Over the last few decades, there have been numerous advancements in cancer treatments and technologies. Historically, cancer has been treated through three modalities:
used to excise visible tumors
used to prevent the proliferation of tumors using drugs
used to stop the dividing of cells through radiation
To address the fact that these procedures are relatively nonspeciﬁc, a new ﬁeld of cancer treatment called immunotherapy has emerged. Immunotherapy harnesses the immune system by helping it to recognize and destroy tumor cells without attacking normal cells. Immunotherapy accomplishes these tasks by stimulating the body’s own immune cells to work smarter and eliminating some of the damage that other treatment modalities may incur.
From a theoretical perspective, developing immunotherapy tools by utilizing T cells has been of interest because:
- T cell responses are speciﬁc in their ability to distinguish between healthy and cancerous tissues.
- T cells have robust responses and upon activation can proliferate dramatically to launch a strong immune response.
- T cells have the ability to travel to remote sites, which is a crucial feature in cases of metastases and cancers that cannot be detected with current imaging technology.
- T cells can confer memory with the help of B cells that can maintain the therapeutic effect for many years after the initial treatment.
Based on the speciﬁc anti-tumor responses, many immunotherapy approaches have been proposed and techniques to isolate tumor-speciﬁc lymphocytes have been developed.
Adoptive Cell Therapy (ACT)
Adoptive Cell Therapy (ACT) is an immunotherapy approach in which anti-tumor lymphocytes (most of which are T cells)Adoptive Cell Therapy (ACT) is an immunotherapy approach in which anti-tumor lymphocytes (most of which are T cells)are harvested from the cancer patient, expanded in vitro, and then re-infused back into the patient. This procedure is often performed in conjunction with vaccines, growth factors that can enhance the in vivo impact of the transferred cells, and lymphodepleting chemotherapy in order to ensure that the body tolerates the tumor targeting cells. Since adoptive therapies physically separate the emerging anti-tumor cells from their host, it is possible to manipulate the cells and their response mechanisms in ways that are clinically relevant. In order for T cells to mount a speciﬁc response to tumor cells, they need to be able to recognize and target antigens on the tumor that are non-existent or poorly expressed in healthy tissue. Tumor-associated antigens (TAAs) were identiﬁed in the 1990s and provided deﬁnitive proof that immune cells can distinguish between cancerous and noncancerous tissue.There are several main cell sources that are able to recognize these tumor-speciﬁc antigens or exhibit anti-tumor activity that can be used for adoptive immunotherapy:
1. Tumor-Inﬁltrating lymphocytes (TILs). Naturally occurring T cells located within the tumors, TILs can be isolated and grown in vitro to be reinfused to the patient after sufﬁcient growth.
2. In a patient who lacks culturable TILs, or in which growing TILs is difﬁcult, one can genetically engineer T cells that can perform the same anti-tumor function by introducing tumor-targeting receptors or other attributes that can help the cells to efﬁciently eliminate cancerous cells. Since this process occurs in vitro, it is possible to engineer qualities that do not occur naturally in these cells. Thus, there are two types of receptors that are used to redirect T cells: physiological T cell receptors (TCRs) and synthetic receptors, known as chimeric antigen receptors (CARs).
3. Dendritic cells, which exhibit an extremely potent ability to present antigens to T cells, have been used as a potential therapy via a vaccine, as they can independently mount a robust immune response.
4. A subset of the population of natural killer (NK) cells known as cytokine-induced killer cells has also been discovered and targeted as a potential immunotherapy method because the cells can be readily grown in vitro and show major histocompatibility complex (MHC)-unrestricted activity against tumors.
5. NK cells have recently advanced as a model for immunotherapy because of their ability to induce antibody-dependent cellular cytotoxicity (ADCC), manipulate receptor-mediated activation, and function as a form of an adoptive immunotherapy with CAR modiﬁcations.
Tumor-Inﬁltrating Lymphocytes (TILs)
Tumor-inﬁltrating lymphocytes are the most natural source of cells that can provide the desired immune response for cancer therapies. These cells were among the ﬁrst cells utilized for ACT in the 1980s, at which point it was demonstrated that TILs, cultured with IL-2, a lymphotrophic cytokine, exhibited cytotoxic activity against cancer cells in vitro. Since these cells were originally located within the tumors, they were found to be more potent than other lymphocytes in the body.
The technologies used to isolate and manipulate TILs are mostly geared toward preparing the patient’s own TILs, growing them, and reintroducing them into the patient to kill cancer cells.The adoptive cell transfer of autologous TILs has been shown to effectively mediate tumor regression in the majority of patients with metastatic melanoma, for example, and thus shows promise in terms of being able to achieve complete regression, which has been observed in a subset of patients with epithelial tumors.
In order to produce therapeutic TILs, resected tumor tissue is cultured in media containing IL-2 for approximately four to six weeks. Once enough TILs are grown from these cultures, they undergo rapid expansion during a two-to-four-week period with the aid of feeder cells, a higher concentration of IL-2, and soluble anti-CD3 antibody for two to four weeks. Next, these TILs are incubated with autologous tumor in order to select for the ones that actually react to the tumor. Once this is done, the levels of interferon (IFN)-γ secreted into the media can be measured using an IFN-γ enzyme-linked immunosorbent assay (ELISA).
Sometimes, the autologous tumor is unavailable or difﬁcult to grow in culture, posing a problem in t selection process. To mitigate this issue, researchers have developed new methods that utilize deep-sequencing technology to identify neoantigens that are presented by the tumor, which can then be synthesized as short peptides and used to identify tumor-reactive TILs.
T Cell Receptors (TCRs)
Since not all tumors yield readily available TILs, the use of TCRs that exhibit tumor- antigen peptides-recognizing properties has emerged as an important strategy for adoptive T cell therapies. Most of the clinical efforts in this realm to date have focused on self-peptides that are upregulated in some cancers, such as the WT1 antigen, differentiation antigens such as gp100 and MART-1, and cancer/testis antigens such as NY-ESO and MAGE-A3. Most of these human “tumor-associated” antigens that are targeted by TCR-engineered T cell therapy are also expressed in normal tissues, albeit at a lower density than on the surface of cancer cells. Therefore, there is a challenge to determine what TCR afﬁnity is necessary to confer therapeutic activity without posing a threat to normal or unrelated tissues, which is hard to anticipate. Many investigational efforts are focused on developing methods to capture neoantigen-reactive TCR genes from the patient’s peripheral blood or other samples.
Though this particular approach allows for the generation of tumor-speciﬁc T cells without the need to isolate TILs from tumors, it has a few limitations. The major drawback of this approach is HLA-restriction, where a given T cell will only recognize and respond to an antigen when it is bound to a particular MHC molecule. Another issue is the competition for pairing with endogenous TCR chains, which can lead to lower levels of tumor-speciﬁc TCRs or possible off-target reactivities of mispaired TCRs that can result in graft-versus-host reactions. To combat mispairing, scientists have started to use cysteines in exogenous TCR-constant domains that promote preferential pairing or gene editing strategies that limit the expression of endogenous TCR chains. Though this concern exists in theory, there have been no reported adverse events related to mispaired TCR formation in clinical trials.
Chimeric Antigen Receptors (CARs)
One alternative to obtaining T cells with anti-tumor reactivity while avoiding the complications that can arise from HLA-restriction is to genetically engineer T cells to express chimeric antigen receptors (CARs). CARs are receptors that have been engineered to give T cells the ability to target a speciﬁc protein by combining antigen-binding and T cell- activating functions into a single receptor. More speciﬁcally, CARs are hybrid receptors formed by the fusion of an extracellular tumor antigen-binding domain, typically a single-chain variable fragment (scFv) of an antibody, fused with intracellular T cell signaling and costimulatory domains.
CARs were originally generated by Zelig Eshhar and colleagues in the late 1980s in order to study TCR signalling. Due to their chimeric construction, CARs can provide non-MHC restricted recognition of cancer cell antigens, which ultimately results in targeted T cell activation. By incorporating chimeric molecules that recognize tumor antigens as well as actively promoting a cascade of signals that could induce further damage to tumor cells, CAR therapy can give patients an alternative that breaks the acquired tolerance of immune cells and bypasses the restrictions of HLA-mediated antigen recognition that are present with TLR-based therapies.
Typically, in order to generate CAR T cells, activated leukocytes are ﬁrst removed from the patient and then processed in order to isolate the autologous peripheral blood mononuclear cells (PBMCs). In order to activate T cells that can effectively ﬁght against cancer, the cells are incubated with IL-2, anti-CD3, and anti-CD28. Subsequently, the T cells are transfected with CAR genes through integration of a gamma retrovirus or lentiviral vectors and expanded using cytokines such as IL-7, IL-15 and IL-21. Since these CAR T cells are further divided into CD4+ and CD8+ subsets, these markers can be used to select these cells; the optimal ratio of CD4+ to CD8+ CAR T cells is of interest for maximum efﬁcacy of this line of treatment. Prior to the introduction of the engineered T cells, the patient often undergoes lymphodepletion chemotherapy. Lymphodepletion serves the purpose of depleting endogenous T cells, including Tregs, which promotes the expansion and survival of the CAR T cells once they have been reinfused.
Due to the success of CAR T cells targeted at CD19 in patients with B cell hematologic malignancies, the U.S. Food and Drug Administration recently approved two CAR T cell therapies. Tisagenlecleucel (Kymriah™ by Novartis), is indicated for the treatment of advanced leukemia in children and young adults up to 25 years of age who have large B cell acute precursor lymphoblastic leukemia (ALL) that has either relapsed or failed to respond to previous conventional treatment. The other, axicabtageneciloleucel (Yescarta™ by Kite Pharma), is approved for treating adults who have either relapsed or refractory cancer that has not responded to previous conventional treatment(s), high-grade lymphoma, diffuse large B cell lymphoma (DLBCL), or DLBCL resulting from follicular lymphoma.
Despite these breakthroughs in the treatment of hematological malignancies, it has been difﬁcult to use CAR T-cell therapy against solid tumors. The poor speciﬁcity and efﬁcacy of CAR T against these tumors can be at least partially attributed to the lack of speciﬁc targetable antigens. In addition, it is difﬁcult for CAR T cells to navigate in the hostile microenvironment of solid tumors, so future efforts are focused on alleviating these problems so that solid tumors can be better treated with this form of immunotherapy.
Recently, researchers have utilized the same CAR technology to equip other immune cells, such as NK cells and even macrophages to recognize tumors. Although these cells are probably not going to replace CAR T-cell therapy, these alternative approaches to ﬁghting cancer could add to the arsenal of therapies that are currently being developed. NK cells, which belong to the innate immune system, act as a ﬁrst line of defense against cancer cells, scanning the other cells in the body and destroying those that are defective or infected, such as tumor cells.
Preliminary studies conducted on chimeric antigen receptor-natural killer cells (CAR-NK cells) have shown that they perform as well as CAR T cells against ovarian tumors and substantially better than unaltered NK cells. In addition, CAR-NK cells have shown less toxicity compared to CAR T cells, which is a signiﬁcant beneﬁt for this new therapy.
Additionally, scientists have observed that NK cells harvested from a donor, engineered with CARs, and then administered to patients do not appear to cause the fatal immune complication of graft-versus-host disease. This phenomenon opens up the possibility to eliminate some of the expenses associated with therapies that rely on the extraction of immune cells from the patient’s blood in favour of approaches that can harvest these cells from umbilical cord blood donations, for example. Thus, one batch of human NK cells derived from induced pluripotent stem cells (iPSCs) could potentially be used to treat thousands of patients, while preventing the need to create a new product for each patient.
Learn more about CAR T-Cell Immunotherapy
Dendritic Cell Vaccinations
Dendritic cells (DCs) are leukocytes that are uniquely potent in their ability to present antigens to T c serving as a bridge between the innate and
adaptive immune systems. Due to this property, dendritic cells have been selected as a potential target for therapeutic cancer vaccines.
Dendritic cells were originally described in the 1970s by Steinman and Cohn, and they are often referred to as “nature’s adjuvant” because of the fact that they are the most potent antigen-presenting cells (APCs) and are capable of activating both naïve and memory immune responses. Since DCs
are able to independently mount a comprehen immune response, they are of particular interest in the formation of vaccines.
In order to form these vaccines, immature DCs are generated from immune cells that are removed from the patient’s blood, using IL-4 and GM-CSF, loaded with tumor antigen ex vivo, and matured. Once the dendritic cells are grown, the loaded DCs are then reinfused into the patient in order to induce protective and therapeutic anti-tumor response by allowing the vaccine DCs to present to T cells in the body.
Pilot clinical trials for patients with non-Hodgkin’s lymphoma and melanoma have shown an induction of anti-tumor immune responses and subsequent tumor regression.
Currently, there are more trials underway for DC vaccination for several other human cancers and some groups are exploring methods for in vivo targeting of tumor antigens to DCs. In addition, it has been shown that pre-conditioning the vaccine site with a potent recall antigen, such as tetanus/diphtheria toxoid, can signiﬁcantly improve the efﬁcacy of DC vaccines.
Thus, by utilizing the antigen-presenting mechanism of DCs, there are several opportunities to develop effective cancer immunotherapy. In fact, Sipuleucel-T (APC8015, trade name Provenge), developed by Dendreon Corporation, was the ﬁrst DC-based cancer vaccine approved by the Food and Drug Administration in 2010 for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer.
Although this vaccine has been shown to be effective, as it improves median survival by 4.1 months, it is still an expensive mode of treatment due to its personalized nature. Additionally, none of the phase III clinical trials found a signiﬁcant difference in the time to disease progression. These circumstances indicate the need for a signiﬁcant improvement of this mode of cancer immunotherapy to become widespread.
Natural Killer (NK) Cell Immunotherapy
NK cells are a part of the innate immune system and are characterized by their lack of CD3/TCR molecules and by the surface expression of CD16 and CD56. As such, they have the distinct ability to mediate cytotoxicity in response to stimulating a target cell. In addition, NK cells interact with other cells of the immune system in several ways: For example, by producing cytokines, such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ, they mediate downstream adaptive immune responses by inﬂuencing the magnitude of T cell responses. On the other hand, NK cells themselves are regulated by cytokines, such as IL-2, IL-12, IL-15, IL-18, and IL-21, and by interactions with other cells, such as dendritic cells and macrophages.
Initially, studies of adoptive NK cell therapy were oriented toward enhancing the anti- tumor activity of the NK cells. Doing so involved using CD56+ beads to select for NK cells and infusing the autologous CD56+ cells into patients, followed by the administration of cytokines IL-2 or IL-15 to encourage additional in vivo stimulation and support their expansion, but this method was found to be ineffective.
NK cell-based immunotherapy can potentially be used as a therapeutic option for solid tumors, which is more difﬁcult for other immunotherapies; however, challenges exist such as trafﬁcking to sites of tumors and penetrating the tumor capsule in order to exert their function. Some strategies to mitigate these setbacks are to target regulatory T cells in order to target the immunosuppressive tumor microenvironments, which could potentially help to treat solid tumors.
The most recent approaches have used a methodology in which NK cells are isolated from a patient and treated with a number of cytokines, initially IL-2, and more recently, other cytokines such as IL-12, IL-15, and IL-18. Once these NK cells have been expanded and activated ex vivo, they are infused into the patient. Studies in experimental models have shown that these cytokine- induced, memory-like (CIML) NK cells have signiﬁcant activity against tumors once they are infused. Expansion of NK cells isolated from PBMCs generally includes using feeder cells in order to provide the NK cells with a stimulatory signal, however, it was also shown that NK cells isolated from cord blood could be efﬁciently expanded by a feeder-free system.
Cytokine-Induced Killer (CIK) Cells
Cytokine-induced killer (CIK) cells are a heterogeneous population of effector CD3+ CD56+ NK cells that can be used in a similar fashion to other immunotherapy methods because they can be easily expanded in vitro from PBMCs. They are an ideal candidate for immunotherapy approaches since they exhibit MHC-unrestricted anti-tumor activity that is both safe and effective.
CIK cells were ﬁrst developed in 1991 by growing PBMCs in the presence of IFN-γ, an anti-CD3 monoclonal antibody, and IL-2. Subsequent studies showed that besides using IL-2, CIK cells could also be generated by using exogenous IL-7 or IL-12. Studies in which DCs were co-cultured with CIK cells showed that they interact with one another which resulted in changes to the surface molecule expression of both cell types and led to an increase in IL-12 expression. From a treatment perspective, combining DCs and CIK cells can be more effective than either one alone in therapies.
In order to generate CIK cells, PBMCs are ﬁrst separated from the blood through centri- fugation and then treated with IFN-γ to activate macrophages. This step promotes the IL-12- and CD58/LFA-3-mediated signaling, both of which enhance the cytotoxicity of CIK cells. After one day, the anti-CD3 antibody and IL-2 are added to the cells. Every 2 days, fresh IL-2 is added to the media; after three to four weeks of culture, the generated CIK cells can be infused back into the patient.
In the last few years, treatment prospects for CIK cells have improved, and a number of therapies have been developed in order to increase cytolytic activity and safety.
Numerous CIK clinical trials are ongoing or completed, and overall the results of these studies seem promising. CIKs can be combined with additional cytokines such as IL-6 and IL-7, DCs, immune checkpoint inhibitors such as CTLA-4 and PD-1, antibodies such as anti-CD20 or anti-CD30, and CARs, which can all improve the efﬁciency of CIK therapy. However, there are still many avenues of CIK therapy, especially in conjunction with other technologies, which need to be explored.
Immune Checkpoint Inhibitors
Inherently, the immune system has a system of inhibitory and stimulatory pathways that adjust their response to inﬂict the most damage on the pathogenic targets, while preventing collateral tissue damage and autoimmunity. Immune checkpoints are often manipulated by tumors in order to escape the protective immune response. Since many of these checkpoint molecules are mediated through ligand-receptor interactions, they can be easily targeted by antibodies or recombinant proteins. By inhibiting the inhibitory checkpoint, one can amplify antigen-speciﬁc T cell responses, which ultimately generates a more robust immune response.
Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are two of the many inhibitory receptors that have been used for clinical beneﬁt. CTLA-4, which counteracts CD28 activity, was the ﬁrst receptor of this nature to be targeted for clinical use. CD28 and CTLA-4 share identical ligands CD80 and CD86, but have opposite effects; when CTLA-4 is present, it competes with CD28 to bind to these ligands, thus dampening T cell activation, which is detrimental to the immune response. Therefore, it seems reasonable that blocking CTLA-4 could result in an increased immune response.
Preliminary studies with CTLA-4 antibodies showed that mice with partially immunogenic tumors demonstrated signiﬁcant anti-tumor responses when treated with CTLA-4, and eventually led to the production of clinical agents. Out of several CTLA-4 antibodies tested in clinical trials, ipilimumab was the ﬁrst therapy to demonstrate a survival beneﬁt for patients, especially with regards to long-term survival with metastatic melanoma and was approved by the U.S. Food and Drug Administration in 2010.
As an immune-checkpoint receptor, PD-1 is another promising target for immune checkpoint therapy. PD-1 limits the activity of T cells peripheral tissues during inﬂammatory responses to infection. This particular mechanism is exploited by many tumors that express the ligand, PD-L1, in order to evade an effective immune response by binding the PD-1 that is expressed on TILs from many cancers.
Thus, therapies targeting checkpoint molecules are promising. Currently, more than 2000 clinical trials are underway for therapies that block this pathway or combine it with some of the other aforementioned therapies.
Immunotherapy Against Other Diseases
Although the majority of articles discuss immunotherapy approaches against cancer, it is important to note that a wide variety of other diseases can be addressed with immunotherapy. Immunotherapy offers the potential to treat numerous conditions in addition to cancer because many diseases invoke immune responses and can manifest in many ways, such as through inﬂammation. For example, HIV-1 peptide-loaded DCs have been shown to be safe and to induce immunogenicity in individuals with HIV-1.
Regarding diseases caused by heightened inﬂammatory responses to otherwise harmless allergens, such as asthma and allergies, allergen immunotherapy has proven effective in controlling symptoms. By repeatedly exposing an individual to the relevant allergen, one can suppress and sometimes resolve the inﬂammatory response to the offending allergens. Through similar mechanisms to those discussed, the tau protein implicated in Alzheimer’s disease can be targeted and eliminated through the use of antibodies and may therefore potentially improve cognition in those who exhibit signs of dementia. All in all, there are numerous applications of immunotherapy that build on the same principles that have driven the development of immunotherapy with regards to cancer.
Immunotherapy harnesses the immune system to ﬁght a variety of diseases by suppression or activation of the immune response. The ubiquity of cancer has made the disease a target for innovations in this ﬁeld, including the development of vaccines and unique therapies that harness the exceptional abilities of immune cells.
Not only can immunotherapy treat cancers, but it can also address several chronic diseases, autoimmune disorders, and allergies. Such therapies generally provide long-term protection, have fewer side effects, and are more targeted than conventional therapies.
However, several challenges still exist for employing immunotherapy treatments. Major challenges include safety issues, developing personalized combination therapies, dose reﬁnement, cost reduction, target speciﬁcity, treatment duration, and disease management. Further research and advances may overcome many of these challenges and the future seems to be very bright for the ﬁeld of immunotherapy.