transfers can be given to patients later, once the graft has been established, creating an ideal platform for adoptive immunotherapy with allogeneic T cells and dendritic cells. Now that older patients can undergo allogeneic adoptive transfers, it will be possible for the first time to determine the efficacy of this approach in patients with the common solid tumors that occur in this age group. Thus, it is likely that a resurgence of interest in allogeneic adoptive cell therapy will occur due to the advent of NMSCT. NMSCT provides a platform to minimize acute GVHD and to circumvent the difficulties with autologous T cell therapy in patients who have had previous repertoire contractions due to chemotherapy.

RANDOMIZED CLINICAL TRIALS

A number of randomized controlled clinical trials testing the efficacy of adoptively transferred cells have been reported (Table 33.2). The first tumors in humans treated with adoptive immunotherapy to be subjected to randomized controlled trials were melanoma and renal cell carcinoma. Renal cell carcinoma and melanoma are relatively highly immunogenic tumors that have proven resistant to standard cytotoxic chemotherapy but have shown reproducible responses to immune-based therapy. Infusions of NK cells, LAK cells isolated from peripheral blood, and polyclonal T cell populations isolated from TILs and nonspecifically expanded in vitro with IL-2 suggested the therapeutic potential of tumor-reactive T cells in humans. However, the low response rates and the severe toxicity due to the high doses of IL-2 injected to maintain cell survival dampened the early enthusiasm. Indeed, randomized studies using this approach have not demonstrated efficacy of the adoptively transferred cell populations. The responses observed can be attributed to cytokine-mediated antitumor effects alone. Similarly, positive effects in randomized trials of patients with advanced renal cell carcinoma treated with anti-CD3–activated peripheral blood mononuclear cells cultured in conditioned medium were also reported;151 however, a larger, multicenter phase III trial failed to confirm the earlier studies. While further trials are ongoing, thus far these approaches have not consistently shown benefit in comparison with standard immune-based treatment with biologic response modifiers, most importantly, high-dose bolus IL-2. In a randomized phase I trial by Fenton and colleagues at the NCI,152 more than half of the patients with advanced renal cell carcinoma entering the trial were found to be anergic to recall antigens, confirming other studies indicating that patients with late-stage tumors can be significantly immunosuppressed and that this may present a significant barrier to overcome if immunotherapy is to be successful. For reasons that remain unclear and may reflect the lack of immunosurveillance and trafficking to the eye, some investigators have reported that metastatic ocular melanoma is much less responsive than cutaneous melanoma to adoptive cellular transfers.153 It is likely that other approaches will be required to establish the usefulness of adoptive immunotherapy in melanoma and renal cell carcinoma, but its promise for these difficult diseases is already evident.

Randomized trials suggest that other tumors may also be responsive to immunotherapy. A single randomized trial of adoptive immunotherapy with TIL and IL-2 infusions indicated a survival advantage for patients with non–small cell lung carcinoma, particularly those with stage IIIB.154 Similarly, in another randomized trial of patients with non–small cell lung carcinoma, LAK cell infusions plus IL-2 therapy in combination with standard therapy was shown to be superior to standard therapy alone.155 In both of these trials, it was not possible to discern if the adoptively transferred cells contributed to the beneficial effects due to the confounding effects of the concomitant IL-2 infusions. Finally, patients with hepatoma may have immunogenic tumors that are targets for adoptively transferred cells.156 Although some intriguing clinical results have been noted, no published randomized controlled trials have convincingly demonstrated clinical benefit from adoptively transferred cells. Currently, adoptive immunotherapy does not represent the standard of care for any disease except for patients with chronic myeloid leukemia who relapse following an allogeneic marrow grafting procedure.

ADOPTIVE CELLULAR THERAPY: TOXICITY, DOSE, AND SCHEDULING ISSUES

Information on the dose and schedule dependence of adoptively transferred cells is widely scattered in the literature, and from this literature one concludes that there is no standardized dose system. In nonlymphopenic hosts, fractionated doses of adoptively transferred T cells are superior to a single infusion of T cells.157 The ideal dose of transferred cells is related to the tumor burden and the homing and persistence (memory) characteristics of the infused cells.158 Doses of adoptively transferred cells are usually reported as the total number of viable cells administered or as the total number of viable cells per kilogram body weight or per square meter body surface area. However, total lymphocyte numbers do not correlate well with body surface area but rather display a stronger inverse correlation with age. Other variables add to the complexity, particularly the fact that, in the case of T cells or other adoptively transferred cells with high replicative potential, the infused dose may not relate well to the steady-state number of cells. Therefore, dose considerations are more complex than in other areas of transfusion medicine, where, for example, the maximal level of transfused red cells or platelets occurs immediately following infusion. In our studies of adoptively transferred autologous CD4+ T cells, we often find that the highest number of cells in the host peaks 2 weeks after infusion of the cells. This is because the engraftment potential and the replicative potential of the infused cells depends on complex host variables such as the number of niches available in the host for engraftment and the antigenic stimulus for clonal expansion or deletion. In most rodent tumor models, T-cell proliferation in the host after transfer is obligatory for therapeutic efficacy (reviewed in ref. 5).

TABLE 33.2 RANDOMIZED CLINICAL TRIALS OF ADOPTIVE IMMUNOTHERAPY

Indication

Description

Reference

Metastatic renal cell cancer and melanoma

Patients were randomly assigned to receive either systemic IL-2 alone or IL-2 plus adoptively transferred lymphokine-activated killer cells. Twenty-three patients with renal cell carcinoma and 20 with melanoma were entered into the protocol. No objective responses were noted.

193

Metastatic renal cell cancer and melanoma

Patients were randomly assigned to receive either systemic IL-2 alone or IL-2 plus adoptively transferred lymphokine-activated killer cells. The results suggested a trend toward increased survival when IL-2 was given with LAK cells in patients with melanoma (P = .09), but no trend was observed for patients with renal cell cancer.

84

Metastatic renal cell cancer

Adoptive autologous polyclonal T therapy after activation with anti-CD3 and conditioned medium. Ninety patients with metastatic renal cell carcinoma were randomly assigned to receive 6 monthly infusions of autologous activated peripheral blood lymphocytes plus oral cimetidine or cimetidine alone. Positive results were reported, but later trials did not confirm the initial results.

151, 194

Advanced renal cell cancer

A randomized phase III trial compared continuous intravenous infusion IL-2 alone with IL-2 plus LAK cell adoptive transfers. Seventy-one patients were treated, 36 on the IL-2 arm and 35 on the IL-2 plus LAK arm. Four patients (6%) had major responses (2 complete, 2 partial). The addition of LAK cells did not improve the response rate (P = .61) or survival (P = .67).

195

Advanced renal cell cancer

A randomized phase I trial was done to determine whether subcutaneous administration of IL-2 in combination with an autologous renal cell vaccine is feasible and can augment tumor immunity. Seventeen patients with metastatic renal cell carcinoma underwent surgical resection with preparation of an autologous tumor cell vaccine. Patients were vaccinated intradermally twice with 10e7 irradiated tumor cells plus bacille Calmette-Guerin and once with 10e7 tumor cells alone. Patients were randomly assigned to one of three groups: no adjuvant IL-2, low-dose IL-2, or high-dose IL-2. Four patients developed cellular immunity specific for autologous tumor cells as measured by DTH responses. IL-2 did not have a major effect on the DTH response. Prospective testing of response to recall antigens indicated that 7 of 12 tested patients were anergic.

152

Metastatic renal cell cancer

A multicenter randomized trial to test the efficacy of TILs in combination with low-dose IL-2 compared with IL-2 alone after radical nephrectomy in 178 patients with metastatic renal cell carcinoma was done. Intent-to-treat analysis demonstrated objective response rates of 9.9% vs. 11.4% and 1-year survival rates of 55% vs. 47% in the TIL/rIL-2 and rIL-2 control groups, respectively. Treatment with TILs did not improve response rate or survival in patients treated with low-dose rIL-2 after nephrectomy.

196

Myeloma

The primary objective of this study was to determine whether T-cell infusions could accelerate lymphocyte reconstitution and restore the ability to immunize patients after high-dose chemotherapy and autologous stem cell rescue. The randomized primary endpoint data show that the groups of patients on day 42 after transplant who were assigned to early (day 14) T-cell infusions had significantly higher CD4 counts than the group assigned to delayed T-cell infusions and that the antipneumococcal humoral and cellular vaccine response to Prevnar (protein-conjugated pneumococcal polysaccharide) was augmented in recipients of adoptively transferred T cells.

197

Hepatocellular carcinoma

Patients were treated with spleen-derived LAK cells cultured for 3 to 30 days in recombinant interleukin-2. These autologous activated spleen cells were administered to patients 2 days after the intraarterial infusion of doxorubicin. Patients randomly assigned to receive splenic LAK cells had a lower recurrence rate that did not reach statistical significance.

156

Non–small cell lung cancer

A phase II randomized study tested the efficacy of TIL plus IL2 infusions in 113 patients with stage II, IIIa, or IIIb NSCLC. Three-year survival was significantly better for patients given TIL therapy than for controls.

154

Non–small cell lung cancer

One hundred and seventy-four patients in a phase III trial were randomly assigned to receive either combined immunotherapy with IL-2 and LAK cells or no adjuvant therapy, or radiotherapy or chemotherapy. There were statistically improved survival rates in the immunotherapy group; however, it is difficult to interpret the results due to changes in study design that occurred during the trial.

155, 198, 199

Cytokines given to the host can also have major impact on the persistence of adoptively transferred T cells. Others have found that the persistence of adoptively transferred CD8+ T cells is enhanced by coadministration of IL-2;159 however, we have found that when autologous human CD4+ T cells are also given, that persistence is not increased by concomitant IL-2 therapy.160 Finally, IL-2 can induce proliferation and maintain effector CD8+ T cells but may actually delete memory cells, while IL-15 and IL-7 appear to select for the persistence of memory CD8+ T cells.19 Thus, it may be desirable to provide IL-2 at early times in immunotherapy when tumor cytoreduction is the issue but to remove IL-2 and/or provide IL-7 and IL-15 signaling later in therapy in order to promote antitumor memory.

Immunotherapy has often been advertised as a “nontoxic therapy” when compared with cytotoxic chemotherapy. However, it is instructive to recall that William Coley's first therapeutic vaccines were accompanied by life-threatening toxicity.1 Furthermore, anyone who has experienced the fatigue and malaise that accompanies systemic viral infections such as infectious mononucleosis would not be surprised to learn that cellular therapies have many of the same toxicities. Thus, one would expect that, in the setting of therapeutic immunization to treat established malignancy, systemic toxicity will be an expected response and that prophylactic immunization strategies in cases with no or minimal residual disease would be accompanied by less toxicity.

Many types of adverse events have been reported following infusion of human autologous or allogeneic lymphocytes or dendritic cells. The toxicities can be classified as (1) those due to extrinsic factors present in the culture process, (2) those due to accompanying cytokines that may be coinfused with the cells, and (3) those that are intrinsic to the cells themselves. With regard to the first type of toxicity, with the earlier cell manufacturing techniques many cell products were cultured in sources of foreign proteins such as fetal calf serum. In such cases, patients often developed febrile transfusion reactions that were sometimes severe and could include anaphylaxis. These reactions were usually encountered in cases of multiple cell infusions to the same patient, but instances have occurred where patients were presensitized to bovine proteins, and the patients have had severe reactions even on the occasion of the first cellular infusion. Hepatitis A has been reported due to the contamination of the culture medium with infectious pooled human serum.84 With the widespread use of serum-free culture medium, a substantial reduction has been seen in the incidence of immediate-type allergic responses, febrile transfusion reactions, and infectious complications. Similarly, with the development of closed cell culturing and manufacturing processes, the potential for microbial contamination has been substantially decreased.161

Many patients have been given infusions of IL-2 at the time of and following cellular adoptive transfer. IL-2 has a well-known dose-dependent and schedule-dependent toxicity. IL-2 given in high doses and by intravenous bolus injection induces multiorgan dysfunction due to a capillary leak syndrome that is directly mediated by local production of nitric oxide by cells of the monocyte-macrophage lineage. In contrast, low doses and subcutaneous injections of IL-2 induce an influenza-like syndrome, and this can be ameliorated by giving the IL-2 at night. Laboratory abnormalities induced at high and low doses of IL-2 include anemia, lymphopenia with rebound lymphocytosis, and eosinophilia.162

The spectrum of adverse effects intrinsic to the cellular therapy is still being defined and appears to be related to whether the cell product is genetically engineered. For cell products that have not been genetically engineered, the adverse effects are limited and are similar to those observed with therapeutic vaccines. Respiratory obstruction has been reported following CTL infusion for EBV-related lymphomas.123 This is probably due to a T cell–induced inflammatory response that results in tumor edema and necrosis. T cells infused under conditions that lead to long-term engraftment produce only mild toxicities. In over 200 infusions of autologous activated CD4+ and CD8+ T cells given to patients with lymphoma or HIV infection in the absence of concomitant IL-2 infusions, we have observed a dose-dependent induction of fever and headaches in a substantial proportion of patients. These symptoms are self-limited, and they typically resolve within 36 hours following the infusion. The onset of the symptoms is delayed and does not occur immediately upon infusion of the cells but rather several hours following the infusion. The etiology of the symptoms is likely related to secretion of cytokines by the infused cells. These symptoms are not due to allergy to the infused cells, because subsequent infusions of cells do not engender more severe adverse effects. Modest eosinophilia occurs in some patients, and this is likely related to an indirect effect of secretion of IL-2 by the infused T cells. As was mentioned previously, eosinophilia occurs in patients treated with systemic IL-2.

Effector functions of infused T cells can be expected to include tissue damage similar to that encountered in T-cell mediated autoimmune diseases. In the case of allogeneic lymphocyte infusions (DLIs), GVHD and marrow aplasia often occur.128 Autoimmune thyroiditis with hypothyroidism has been reported to occur following LAK cell and IL-2 infusions.163 The passive transfer of antibodies with shared specificities between normal and malignant tissues can also induce autoimmune pathology.164 Theoretic toxicities associated with T-cell transfer also include leukemia or lymphoma if transformation is induced consequent to the in vitro culture process. T-cell lymphomas have developed in nonhuman primates following transplantation with gene-modified stem cells.165 The etiology of the lymphomas appears to be due to insertional mutagenesis from the presence of replication-competent retrovirus that was generated from recombination from the viral vector that was used to transduce the stem cells. T-cell leukemia due to insertional mutagenesis in the LMO-2 oncogene has occurred in children following retrovirally modified CD34 cell infusion to correct common gamma chain deficiency.166 In human trials involving genetically modified T cells, no cases of malignant transformation of the infused T cells have been reported to date.

Schedule-dependent efficacy and adverse effects from adoptively transferred cells have been reported. Many studies in rodent tumor models show that the administration of cytotoxic therapy can enhance the effects of adoptively transferred cells. Cyclophosphamide is the preferred drug, and the mechanism is not thought to be consequent to tumor cytoreduction. The mechanism is likely due to multiple causes, including (a) killing of host regulatory lymphocytes that suppress antitumor immune responses,7, 110 (b) creating “space” in the host so that the adoptively transferred cells can engraft,158 and perhaps (c) enhanced cross-priming of tumor antigens. Cyclophosphamide and/or fludarabine is generally given several days before the adoptively transferred T cells.31, 110 Curti and colleagues96 have examined a related issue concerning the optimal time to harvest autologous CD4+ T cells in relation to the timing of cyclophosphamide administration in patients with advanced cancers. T cells were harvested at steady state, when on the decline, or when on recovery from the cyclophosphamide-induced leukopenia. From that study, they concluded that the best time to harvest autologous T cells was not at steady state but rather just before the leukopenic nadir that occurred following administration of cyclophosphamide. The best in vivo expansion of the infused CD4+ T cells occurred when the cells had been harvested as patients entered the cyclophosphamide-induced nadir. Most of the clinical antitumor responses also occurred in patients treated on this schedule. These results are generally consistent with animal models that predicted a need to ablate immunosuppressive lymphocytes for efficient engraftment and subsequent in vivo expansion of adoptively transferred CD4+ T cells. In a study of patients with stage III non–small cell lung cancer, investigators tested the sequence of adoptive therapy with autologous TIL and IL-2, followed by standard chemotherapy and radiotherapy; perhaps not surprisingly, they found that the sequence of immunotherapy followed by chemotherapy is not effective.167

In patients with early-stage cancers who have not yet had cytotoxic chemotherapy, it is probably best to harvest autologous T cells before initiation of chemotherapy. Adults have limited capacity to generate new T cells from the thymus, and therefore the repertoire remains contracted for long periods of time and in many cases never recovers.168, 169 Naïve T cells are most sensitive to the effects of cytotoxic chemotherapy, and their numbers are severely depleted in heavily pretreated patients. It is not yet known whether the tumor-specific T cells are derived from primed or naïve T cells in the host, and this likely varies depending on the intrinsic immunogenicity of the tumor. Studies with tetramers that can identify tumor antigen–specific T cells show that in some patients chemotherapy can ablate the tumor-specific T cells that have an effector phenotype while sparing memory cells.14 The mechanism for this is unknown, but the authors speculated that the effector cells were in the active phases of the cell cycle and were therefore relatively susceptible to the cytotoxic effects of the chemotherapy. If these results are confirmed, they would argue that patients should have their repertoire “archived” by apheresis before undergoing chemotherapy.

Anecdotal evidence suggests that immunotherapy may in some circumstances restore tumors to a chemotherapy-sensitive state. Several of our patients had platinum-resistant ovarian cancer and were then treated with adoptive cellular therapies or therapeutic vaccines. The patients had responses to the immunotherapy; however, they subsequently experienced disease progression. Interestingly, when chemotherapy was recommenced, they appeared to have tumor that was more sensitive to the drugs than when they had previously been treated. If this observation is confirmed, this would suggest a need for further study of this interesting scheduling issue between cytotoxic chemotherapy and immunotherapy. Data in mouse syngeneic tumor models support this concept.170, 171

Finally, there are dose-dependent and schedule-dependent effects that have been observed with DLI in connection with the induction of GVHD. Early studies showed that the infusion of donor T cells soon after a myeloablative transplant conditioning regimen resulted in the marked augmentation of acute GVHD.172 It has been well established by the work of O'Reilly and colleagues that the initial dose of infused T cells in the setting of allogeneic marrow transplantation has a major effect on the incidence and severity of acute GVHD.173 As was discussed earlier, it has only recently been appreciated that donor T cells can be infused with relative freedom from acute GVHD in the setting of NMSCT.148 Studies by Goldman and colleagues showed that in the steady-state setting of relapsed CML, infusions of resting T cells result in a decreased incidence of GVHD when given by dose fractionation, starting with low doses of donor cells and escalating subsequent doses as required.130 In a nonrandomized trial, the researchers compared a bulk, single-infusion DLI (average 1.5 × 108 CD3+ cells/kg) to an escalating dose regimen of DLI, where increasing numbers of cells (average total 1.9 × 108 CD3+ cells/kg) were given at 20-week average intervals between infusion. They found that antileukemic effects were preserved but that the incidence of GVHD was much lower using the escalating dose regimen of DLI.

T REGULATORY CELL ADOPTIVE THERAPY

CD4+CD25+ T regulatory (Treg) cells have been shown to regulate self-tolerance in mice. Initially they were described to be critical for the control of autoimmunity174 and were found on adoptive transfer to prevent experimental autoimmune diseases.175 More recently, Treg cells have been shown to suppress allogeneic immune responses176 and can prevent transplant rejection.177 Studies of human Treg have been hindered by the low numbers present in peripheral blood and the fact that the cells were initially thought to have poor replicative capacity in vitro.178 However, it was later shown that Tregs have substantial replicative capacity in vivo.179 We and others have developed improved ex vivo culture conditions that should permit pilot trials of Treg adoptive immunotherapy for the prevention or therapy of GVHD.180

GENETICALLY MODIFIED CELLULAR THERAPY

Genetic modification of T cells and dendritic cells ex vivo to engineer an improved antitumor effect is an attractive strategy for many settings. Unlike hematopoietic stem cells, currently available vectors provide high-level expression of transgenes in T cells and dendritic cells. The first use of genetically modified T cells was to demonstrate that adoptively transferred cells could persist in the host and traffic to tumor, albeit with low efficiency.92 A principal limitation of immunotherapy for some tumors is that the tumors are poorly antigenic, in that no T cells are available that have high avidity for tumor-specific antigens or that no T cells remaining in the patient after chemotherapy have the desired specificity. To address this problem, some clinical trials attempt to endow T cells with novel receptor constructs by the introduction of “T bodies,” chimeric receptors that have antibody-based external receptor structures and cytosolic domains that encode signal transduction modules of the T-cell receptor.181 These constructs can function to retarget T cells in vitro in an MHC-unrestricted manner. The major issues with the approach currently involve improved receptor design and the immunogenicity of the T body construct. T cells are also being transduced to express natural TCR receptor alpha beta heterodimers of known specificity and avidity for tumor antigens182; however, this approach is of limited general value for humans because each TCR will be specific for a given MHC allele such that each vector would be patient-specific.

A major limitation to adoptive transfer of CTLs is that they have short-term persistence in the host in the absence of antigen-specific T helper cells. Greenberg and coworkers have transduced human CTLs with chimeric GM-CSF/IL-2 receptors that deliver an IL-2 signal on binding GM-CSF. Stimulation of the CTLs with antigen caused GM-CSF secretion and resulted in an autocrine growth loop such that the CTL clones proliferated in the absence of exogenous cytokines. This type of genetic modification has potential for increasing the circulating half-life and, by extension, the efficacy of ex vivo–expanded CTLs. A related strategy to rejuvenate T cell function is to engineer T cells to ectopically express CD28 or the catalytic subunit of telomerase.183, 184 To date, there is limited clinical experience with engineered T cells; however, in certain instances such T cells have been shown to persist after adoptive transfer in humans for years.160, 185

As was noted, severe and potentially lethal GVHD represents a frequent complication of allogeneic DLI. The promising results with DLI have created increased interest in developing T cells with an inducible suicide phenotype. Expression of herpes simplex virus thymidine kinase (HSV TK) in T cells provides a means of ablating transduced T cells in vivo by the administration of acyclovir or ganciclovir.186 Using this strategy, Bordignon and colleagues infused donor lymphocytes into 12 patients who, after receiving allogeneic bone marrow transplants, had suffered complications such as cancer relapse or virus-induced lymphomas.187 The lymphocytes survived for up to a year, and complete or partial tumor remissions in five of the eight patients were achieved. Tumor regressions coincided with the onset of GVHD, and in most cases the GVHD was abrogated when ganciclovir was given. Thus, GVHD associated with the therapeutic infusion of donor lymphocytes after allogeneic marrow transplantation could be efficiently controlled by these novel suicide gene strategies in allogeneic lymphocytes. However, subsequent studies have indicated problems with this approach, in that the HSV TK gene confers immunogenicity to the transfused cells, leading to impaired survival and the inability to retreat a patient with DLI should the tumor recur. Future experiments will be required to develop vectors that are less immunogenic and able to confer even higher ganciclovir sensitivity to transduced human lymphocytes. Investigators have developed suicide systems composed of fusion proteins containing a Fas or caspase death domain and a modified FKBP.188, 189 These approaches have the advantage that the suicide switches are expected to be nonimmunogenic. T cells expressing these modified chimeric proteins are induced to undergo apoptosis when exposed to a drug that dimerizes the modified FKBP.190, 191 Finally, the advent of lentiviral vectors has greatly increased the efficiency of T-cell engineering, and it is likely that adoptive therapies with lentiviral engineered T cells will become a clinical reality.192

SUMMARY

The basis for tumor immunology is the premise that the immune system is capable of recognizing tumor cells together with the premise that the activated immune system can lead to the subsequent rejection of tumors. While the former premise is now well accepted, the latter remains controversial. As we complete nearly 50 years of research into adoptive immunity for tumors, there are no forms of cellular therapy that have been approved by the Food and Drug Administration. Allogeneic DLI is being incorporated into the practice of medicine as a valuable and potentially curative indication for selected patients with CML. However, there are several obstacles that investigators must overcome before adoptive immunotherapy can become a more generally applicable and successful a form of prophylaxis or treatment for human tumors. Concerns about the costs of cellular therapy should eventually be overcome if it achieves curative potential or long-lasting tumor immunity for patients with indications that are otherwise chemotherapy refractory. Finally, it is likely that adoptive immunotherapy will be used not alone but in combination with other forms of immunotherapy and chemotherapy.

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