21.5.1 Introduction to Immunotherapeutic Approaches
Our improved understanding of tumor immunology has led to a growing list of immunotherapeutic agents being approved for use against cancer. One broad category of agents consists of monoclonal antibodies (mAbs). Although the mechanisms of action of various mAbs are not always related to the antitumor T-cell response that is discussed above—many function in a manner completely independent of T cells—they are, nevertheless, important players in cancer therapy.
Immunotherapy that is aimed at augmenting antitumor T-cell immunity can be broadly categorized into (a) nonspecific immunotherapy, (b) specific immunotherapy, and (c) adoptive cell therapy. Each category encompasses many different strategies—some investigational and some approved for therapy. The small number of these strategies selected for discussion in this chapter are listed in Table 21–1.
TABLE 21–1Selected approaches to cancer immunotherapy. ||Download (.pdf) TABLE 21–1 Selected approaches to cancer immunotherapy.
|Monoclonal antibody therapies ||(See Table 21–2) |
|Nonspecific immunotherapies || |
Bacillus of Calmette and Guérin (BCG)*
Anti-CTLA-4 blockade (Ipilimumab)*
|Specific immunotherapies (vaccination) || |
Irradiated tumor cells
|Adoptive cell therapies || |
Nonspecific immunotherapy refers to strategies that augment general T-cell responses, in a "nonspecific" or "polyclonal" manner. Nonspecific immunotherapy includes the use of cytokines (eg, INF-α, IL-2), immunological adjuvants (Imiquimod), and agents that target immunomodulatory molecules (anti–CTLA-4 antibody). Specific immunotherapy focuses on the activation and enhancement of the number of T cells that can recognize TAAs by using vaccine strategies. Although the induction of an immune response by vaccination often refers to a prophylactic strategy, such as that used to limit viral infection, vaccination may also refer to therapy aimed at eliciting antitumor immune responses to eliminate established cancers. The field of prophylactic cancer vaccines will not be addressed in this chapter but is reviewed in Lollini et al (2006). Common vaccine approaches include the use of TAAs or peptides derived from TAAs. Other approaches are based on vaccination using whole tumor cells (autologous or allogeneic) that have been irradiated prior to infusion to prevent their proliferation following infusion. In adoptive cell therapy, autologous immune cells such as DCs and T cells are manipulated ex vivo and then reinfused.
21.5.2 Monoclonal Antibodies for Cancer Therapy
Monoclonal antibodies (mAbs) bind specifically to their target protein and can block the target protein's function, trigger signaling downstream of the target protein, or deliver conjugated toxins to cells expressing the target protein. Current Food and Drug Administration (FDA)-approved mAbs include those that target antigens expressed by tumor cells (eg, CD20 on non-Hodgkin lymphoma and chronic lymphocytic leukemia), molecules that promote tumor growth (eg, epidermal growth factor receptor), or angiogenic molecules (eg, vascular endothelial growth factor). Table 21–2 lists examples of antibodies approved for cancer therapy.
TABLE 21–2Table of monoclonal antibodies approved for cancer therapy. ||Download (.pdf) TABLE 21–2 Table of monoclonal antibodies approved for cancer therapy.
|Monoclonal Antibody ||Cancer Targeted ||Target Molecule ||Type of mAb |
|Alemtuzumab ||CLL ||CD52 ||Humanized IgG1 |
|Bevacizumab ||Colorectal, lung cancer ||VEGF ||Humanized IgG1 |
|Cetuximab ||Colorectal cancer ||EGFR ||Chimeric IgG1 |
|Gemtuzumab ||AML ||CD33 ||Humanized IgG4 |
|Ibritumomab tiuxetan ||NHL ||CD20 ||Mouse |
|Ofatumumab ||CLL ||CD20 ||Human IgG1 |
|Panitumumab ||Colorectal cancer ||EGFR ||Human IgG2 |
|Rituximab ||NHL, CLL ||CD20 ||Chimeric IgG1 |
|Tositumomab ||NHL ||CD20 ||Mouse |
|Trastuzumab ||Breast cancer ||HER-2/neu ||Humanized IgG1 |
188.8.131.52 Production of Monoclonal Antibodies
A mAb that recognizes a given protein target is derived from an antibody molecule that was produced by a single B cell. Originally, mAbs were produced by fusing B cells from the spleen of an animal that had been immunized with the target protein, with a myeloma cell line that was selected for the inability to produce immunoglobulin. Primary spleen cells cannot survive ex vivo in unsupplemented culture medium. The myeloma cell line was also selected for a deficiency in an enzyme (hypoxanthine-guanine phosphoribosyltransferase [HGPRT]) that renders the cells unable to grow in culture medium containing hypoxanthine, aminopterin, and thymidine (HAT). Successful cell fusions between spleen cells (expressing HGPRT) and myeloma cells could therefore be selected by culturing in HAT-containing medium. Fused cells were then cloned: Cells were plated at limiting numbers and individual cells were expanded separately. These fused cells (called hybridomas) were then screened for secretion of antibodies that could bind the target protein of interest. Once a hybridoma with the desired antibody specificity is identified, large batches of antibodies can be produced by expanding the hybridoma in culture, or expanding the hybridoma in the peritoneal cavity of animals (mice, rabbits) and harvesting the ascites. A schematic of this classical method of antibody production, and an example of a current method based on a genetic approach, described below, are depicted in Figure 21–13.
Methods of monoclonal antibody production. A) mAbs were initially produced by the fusion of B cells with a specialized myeloma cell line. Fused cells were cloned and then the clone that produced the antibody of interest was selected for antibody production. Further details are provided in the text. B) Other methods are based on recombinant technologies, where the desired antibody genes are transfected into cells such as Chinese hamster ovary (CHO) cells and antibodies are harvested from culture supernatants. In this example, the antibody genes of interest are identified by screening bacteriophages for binding to the antigen of interest. The bacteriophages have been engineered to express antibodies that are the product of random rearrangement of immunoglobulin genes.
184.108.40.206 Types of Therapeutic Antibodies
Antibodies that recognize human proteins were generated initially by immunization of animals (eg, mice) with human proteins and isolation of specific antibodies, as described in the hybridoma approach above. However, mouse antibodies have limited therapeutic value for several reasons. First, mouse Fc domains (which is the "constant" domain of an antibody that is conserved amongst all antibodies of a particular class) are not recognized as efficiently as human Fc domains by human immune cells, and such recognition is required for some of the mechanisms of action of antibody therapy, described below. Also, mouse antibodies are recognized as foreign proteins by the human immune system and thus repetitive antibody administration would result in an immune response against the therapeutic antibody. One solution has been the generation of chimeric antibodies, which are engineered to contain the variable (Fv) region from a mouse antibody and the human constant (Fc) regions. An alternate approach is to generate humanized antibodies where the hypervariable regions of the variable antigen-binding domain (Fv) is derived from a mouse antibody and the rest of the Fv and the entire Fc region is derived from human sequences. To completely circumvent neutralizing antibodies with xenogeneic sequences, many current therapeutic antibodies are fully human. These antibodies can be isolated from mice that have been engineered to express only human antibodies, or they can be isolated by a phage display approach, where libraries of random recombinations of human antibody genes are expressed in bacteriophages. The antibody-expressing phages are then screened for the ability to bind the target protein. Recombinant antibodies are often produced by transfecting Chinese hamster ovary (CHO) cells with the desired antibody genes, as CHO cells grow well in suspension culture. Figure 21–14 illustrates these types of antibodies.
Chimeric and humanized antibodies. In chimeric antibodies, the variable fragment (Fv) of the antibody molecule is of mouse origin and the constant regions (Fc) is of human origin. In humanized antibodies, only the hypervariable region of the Fv is of mouse origin; the rest of the antibody molecule is of human origin.
220.127.116.11 Mechanisms of Action of Antibodies
Antibodies that bind to receptors on the cell surface can block receptor signaling (by steric blockade of ligand binding) or can trigger receptor signaling (by aggregation of multiple receptors). Antibodies can also lead to lysis of cells that express the molecule targeted by the antibody. Target-cell lysis can occur by activation of the complement protein cascade or initiation of antibody-dependent cell-mediated cytotoxicity (ADCC) (Fig. 21–15). The complement cascade is initiated by binding of the Fc domain of immunoglobulin (Ig)G to a complement protein, which then leads to a cascade of cleavage of various complement system proteins. The end result of this cascade is the formation of a protein complex called the membrane attack complex (MAC) on the target cell (in this case, the tumor cell) that leads to pore formation and lysis of the target cell. Other proteins activated by the complement cascade are able to mediate chemoattraction of various immune cells to the site of tumor. ADCC, on the other hand, is initiated upon binding of the Fc region of antibodies by Fc receptors (FcRs). FcRs are expressed on various innate immune cell types, and the activation of FcRs leads to activation of cytotoxic activity against target cells. For example, FcγRIIIA is expressed by natural killer (NK) cells, and binding of this receptor by the Fc region of an IgG molecule activates NK cell-mediated lysis of the target cell. Trastuzumab (Herceptin), which is principally thought to bind and inhibit signaling through the HER-2/neu growth receptor (see Chap. 7, Sec. 7.5.3 and Chap. 20, Sec. 20.3.3), also has been shown to mediate antitumor activity via ADCC (Clynes et al, 2000). Because ADCC is a potentially important mechanism mediating the action of therapeutic antibodies, the most effective antibodies should be engineered or selected to bind well to activating FcRs and to bind poorly to the inhibitory FcγIIB (Nimmerjahn and Ravetch, 2008).
Tumor-specific antibodies can promote tumor regression by multiple mechanisms. In addition to either blocking or triggering cell-surface receptors, antibodies can lead to immune-mediated elimination of tumor cells. A) ADCC is induced when antibodies bound to tumor antigens (TAA) on the surface of tumor cells are then bound by Fc receptors expressed by natural killer (NK) cells. Triggering of the Fc receptors activates the cytolytic activity of NK cells. B) Complement activation is induced by binding of complement proteins to the Fc domain of antibody/antigen complexes. One of the end results of the complement cascade is the formation of the membrane attack complex (MAC) in the cell membrane of target cells, leading to cell death. C) Antibodies can also bind to tumor antigens present on apoptotic bodies formed when tumor cells undergo apoptosis during the normal cycle of proliferation and cell death. By this route, tumor antigens are taken up by DCs via Fc receptors. Tumor antigens can be processed by the DCs and presented as peptide/MHC complexes to T cells. Tumor-specific T cells can then be activated and mediate antitumor activities including cytotoxic activity against tumor cells.
Antibodies may also lead to enhanced T-cell priming. In the natural course of tumor progression or during antibody-mediated lysis of tumor cells, fragments of dying tumor cells can be recognized and bound by TAA-specific antibodies (see Fig. 21–15). These complexes can then be recognized by FcRs expressed on DCs, internalized, and the TAA-derived peptides presented to tumor-specific T cells via cross-presentation. The relative contribution of each of these different mechanisms for various antibodies is unclear.
18.104.22.168 Modified Antibodies
Antibodies can be modified in various ways in order to alter their therapeutic function. For example, bispecific T-cell engager (BiTE) molecules consist of 2 Fv fragments, each with different specificities, linked together with a flexible linker (Wolf et al, 2005). For example, a BiTE has been generated that is specific for CD3 and epithelial cell adhesion molecule (EpCAM). The EpCAM-specific component binds tumor cells expressing this common tumor adhesion molecule and the CD3-specific component functions to recruit T cells to EpCAM-expressing tumor cells. Another example is Blinatumomab, which has been shown to mediate regression of non-Hodgkin B-cell lymphomas (Bargou et al, 2008). This agent is specific for CD19 (expressed on non-Hodgkin lymphoma cells) and CD3 (to recruit T cells to tumor cells).
Three FDA-approved antibodies for cancer have been modified to deliver toxic or radioactive molecules to tumor cells. Gemtuzumab is fused to the toxin ozogamicin, Ibritumomab tiuxetan is conjugated to yttrium-90, and Tositumomab is conjugated to iodine-131 (Bross et al, 2001; Fisher et al, 2005; Witzig et al, 2002).
21.5.3 Nonspecific Immune Modulators
22.214.171.124 Bacillus Calmette-Guérin
Bacillus Calmette-Guérin (BCG) is a live-attenuated strain of Mycobacterium bovis and is effective in preventing the recurrence of superficial bladder carcinoma. BCG is administered intravesically, and its mechanism of action is related to its immunostimulatory activity (Patard et al, 1998).
After instillation in the bladder, BCG is processed and presented locally by APCs. These APCs then stimulate CD4 and CD8 T-cell responses. In addition, upregulation of MHC class I and II on urothelial cells is observed. During BCG treatment, levels of various cytokines are found in the urine: IL-1, IL-6, IL-8, and IL-10 early during treatment, and IL-2, TNF-α, and IFN-γ later during treatment. Infiltration of T cells into the bladder wall is also observed after treatment. Overall, BCG instillation leads to a local T-cell response, which appears to have features of a Th1-type response. The mechanism whereby tumor cells are eliminated is unclear. It is possible that mycobacteria-specific T cells are activated and lyse tumor cells that have been infected with the mycobacteria. It is also possible that the immune stimulation induced by BCG promotes the activation of T cells specific for bladder tumor antigens and that these T cells kill tumor cells.
IFN-α is produced by many cell types, including cells of the immune system such as T cells, B cells, NK cells, DCs, and macrophages, and has direct antitumor effects, as well as a role in immunomodulation (Dunn et al, 2006). IFN-α can directly inhibit proliferation of tumor cells, downregulate expression of oncogenes, and induce tumor-suppressor genes. It also possesses antiangiogenic activity.
IFN-α therapy induces responses in more than 90% of patients with hairy cell leukemia, although most patients will relapse within 2 years. IFN-α also shows some activity in patients with other hematological cancers, such as chronic myelogenous leukemia, myeloma, and low-grade non-Hodgkin lymphoma. Although approved by the FDA for patients with melanoma and renal cell carcinoma, the overall clinical response rate in these patients to IFN-α therapy is relatively low. Furthermore, high-dose IFN-α therapy is associated with severe toxicities, including hypotension, vomiting, fever, and diarrhea (Kirkwood et al, 1996; Motzer et al, 2002).
Although the utility of IFN-α therapy is limited, IFN-α is an important enhancer of antitumor immunity. IFN-α induces upregulation of MHC class I and contributes to maturation of DCs. It also is important for promoting CD8 T-cell survival and enhances migration of T cells into tissues. For example, mice treated with an IFN-α receptor-blocking antibody could not reject MCA-induced sarcomas that could be rejected in untreated mice. In addition, IFN-α receptor 1-deficient mice were more susceptible to MCA-induced tumor formation (Dunn et al, 2006). IFN-α may have a therapeutic role in combination with other types of immunotherapy. For example, in a murine model of colon carcinoma, combination therapy using an immunostimulatory antibody (agonistic anti-CD137 mAb) and IFN-α resulted in synergistic antitumor immunity (Dubrot et al, 2011).
IL-2 is a well-characterized cytokine that acts primarily to stimulate proliferation of T cells and NK cells. High-dose IL-2 therapy is an approved treatment for renal cell carcinoma and melanoma, but the clinical response rate for this therapy is only in the range of 15% for both cancer types (Atkins et al, 1999; Fyfe et al, 1995). High-dose IL-2 therapy is also associated with substantial toxicity, including cardiac arrhythmias, capillary leak syndrome leading to hypotension, central nervous system toxicity leading to confusion and gastrointestinal toxicity such as diarrhea (Rosenberg et al, 1989). Although IL-2 promotes effector T-cell responses and can function to reverse anergy of effector T cells, IL-2 may not represent an ideal agent to promote immunity because it also promotes the expansion of Tregs.
Imiquimod is a synthetic compound that triggers the TLR7 molecule. TLRs are a family of molecules that are stimulated by conserved molecular motifs that are present on various microorganisms (Akira et al, 2006; see also Sec. 21.2.2). For example, the natural ligand for TLR7 is single-stranded RNA, which is present in some viruses. The TLRs are expressed by many cell types, including cells of the innate immune system such as macrophages and DCs. Interactions between TLRs and corresponding ligands lead to activation of innate immune cells, which promote elimination of the microorganism through various mechanisms, including production of interferons and proinflammatory cytokines.
Because TLRs activate APCs such as DCs, they can enhance the ability of DCs to stimulate antitumor T-cell responses. Various TLR ligands are under investigation in animal models and early clinical trials for cancer immunotherapy. These agents are often used in combination with vaccination strategies to enhance T-cell responses induced by vaccination.
Imiquimod is approved for use in the treatment of superficial basal cell carcinoma, and for treatment of genital warts and actinic keratosis. Treatment is generally effective for superficial basal cell carcinoma, with complete clearance rates of approximately 75% or higher (compared to a few percent in placebo-treated groups). Imiquimod has been shown to induce proinflammatory mediators such as IFN-α, TNF-α, and IL-6 by various innate immune cell types, including macrophages and a specialized subset of DCs called plasmacytoid DCs. Imiquimod also upregulates costimulatory molecules and chemokine receptors on plasmacytoid DCs, thus enhancing their ability to activate T cells and home to sites of T-cell activation, respectively. Histological studies have shown an increase in infiltration of tumor lesions with T cells and DCs following Imiquimod treatment, further supporting its immunological mechanism of action.
21.5.4 Other Approaches to Immunotherapy
126.96.36.199 Dendritic Cell Vaccines
Mature DCs are highly efficient APCs and therefore are central players in the induction of T-cell responses. Therefore, adoptive cell therapy using DCs that present TAAs to induce antitumor T cells in vivo is of major interest (Tacken et al, 2007). In general, these DCs are obtained by in vitro differentiation of bone marrow progenitor cells (for mice) or peripheral blood monocytes (for humans). Granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 are commonly used to stimulate differentiation. Figure 21–16 outlines the generation of DCs for infusion. Some of the methods for loading DCs with TAAs include loading peptides derived from TAAs into surface MHC molecules, incubating DCs with whole TAA proteins, and transfecting or transducing DCs with DNA encoding TAAs. TAA-loaded DCs may also be exposed to a variety of maturation stimuli in order to enhance their immunogenicity. These stimuli include agonistic anti-CD40 antibody, various TLR ligands (eg, CpG oligodeoxynucleotides, poly I:C, LPS), and cytokine cocktails (eg, a cocktail of IL-1β, IL-6, TNF, and prostaglandin E2).
Generating DCs for therapy. Common methods for generating DCs for vaccination are shown. A) For mouse studies, DC precursors are obtained from bone marrow cells and differentiated into immature DCs in vitro using GM-CSF. (B) For human studies, monocytes are obtained from peripheral blood mononuclear cells by a variety of methods, including elutriation, plastic adherence, or isolation of CD14+ cells. Monocytes are then differentiated into immature DCs in vitro using GM-CSF and IL-4. For both mouse and human studies, immature DCs are manipulated to present tumor-associated antigens. This can be done by a variety of approaches, including exposing them to peptides or RNA, or fusing the DCs with tumor cells. DC maturation is induced by stimuli such as TLR ligands (eg, LPS or CpG oligodeoxynucleotides) or agonistic anti-CD40 antibody. Mature DCs presenting TAAs are then ready for infusion.
Clinical trials based on DC vaccination have demonstrated that this approach is associated with minimal toxicity, and there is some evidence for clinical effectiveness (Tacken et al, 2007; Palucka et al, 2012).
Sipuleucel-T was the first autologous cellular therapy for cancer to receive FDA approval. The therapeutic product is produced by leukapheresis from a patient with prostatic cancer. The leukapheresis product is transported to a central facility where the cells are processed and incubated with a fusion protein of a prostate TAA (prostatic acid phosphatase [PAP]) and the cytokine GM-CSF (Small et al, 2000). The cellular product is then reinfused into the same patient. The premise of this therapy is APCs, such as DCs, will take up the fusion protein. The PAP will be processed and presented by MHC molecules on the surface of APCs and will thus be able to stimulate PAP-specific T cells to mediate antitumor activity. The functions of GM-CSF include promoting the differentiation of DCs. Presentation of a prostate TAA by efficient APCs such as DCs should activate endogenous prostate-specific T cells upon reinfusion. However, it remains unclear how the other cell types present in the leukapheresis product may contribute to the effectiveness of the treatment. In vitro biological potency tests as well as clinical trial data indicate that the activity can be attributed to the cell fraction expressing CD54, which is a cell adhesion molecule expressed on a variety of cell types, including APCs. An integrated analysis of 2 Phase III trials showed a survival benefit for advanced prostate cancer patients treated with sipuleucel-T compared to placebo (Higano et al, 2009), with a 23.2-month median survival in the Sipuleucel-T arms and an 18.9-month median survival in the placebo arms. Common adverse events included chills, pyrexia, and headache, and were mostly low grade. An additional double-blind, multicenter Phase III trial showed similar results. In this trial, 512 men with metastatic castration-resistant prostate cancer were randomized at a 2:1 ratio to receive Sipuleucel-T or placebo (Kantoff et al, 2010). Interestingly, the time to objective disease progression was similar in both arms, but the relative risk of death was reduced by 22% in the Sipuleucel-T arm compared to the placebo arm, leading to a 4-month improvement in median survival. However, this trial has been criticized because the leukapheresis procedure was used in control patients (but leukocytes were not re-infused into all of the control patients), and loss of white cells might be harmful, particularly to older adults, leading (in part) to a difference in survival because of poorer outcome in controls (Huber et al, 2012).
CTLA-4 is expressed on activated T cells and also Treg cells. Initial experiments in animal models demonstrated that blockade of CTLA-4 induces tumor regression. Two therapeutic anti–CTLA-4 blocking antibodies have been developed. One is a fully human IgG2 antibody (Tremelimumab) (Ribas, 2008), the other is a fully human IgG1 antibody (Ipilimumab) (Weber, 2008). These agents have been tested mainly in patients with metastatic melanoma.
The clinical activity of these agents, when assessed initially using objective response (ie, tumor shrinkage) to evaluate clinical responses, appeared to be relatively low. However, this type of immunomodulatory agent may induce unconventional response patterns, such as tumor progression that precedes regression, or mixed responses of different lesions but with overall decreases in tumor burden (Wolchok et al, 2009). Indeed, a Phase III randomized trial in patients with unresectable Stage III or IV melanoma demonstrated that patients that received Ipilimumab had a significantly longer median overall survival (10.0 months) compared with patients not receiving anti–CTLA-4 blockade (6.4 months) (Hodi et al, 2010). Because anti–CTLA-4 blockade is not an antigen-targeted approach, it is associated with some grade 3 or 4 autoimmune toxicities, the most common being colitis. In 2010, Ipilimumab received FDA approval for treatment of patients with unresectable or metastatic melanoma.
Although the negative regulatory role of CTLA-4 signaling is well established, the mechanism(s) whereby anti–CTLA-4 blockade induces antitumor T-cell immunity remains under investigation. There remains some debate whether the predominant target of anti–CTLA-4 blockade is to "release the brakes" on effector T cells or to inhibit the suppressive activity of Tregs. CTLA-4 blockade is thought to enhance effector T cells by the following mechanisms: Anti–CTLA-4 antibodies may prevent the interaction of CD80 and CD86 with CTLA-4, thereby allowing for prolonged interaction of CD80 and CD86 with the positive costimulatory receptor CD28. In addition, CTLA-4 blockade may inhibit the CTLA-4–mediated negative intracellular signals that shut down effector T cells. There is also strong evidence that CTLA-4 suppresses effector T cells in a non–cell-autonomous fashion, that is, by inhibiting the suppressive activity of Tregs (Bachmann et al, 1999; Read et al, 2000). Evidence suggests that the effects of CTLA-4 on both effector T cells and Tregs contribute to its ability to enhance antitumor immune responses (Peggs et al, 2009).
188.8.131.52 Adoptive T-cell Therapy
Infusion of T cells that can recognize and destroy tumor cells is a major area of interest for immunotherapy. That transferred T cells can mediate potent antitumor effects has been conclusively demonstrated by donor lymphocyte infusions (DLIs), which are the standard treatment for patients with relapsed leukemia following allogeneic bone marrow transplantation. For therapy using DLI, lymphocytes from allogeneic bone marrow transplantation donors are stored in a sample bank. If the leukemia patient relapses following a bone marrow transplantation, then the lymphocytes from the same donor are infused into the patient. These lymphocytes mediate regression of the relapsed leukemia (a so-named graft-versus-leukemia effect). For patients with relapsed chronic myelogenous leukemia, 70% of patients treated with DLI experience complete remission (Kolb et al, 2004). This approach demonstrates that T cells have the potential to mediate antitumor activity.
Adoptive T-cell therapy is based on ex vivo expansion and manipulation of patient-derived tumor specific T cells followed by reinfusion in an autologous manner. General approaches to generate T cells for adoptive cell therapy are depicted in Figure 21–17 and described below.
Generating T cell clones or tumor-infiltrating lymphocytes (TILs) for adoptive cell therapy. A) To generate T-cell clones, peripheral blood mononuclear cells are isolated and the bulk T-cell population is stimulated with DCs that have been exposed to peptides derived from tumor-associated antigens. Through successive rounds of stimulation and expansion, tumor-specific T cells are selectively expanded. These T cells are cultured under "limiting dilution" conditions to generate cultures originating from 1 parent cell (a "clone") consisting of T cells with the same T-cell receptor. B) To generate TILs, tumor tissue is dissociated and the bulk population of TILs is expanded in vitro using a T-cell growth factor (eg, IL-2). In theory, the resulting T-cell population is enriched for tumor-specific T cells.
184.108.40.206.1 Adoptive Cell Therapy with T-cell Clones
One source of T cells for adoptive cell therapy is expanded T-cell clones from the peripheral blood of cancer patients. This approach is advantageous in that the peptide specificity of the transferred T cells is well defined, and peripheral blood T cells have not been subjected to immunosuppressive factors present within the tumor microenvironment and therefore may be more responsive. Potential drawbacks for this approach include the possibility that these T cells may not home to the tumor site. It is also possible that tumor cells do not express the peptide recognized by the T-cell clones and will escape detection by the T cells.
The general approach to generating T-cell clones for therapy involves stimulation of bulk peripheral blood T cells with peptides derived from TAAs of interest. The most well-studied TAA-derived peptides are those that bind the HLA-A*0201 MHC molecule, and therefore trials of adoptive cell therapy using clones are generally limited to HLA-A*0201–positive patients. Peptide-stimulated T cells are then cultured under limiting dilution conditions in order to expand a population of T cells derived from a single clone. All T cells expanded from a single T cell will express identical TCRs and therefore can be considered clones. CD8+ T-cell clones and, more recently, CD4+ T-cell clones have been used in early phase clinical trials that have demonstrated this approach is associated with low toxicity (Yee, 2010).
220.127.116.11.2 Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes
Many solid tumors are infiltrated with T cells. Because, under normal circumstances, T cells do not infiltrate tissues, the presence of tumor-infiltrating T cells (TILs) suggests that an antitumor response is occurring and the T-cell infiltrate is likely enriched for those that can recognize TAAs. Therefore, TILs represent a source of tumor-specific T cells for use in therapy. TILs can be obtained following dissociation of tumor tissue and expansion of TILs can be undertaken ex vivo in the presence of various T-cell growth factors such as IL-2. Adoptive transfer of bulk populations of TILs has been performed in trials for various cancers, including melanoma, ovarian cancer, renal cell carcinoma, and non–small cell lung cancer. Collectively, these trials demonstrate that adoptive transfer of TILs is associated with minimal toxicity and some of these studies provide evidence that TILs are clinically active.
High clinical response rates have been observed when patients with metastatic melanoma were treated with TIL-based protocols in a particular series of trials. In these protocols, patients were given nonmyeloablative lymphodepleting chemotherapy (cyclophosphamide and fludarabine) immediately prior to infusion of TILs (1010 to 1011 cells) and high-dose IL-2 therapy. Using this protocol, the objective clinical response rate by RECIST (Response Evaluation Criteria in Solid Tumors) criteria was approximately 50% (21/43 patients) (Dudley et al, 2002, 2005). When total body irradiation was added to the treatment protocol, a trend to higher clinical response rates with increasing intensity of lymphodepletion was observed (Dudley et al, 2008). Therefore the combination of TIL transfer with other therapeutic interventions has the potential to improve disease outcomes, although this has not yet been tested in Phase III trials.
The Immune System
The two arms of the immune system are innate immunity and adaptive immunity. One of the functions of the innate immune system is to detect the presence of pathogens and present antigens to cells of the adaptive immune system. Dendritic cells (DCs) are efficient antigen-presenting cells (APCs). When DCs encounter pathogens, they differentiate from an immature to a mature state. Mature DCs have the ability to efficiently activate T cells.
Antigens are presented to T cells by major histocompatibility complex (MHC) molecules. The antigens presented by MHC molecules are peptide fragments that can be derived from self or foreign proteins. There are 2 main types of MHC molecules: MHC class I and MHC class II. In general, MHC class I molecules present antigens from intracellular proteins ("endogenous pathway"), and MHC class II molecules present antigens from extracellular proteins ("exogenous pathway"). The exception to this paradigm is a pathway called "cross-presentation," where MHC class I molecules can present antigens derived from exogenous sources.
B cells recognize antigens in their native form via a B cell receptor (BCR). The BCR is a membrane-bound form of the soluble immunoglobulin (antibody) that will be produced by that B cell upon activation. T cells recognize antigens as peptide fragments that are presented by MHC molecules via the T-cell receptor (TCR). The TCR is expressed on the surface of T cells and is not secreted. The diversity of BCR and TCR specificities is partly a result of a DNA rearrangement process called V(D)J rearrangement. In this process, selected gene segments from a large number of possible gene segments are spliced into a combined exon.
A T-cell response is induced when TCRs are engaged by their cognate peptide/MHC ligand. Productive T-cell activation also requires signaling through co-stimulatory molecules (eg, CD28). T-cell activation leads to proliferation of the T cell as well as the acquisition of effector functions. After T-cell activation, most of the activated T cells disappear, leaving only a small pool of memory T cells behind. T-cell responses are subject to suppression by various mechanisms, including down-regulation via molecules such as cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed death (PD)-1.
There are various types of T cells. Cytotoxic T lymphocytes (CTLs) can kill target cells and generally express the CD8 co-receptor and recognize peptides presented by MHC class I molecules. T helper cells (Th cells) secrete various cytokines depending on their particular Th cell subtype (Th1, Th2, Th17, etc). T helper cells express the CD4 co-receptor and recognize peptides presented by MHC class II molecules.
Various mechanisms help to establish T-cell tolerance to self antigens. For example, during T-cell development in the thymus, T cells that strongly recognize self antigens are eliminated. Self-reactive T cells that escape thymic development can be suppressed by various peripheral tolerance mechanisms including regulatory T cells (Tregs).
Tumor Immunology and Immunotherapy
Tumor-associated antigens (TAAs) are proteins that are expressed preferentially (or uniquely) by tumor cells. Types of TAAs include mutated self proteins, cancer-testis antigens, proteins normally expressed during differentiation, overexpressed self proteins, and viral antigens. They may also have unique post-translational modifications. Some TAA-derived peptides can be recognized by T cells. Peptide/MHC multimer reagents can aid in the detection of TAA-specific T cells.
The immune system can survey the body for tumor cells and eliminate them, a concept termed "cancer immune surveillance". Immune surveillance was in part demonstrated in studies where immunodeficient mice developed tumors at a higher frequency and with faster kinetics than did immunocompetent mice. A current model for tumor progression and the relationship with the immune system includes three phases: elimination, equilibrium, and escape. The importance of the immune system in controlling tumor growth is in part demonstrated by studies that show a correlation between T-cell infiltration in tumors and good prognosis.
Barriers that inhibit the T-cell response against tumors include the tolerance mechanisms that also inhibit the T-cell response against self antigens. In addition, immunosuppressive factors in the tumor microenvironment may be present such as transforming growth factor (TGF)-β and indolamine 2,3-dioxygenase (IDO).
Monoclonal antibodies (mAbs) bind specifically to their target protein. The classical method of mAb production involves the generation of hybridomas. Monoclonal Abs can be completely of animal origin (eg, mouse) or can be chimeric, humanized, or fully human. Monoclonal Ab therapy has various possible mechanisms of action: blockade of receptor signaling, triggering of receptor signaling, activation of the complement cascade leading to target cell lysis, or target cell lysis via antibody-dependent cell-mediated cytotoxicity. In addition, mAb therapy may enhance T-cell priming due to enhanced uptake and presentation of antigens.
Approved immunotherapies for cancer include Bacillus Calmette-Guerin for superficial bladder cancer, interferon-α for some hematological cancers, interleukin-2 for renal cell carcinoma and melanoma, Imiquimod for superficial basal cell carcinoma, and Ipilimumab (amonoclonal antibody that blocks the negative regulation of T-cell responses) for metastatic melanoma. These are "nonspecific" approaches that augment T-cell responses in a polyclonal manner.
Other immunotherapeutic approaches are at various stages of development. Vaccination with DCs involves the in vitro production of autologous DCs. Generally, these DCs are loaded with TAA-derived peptide(s) and matured with various stimuli before administration. The first autologous cellular therapy for cancer to receive FDA approval was Sipuleucel-T for prostate cancer. Other types of adoptive cell therapies under investigation include immunotherapy with tumor-specific T cell clones or with tumor-infiltrating lymphocytes.