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Review ARTICLE

Front. Immunol., 18 August 2017 | https://doi.org/10.3389/fimmu.2017.01001

Therapeutic Antibodies against Intracellular Tumor Antigens

imageIva Trenevska, imageDemin Li* and imageAlison H. Banham*
  • Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom

Monoclonal antibodies are among the most clinically effective drugs used to treat cancer. However, their target repertoire is limited as there are relatively few tumor-specific or tumor-associated cell surface or soluble antigens. Intracellular molecules represent nearly half of the human proteome and provide an untapped reservoir of potential therapeutic targets. Antibodies have been developed to target externalized antigens, have also been engineered to enter into cells or may be expressed intracellularly with the aim of binding intracellular antigens. Furthermore, intracellular proteins can be degraded by the proteasome into short, commonly 8–10 amino acid long, peptides that are presented on the cell surface in the context of major histocompatibility complex class I (MHC-I) molecules. These tumor-associated peptide–MHC-I complexes can then be targeted by antibodies known as T-cell receptor mimic (TCRm) or T-cell receptor (TCR)-like antibodies, which recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. Advances in the production of TCRm antibodies have enabled the generation of multiple TCRm antibodies, which have been tested in vitro and in vivo, expanding our understanding of their mechanisms of action and the importance of target epitope selection and expression. This review will summarize multiple approaches to targeting intracellular antigens with therapeutic antibodies, in particular describing the production and characterization of TCRm antibodies, the factors influencing their target identification, their advantages and disadvantages in the context of TCR therapies, and the potential to advance TCRm-based therapies into the clinic.

Introduction

Historically the consensus in the immunotherapy field has been that antibody therapy is amenable to targeting only extracellular antigens that are accessible for antibody binding. This is due to the fact that the high molecular weight of antibodies prevents them from crossing the cell membrane to access intracellular targets. Consistent with this train of thought, the targets of approved antibody therapies are predominantly extracellular antigens (1). By contrast, small molecules have been used to target those intracellular antigens with a functionality that is suitable for drug screening. In comparison to antibodies, small molecules tend not to be as selective for their targets. They can exhibit unpredictable off-target activities, which consequently lead to adverse side effects and may require a more individualized clinical development pipeline.

More recently, there are three broad approaches whereby antibodies have been used to target intracellular antigens.

(1) It is possible for antibodies (or their derivatives) to target antigens that are normally intracellular but become externalized (for example, during disease).

(2) It is also possible to engineer antibodies or antibody fragments that penetrate into cells, or those that are directly expressed within cells using a gene therapy style approach.

(3) Antibodies can also be generated that bind cell surface major histocompatibility complex class I (MHC-I)-presented peptides that are derived from intracellular proteins.

With further developments in this field, it is becoming clear that the dichotomy between the antibody targeting of intracellular and extracellular targets is not as rigid as originally thought. Antibodies with novel mechanisms of action are challenging this belief and are re-defining the selection of suitable targets for antibody therapy. Antibodies that target intracellular antigens could open the door to a whole new realm of therapeutic targets, with potentially immense clinical benefits. While antibodies targeting intracellular antigens have broad clinical potential, this review will focus primarily on their application for cancer therapy.

Antibodies Targeting Externalized Antigens

Intracellular antigens can become externalized on the cell surface or secreted and can, therefore, be targeted by antibodies. The Zeng group has further explored the possibility of developing antibodies to intracellular oncoproteins. After an initial proof-of-concept study investigating intracellular proteins targeted by both antibody and vaccine therapy, they focused on phosphatase of regenerating liver 3 (PRL-3) and developed a humanized anti-PRL-3 antibody (2, 3). PRL-3 is a cancer-related phosphatase (4) that is reported to be involved in malignant transformation and metastasis, as well as its expression correlating with poor prognosis (5). It is undetectable in most normal human tissues, is involved in colorectal cancer and uveal melanoma, and is overexpressed in 85% of gastric cancers (but not patient-matched normal gastric tissue), which is the cancer model that has been further studied (3). Importantly, intracellular PRL-3 can be externalized by tumor cells, thus enabling its targeting using classical antibody technology.

It is not the first time that secreted or externalized intracellular proteins have been observed on cancer cells or within the tumor microenvironment and identified as potential therapeutic targets. One such example is the intracellular melanosomal membrane glycoprotein, gp75, which is normally expressed in the melanosome, a specialized organelle present in melanocytes. In melanoma, gp75 is expressed on the cell surface of malignant melanocytes and can be targeted by antibodies in mouse melanoma models (6). In addition, heat-shock proteins 70 and 90 are chaperone proteins, which are further examples of targets that are intracellular in normal cells but become presented on the cell surface, or secreted into the extracellular environment, in transformed cells (7, 8). Tumor cells have been previously shown to shed intracellular material into the tumor microenvironment and extracellular space. This is believed to be a consequence of the inflammatory reaction that surrounds tumor tissues, where immune surveillance can provoke apoptosis and necrosis of tumor cells, thus releasing intracellular components into the extracellular space (9). It has also been suggested that typically intracellular antigens can also be externalized through unconventional secretion pathways (10). This is corroborated by the observation that antibodies against gp75 can reject tumors where there is no necrosis, suggesting an alternative pathway enabling antigen externalization (6). It is the restricted expression profile and the secretion and externalization of PRL-3, by cancer cells, that make it possible to selectively target this oncoprotein with antibody therapy. In this context, it is possible to target an intracellular oncoprotein, which has become externalized onto the cell surface, with an antibody in the same manner as targeting a classical cell surface target.

Several observations have been made on the possible mechanisms of action that mediate the therapeutic effect of targeting extracellularized antigens with a non-neutralizing antibody. It is postulated that, in vivo, the Fc portion of these antibodies can be recognized by immune effector cells that have immunoglobulin (Ig) receptors (FcRs), such as macrophages, B cells, and natural killer (NK) cells (11). Therefore, the mechanisms of action could involve a combination of the following:

(1) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells,

(2) antibody-dependent cellular phagocytosis by macrophages,

(3) secreted antigens bound to antibody can form immune complexes that can be processed by dendritic cells, which then proceed to activate NK cells (12).

The importance of immune effector cells to the therapeutic efficacy and the aforementioned hypotheses are corroborated by the Zeng group’s previous findings, which showed that anti-PRL-3 antibodies have no therapeutic activity in immunocompromised SCID mice or in vitro against PRL-3-expressing cancer cells where no effector cells are present (2, 13). Such engagement with innate immune effectors is a common mechanism of action of therapeutic antibodies that do not modify the activity of the target antigen, including those against cell surface targets.

Intracellular Antibodies

Intracellular antibodies, which may also be called intrabodies, are antibodies that are produced in the cell, and bind an antigen within the same cell. This is a different delivery strategy from antibodies that are produced extracellularly, and are engineered to then penetrate the cell to access their intracellular target.

Antibodies are soluble proteins that are normally found circulating the body within the serum. They are synthesized in the endoplasmic reticulum (ER) of B cells as separate heavy chain and light chains, which are then linked by disulfide bonds in the mature Ig. However, the full-length antibody is not functional in the cytosol, prior to secretion, due to its reducing conditions, which affect protein folding and the intramolecular disulfide bonds that are required to maintain the antibody’s conformation and stability (14). Fortunately, the complementarity-determining regions that endow an antibody with its exceptional target specificity are located in the variable regions of both the heavy and light chains. Therefore, it is possible to use antibody fragments incorporating the specificity-providing regions within a single-chain variable fragment (scFv), which can be further engineered for cytosolic stability, to target intracellular antigens (15, 16). The scFv is a single polypeptide, which is a favorable characteristic for in vivo expression, and it has been studied as a therapeutic for viral infections and cancer, among other diseases.

Furthermore, the variable (V) region domain can be used by itself to form a domain antibody or Dab (17). These can be engineered from conventional human Igs, or also from those from camelids (camel or llama) and cartilaginous fish (carpet or nurse sharks), whose immune systems were found to have evolved high-affinity V-like domains fused to a conserved framework that is reflective of the constant Fc region found in human Ig (18, 19). It has been reported that single heavy chain V regions or light chain V regions can be expressed inside cells. These are referred to as intracellular domain antibodies, which do not require intramolecular disulfide bonds for stability, hence representing the smallest format of the antibody that retains target specificity while minimizing size—a crucial factor for intracellular targeting (20).

There are several critical aspects to generating functional intracellular antibodies. The first is designing an antibody format that will retain its stability and antibody binding capacity within the cell and the second is the ability to introduce or express the antibody within cells. Furthermore, as intracellular antibody fragments do not possess an Fc region (and full length intracellular antibodies cannot recruit extracellular immune effector cells from within the cell), different strategies must be employed to equip them with effector functions unless they have directly neutralizing activity against the target. Examples include ER targeting to cause degradation of the target protein, antibody–antigen interaction-dependent apoptosis that is used to induce programmed cell death through the activation of caspases, and suicide intrabody technology that causes proteolysis of the target protein (21, 22).

In cancer, some of the proteins that are key players in signaling pathways leading to malignant transformation have thus far been inaccessible to small molecule inhibitors (23). In particular, some of these are large, intracellular proteins that act as molecular scaffolds and function primarily through facilitating protein–protein interactions (PPIs). Due to their size, small molecules cannot physically block the large surface of such proteins, nor interfere in the protein–protein interfaces they form, which are typically hydrophobic, flat surfaces, presenting few possibilities for small molecule anchorage (24). This is where technologies that enable the use of antibodies within the cell can bridge the gap between small molecule inhibitors and large protein targets. In this context, the proteins themselves are not the target, but it is the interactions they form with other proteins or nucleic acids that are the therapeutic targets as they contribute to the diseased state. One example is the use of an anti-RAS intrabody, which is composed of a single variable heavy region domain that targets activated GTP-bound RAS. This antibody competitively blocks RAS-effector functions within the tumor cell and while able to prevent in vivo tumor initiation and further tumor growth in murine models, it was not curative (25, 26). Thus, some antibodies may enable control of tumor growth and require combinations with additional agents to potentially achieve a cure.

Intrabodies can also be used to characterize the expression of their target proteins and study the in vivo knockdown of protein function, and can represent an alternative to generating gene knockout animal models. There are different types of intrabodies that can be tailored to target proteins within subcellular compartments, primarily the cytoplasm or the ER, but the addition of a signal peptide also allows targeting to the mitochondria or the nucleus. This can be used to confer additional subcellular specificity on their intracellular targeting. Importantly, antibodies retained in the ER do not experience the problems with conformation that are caused by the reducing conditions within the cytosol and can be active without neutralizing function against their target (27). For example, using intrabodies targeted to the ER (using a “KDEL” or “SEKDEL” sequence) allows the knockdown of proteins that are passing through the ER, thus abrogating their downstream function in a similar way to RNA interference and providing an alternative strategy for silencing gene products. It has also been proposed that ER-targeting intrabodies may maintain silencing more effectively than short interfering RNA (siRNA) and their specificity may be easier to predict than the off-target effects of an siRNA. An intradiabody that simultaneously enabled the knockdown of VEGF-R2 and Tie-2 was able to reduce both tumor growth and angiogenesis in vivo (28). Intrabody technology is overviewed in depth by recent reviews including Marschall and Dübel (29).

Delivery of Antibodies to the Intracellular Compartment

Despite the general consensus that antibodies can only be used to target extracellular or secreted antigens, the cellular uptake of antibodies (by processes such as endocytosis) has been observed both clinically and experimentally in the case of autoimmune disease. It has been reported that once autoantibodies bind their intracellular target, they can cause apoptosis of the cell (3032). Therefore, the idea of using intracellular antibodies therapeutically represents a logical expansion of such observations. At present, the use of intracellular antibodies is still limited by the technology needed for antibody delivery and they are used primarily as research tools. A number of different methods are being investigated for the delivery of antibodies to the intracellular compartment within target cells. Some of these strategies are illustrated in Figure 1 and they fall into two broad strategies:

(1) The first is a type of “gene therapy” approach using vectors that enable expression of the intracellular antibodies within the target cell—these can be either viral vectors or plasmids.

(2) The second is direct administration of the antibody-based therapeutic—either alone, using electroporation or with dendrimers, liposomes, nanoparticles, or by fusing the antibody to protein-transduction domains that enable it to penetrate the cell (33).

FIGURE 1
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Figure 1. Strategies for targeting intracellular tumor antigens with antibody therapy. Some of the methods for targeting intracellular tumor antigens are illustrated. (A) Intracellular antigens can be externalized on the cell surface or secreted, allowing targeting by antibodies. (B) Plasmids or viral vectors can be used to deliver antibody-encoding genes into the cell. Once internalized, the DNA is transcribed into the targeting antibody, which can be designed to translocate to the nucleus, mitochondria, endoplasmic reticulum (ER), or cytoplasm. (C) Nanoparticles, dendrimers, or liposomes can be used to deliver an antibody or an expression vector encoding the intracellular antibody into the target cell. (D) Antibodies can be fused to cell-penetrating peptides, which allow internalization of the antibody. (E) T-cell receptor mimic (TCRm) antibodies can be used to target peptides bound to major histocompatibility complex class I (MHC-I) molecules on the cell surface. The peptides are derived from intracellular proteins, which have been degraded by the proteasome into short peptides. Peptides are loaded onto MHC-I molecules in the ER, transported through the Golgi apparatus, and finally presented on the cell surface. (F) The antibody depicted on the diagram could represent a full-length IgG, a Fab fragment, scFv or a single domain antibody.

Viral vectors that can be used to deliver the genetic information for expression of intracellular antibodies include adenovirus, adeno-associated virus (AAV), and retrovirus (including lentivirus), which have all been studied extensively in the pre-clinical setting as gene therapy delivery vehicles (34). Retroviruses integrate the antibody fragment expression cassette into the host genome, allowing long-term expression of the intracellular antibody fragment. Despite this advantage, a safety concern with the use of lentiviruses is the risk of integration of the expression cassette in the proximity of an oncogene in the host genome, thereby triggering secondary cancers. By contrast, AAV releases the DNA as an episome, avoiding such safety concerns, however, there is always the possibility of loss of expression, which means relatively shorter term expression.

A non-viral strategy for delivering genes or proteins to the intracellular compartment involves the encapsulation of DNA or proteins in cationic lipid structures called liposomes (3537). Liposomes form a closed, spherical particle that is amphiphilic and composed of one or more lipid bilayers with an aqueous center. In addition to delivering antibodies, they can also be coated with antibodies that bind cell surface proteins on the target cells (38). Thus, they are targeted to a specific cell type and can deliver an antibody, or an expression vector encoding the intracellular antibody, to the target cell without employing a viral delivery method. Liposomes are internalized via endocytosis following interaction with the plasma membrane, which is based on multiple factors, including particle size and charge interactions (39). Nanoparticles are an alternative non-viral method for delivering DNA or antibodies intracellularly (40, 41). They are made of polymers such as poly lactic-co-glycolic acid (PLGA), which is an FDA-approved polymer that has been studied extensively for therapeutic applications (42). PLGA-based nanoparticles have been used to improve the endocytic cellular uptake of antibody fragments such as 3D8 scFv (43). Similarly, antibody-coupled delivery can be used, wherein the expression vector DNA is coupled to the C-terminus of an antibody that binds a cell surface target. The vector DNA is internalized upon internalization of the delivery antibody, and the therapeutic antibody it encodes is then expressed intracellularly (44). Expression vectors and antibodies can also be conjugated to dendrimers (synthetic polymers with a branching tree-like structure) for delivery into target cells (45).

Fusion to cell-penetrating peptides may be an alternative method for delivering antibody fragments into cells through protein transduction. Antibodies that have cell-penetrating peptides fused to them can be referred to as TransMabs (46). The first TransMab that was generated was composed of an anti-caspase-3 antibody fused to a 17 amino acid peptide that could translocate the antibody across the plasma membrane of target cells (47). This then blocked events related to apoptosis, such as caspase-3 activity and DNA fragmentation. Using this method, the antibody–peptide fusion protein enters the cell through endocytosis (48). However, it is difficult to predict whether sufficient macrodrug will enter the cell in order for it to mediate a therapeutic effect. Particularly as cell-penetrating peptides fused to macromolecules have been reported to be at risk of being trapped within endosomes (49). Another disadvantage of this method is that antibody fragments will undergo degradation in the intracellular compartment, as would any protein, therefore, continuous re-administration would be required to maintain any therapeutic activity. A cell-penetrating IgG1 antibody targeting activated GTP-bound RAS (RT11) has recently been shown to block oncogenic signaling and inhibit tumor growth in mouse xenograft models with mutated but not wild type Ras. This iMab (internalizing and PPI interfering monoclonal antibody) has successfully blocked the activity of a highly desirable oncogenic target that lacks effective small molecule inhibitors (50).

T-Cell Receptor Mimic (TCRm) Antibodies

Immunotherapies targeting intracellular proteins can also exploit the immune system’s own intracellular surveillance mechanism. Intracellular proteins are degraded by the proteasome to form short peptides of specific lengths. These peptides are then presented on the cell surface of most nucleated cells, in a complex with MHC-I molecules (51). CD8+ T cells recognize peptide–MHC-I complexes through their clonotypic T-cell receptor (TCR) and become activated to kill malignant or virus-infected cells that present tumor or viral peptides (51). Significantly these MHC-presented peptides do not have a functionality that would make them suitable targets for small molecule drug screening.

Antibodies targeting disease-associated peptide–MHC-I complexes, the so-called TCRm antibodies or TCR-like antibodies, are similar to the TCR in that they bind both the peptide and the MHC-I molecule and, therefore, their binding is both peptide-specific and MHC-restricted (52, 53). TCRm antibodies have expanded the range of targetable antigens to include intracellular proteins without the delivery complications associated with intracellular antibodies. Another advantage of TCRm antibodies is that they combine the intricate tumor specificity of TCRs with the biological properties of antibodies, which do not succumb to immune regulatory mechanisms that obstruct T-cell function in the tumor microenvironment (54). Like conventional monoclonal antibodies, TCRm antibodies have been shown to cause tumor killing through antibody-dependent mechanisms such as cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (55, 56). Furthermore, studies have shown a TCRm antibody to cause apoptosis in breast cancer cells through a caspase-dependent pathway (55). In addition to the success of using naked TCRm antibodies, there have also been reports of anti-tumor activity when they are conjugated to toxins (57, 58). The ability of TCRm Abs to target intracellular antigens has also been applied to cellular therapies in the development of chimeric antigen receptor T cells (59, 60).

TCRm Antibodies Published to Date

Since the advent of the necessary techniques and technologies, there has been an increase in the production of TCRm antibodies and constructs derived from them. The target peptides of such reagents have typically derived from either viral antigens (including HIV and Hepatitis B antigens) or cancer antigens, and they are commonly presented by either the HLA-A*0201 or the HLA-A*2402 MHC-I haplotype (61, 62). While TCRm antibodies can be used for therapeutic purposes, they are also widely used as research tools for the study of antigen presentation and recognition, as well as for structural studies. Some of the TCRm antibody therapeutics have shown promise in both in vitro and in vivo studies, however, none of them have advanced to clinical studies. Information on the TCRm and TCR-like antibodies generated to date is summarized in Table 1.

TABLE 1
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Table 1. TCRm antibodies for cancer immunotherapy.

Production of TCRm Antibodies

T-cell receptor mimic antibodies are not as commonly available as traditional antibodies; this may be a consequence of the difficulty of their production in addition to the technology being less established. Recently, there has been an increase in the generation of TCRm antibodies targeting a variety of cancer or viral T-cell epitopes due to advances in the necessary technologies and techniques. TCRm antibodies have been produced either by immunization or by phage display, with both strategies presenting their respective pros and cons. One of the main limitations in the production of TCRm antibodies by both strategies was the correct refolding of recombinant peptide–MHC complexes and their purification (53). Recombinant peptide–MHC complexes are made by using bacterial expression to generate inclusion bodies containing the extracellular domains of the heavy chain of human leukocyte antigen (HLA) and β2-microglobulin. These are then refolded with the MHC-restricted peptide to generate correctly refolded monomers of high purity, in quantities that are sufficient for downstream applications. The correct refolding can be verified by structural and functional experiments, and the monomers can then be biotinylated for specificity and affinity characterization, and for antibody isolation (8688).

Initially, TCRm antibodies were produced using hybridoma technology. The immunization methods used in these experiments limited the successful generation of TCRm antibodies. Antigen-presenting cells harboring immunogenic peptides in the groove of their MHC molecules were used as immunogens (89, 90). Obtaining TCRm antibodies of the correct specificity by employing this method yielded very few antibodies and many efforts proved to be unsuccessful (91). Since then, more successful attempts have been made by using recombinant peptide–MHC complexes, such as tetramers, in the immunization protocol, followed by high-throughput screening in order to isolate specific TCRm antibodies out of a pool of thousands of clones (62, 71, 81). This requires stable peptide–MHC-I binding and has resulted in the production of TCRm against tumor and viral T-cell epitopes.

While the traditional strategy for making TCRm antibodies is hybridoma technology, in the mid 1990s, it was shown that phage display technology could also be used to isolate antibodies (87). In this method, libraries of phage particles are generated, where each phage displays a unique antibody (a scFv or a Fab fragment) as a fusion protein on their surface. Each phage particle has the genes that encode the particular antibody that is expressed on its cell surface. Therefore, it is possible to select different phage particles by assessing whether they bind a target and thereby isolate the antibodies that have the desired specificity. The bound phages are then eluted and amplified in bacteria. Phage display technology has been used to isolate various TCRm antibodies against cancer antigens (63, 69, 92, 93).

Most TCRm antibodies published thus far have used phage display libraries for antibody production. Investigators argue that the main advantage of phage display is that it is efficient while being a relatively fast method (53). On the other hand, hybridoma technology is a relatively slower strategy, and it requires the immunogenic peptide and the MHC complex to bind with high affinity and form a very stable complex in order for the complex to persist throughout the immunization and in vivo IgG maturation. Nevertheless, the advantages of hybridoma technology include the isolation of antibodies that have a high affinity (in the low nanomolar range) for the peptide–MHC complex. This is due to the fact that antibodies undergo multiple antigen challenges and affinity maturation in vivo. Whereas affinities of TCRm antibodies produced through phage display tend to lie in the moderate nanomolar range (≈50–300 nM) and many require further in vitro affinity maturation (94, 95).

Furthermore, the antibodies produced through hybridoma technology are bivalent IgG isotype antibodies, whereas antibodies isolated using phage display are either scFv or Fab fragments (i.e. in the monovalent form with no Fc region). The Fc portion of the antibody is crucial in recruiting components of the immune system for cytotoxic effects mediated through ADCC and CDC. Antibodies in the monovalent form have reduced avidity (functional affinity) and increased turnover rates, which are undesirable when targeting epitopes that may be expressed at low densities, such as epitopes on tumor-associated peptide-MHC complexes (53). To circumvent this difficulty, further engineering can be undertaken to address these limitations. For example, scFv or Fab tetramers can be generated through biotinylation, thereby increasing their avidity or antibodies can be engineered to have a classical Fc region. On the other hand, monovalent antibody fragments are ideal for studies of epitope presentation and structure, as well as being used as the targeting moieties that deliver a conjugated toxin to target cells. One advantage over immunization of mice to generate antibodies (unless using those genetically engineered to have a human B-cell repertoire) is the possibility to generate fully human antibodies from display libraries.

Considerations for Selecting TCRm Antibody Targets

The ideal target for a TCRm antibody would be a disease-specific peptide–MHC complex that is present at high density on the target cell surface while being absent from other normal cells. When considering TCRm antibodies against tumor targets, such peptides are most likely to arise from overexpressed proteins, which have a short half-life and, hence, a high turnover rate (96). Targeting an antigen with a functional role in tumor biology will also help avoid loss of the antigen under subsequent therapeutic selection pressure. The peptides must also have a high affinity for the patient’s MHC and form a stable complex that persists on the cell surface, allowing recognition by TCRm antibodies.

Antigens that could be promising therapeutic targets include peptides processed from mutated proteins, which are tumor specific, such as KRAS G12V/D or oncogenic fusion proteins (97, 98). Over-expressed genes, cancer testis antigens, and re-expressed oncofetal proteins are also potential tumor targets, for example, CEA and WT1 (99). The expression of these targets on normal healthy tissues must be considered when developing these as therapeutics (100). TCRm antibodies could also have use in targeting cells of the tumor microenvironment, such as regulatory T cells, tumor-associated macrophages, or cells with a role in angiogenesis (101, 102).

A key factor that needs to be considered when choosing a target antigen for TCRm antibody therapy includes the epitope expression on the cell surface. It is important to consider that it is the presentation of the epitope, and not expression of the antigen per se, that will determine the availability of antibody binding sites. Epitope density of TCRm antibody targets has been reported to be as low as 100–1,000 sites per cell, which is significantly lower than some epitope densities reported for traditional mAb cell surface targets at 20,000–500,000 sites per cell. Nevertheless TCRm can activate ADCC against low-density targets (82, 103, 104). Before being presented on the MHC molecule, the peptide undergoes various steps of processing from its original protein. Therefore, events at any of these steps could affect the epitope density observed at the cell surface, including the level of protein expression and its half-life, the peptide processing, the MHC levels, and the presentation of the peptide in the context of MHC at the cell surface. Proteins must be stable and translated in sufficient quantities to allow peptide processing, and it has been shown that proteins with shorter half-lives are more likely to be presented than ones with longer half-lives (105). Furthermore, it has been reported that tumors downregulate their surface MHC expression as an immune evasion mechanism, suggesting that such tumors will be less susceptible to TCRm therapy (106, 107). The possibility of this evasion mechanism must be considered when selecting both target antigens and disease indications.

Target Epitope Discovery

Progress in our understanding of peptide processing and presentation on MHC has facilitated the discovery and evaluation of novel peptide–MHC epitopes. Initially, expression profiling was used to identify epitopes on tumor-associated antigens (TAAs) found on tumor cells—a process also called “direct immunology.” Using this method, the isolation of tumor-specific CTLs from melanoma patients led to the discovery of the first tumor-specific CTL epitope, which was encoded by the MAGE-1 gene. A cDNA library of the melanoma was generated, and melanoma-specific CTLs were used to identify the cDNA that encoded the CTL epitope (108, 109). Since this initial discovery, the use of direct immunology has led to the identification of other epitopes, including ones from the MAGE, BAGE, and GAGE families, as well as Melan-A/MART-1, tyrosinase, and gp100.

Bioinformatics techniques using algorithms to predict peptide binding to specific MHC molecules are often used to predict TAA epitopes. This process is known as “reverse immunology” and is a systematic method of identifying TAA epitopes from a defined antigen that has emerged from the recent progress in genome sequencing and in silico techniques. It involves a prediction phase, where potential epitopes are predicted in silico using algorithms. The prediction of epitopes is based on proteasome processing, binding to MHC and TAP translocation. This is followed by the validation phase, where the predicted epitopes must then be verified by MHC-I peptide binding assays or mass spectrometry to confirm that they are found on the cell surface (110).

There are a significant number of peptides that while being capable of binding MHC-I are either not presented on cancer cells or are altered, for example, by post-translational modification. Thus, there has been considerable interest in performing cancer HLA peptidome analysis to identify MHC-I bound peptides within both normal and malignant cells and tissues (111). In this approach, the HLA-complexes are immunoaffinity purified, the bound peptides are isolated and then analyzed by mass spectrometry. By comparing the MHC-I bound peptides in normal and diseased tissues, it is possible to prioritize those epitopes that are most suitable for therapy. Interestingly a meta-analysis of the HLA peptidomes from 83 mass spectrometry-based datasets from four major hematological malignancies found very few common “pan-leukemia” epitopes and these exhibited low presentation frequencies within each cohort of patients (112). Thus, in hematological malignancy, the epitopes selected for therapy are likely to be disease specific and, thus, multiple TCRm antibodies will be needed to exploit this therapeutic approach.

TCRm Antibodies and TCR-Based Therapies

Both TCRm antibodies and recombinant TCRs can bind MHC-I presented peptides. Traditionally, those employing TCR-based therapies have compared their technology to the desirable qualities of antibodies but commented on the inability of antibodies to target intracellular antigens. Those generating TCRm/TCR-like antibodies have promoted antibodies having higher affinity and specificity than TCRs (82, 113) and an easier development route and lower cost than TCR-targeted cellular therapies. However, advances in the engineering and production of soluble high-affinity TCRs and the production of TCRm antibodies have now made these approaches much more interchangeable.

T-cell receptor mimic antibodies can be used in place of a TCR as the targeting moiety for cellular therapies, such as CAR T cells (59, 60). Alternatively, a TCR can be fused to an Ig Fc region to enable TCR-directed antibody-dependent cytotoxicity (114). ImmTACs (immune-mobilizing monoclonal TCRs against cancer) are engineered high-affinity soluble TCRs bispecifically linked to anti-CD3 that can drive an anti-tumor T-cell response (115). Some studies have reported that the orientation of binding is similar for TCRs and TCRm antibodies, with both binding their peptide–MHC target in a diagonal orientation (116, 117). TCRm antibodies can also bind in additional conformations, gaining access to epitope regions that are not naturally targeted by TCRs (94, 118).

It seems likely that both TCRm antibodies and TCRs will be used to effectively target intracellular antigens using both soluble drugs and cellular therapies. The specificity of binding of high-affinity TCRs and TCRm antibodies to the target peptide presented by MHC-I is likely to be a crucial determinant of the suitability of individual reagents for therapy. Comparative studies using TCRs to the tumor-associated antigen survivin effectively highlighted the importance of specific peptide binding. High-affinity TCRs against a survivin peptide presented by HLA-A2 isolated from an allogeneic HLA-mismatched TCR repertoire lacked the ability to distinguish high levels on tumor cells from low expression in normal tissues. This included activated T cells, leading to fratricide when the engineered T cells targeted each other for destruction. However, an autologously derived TCR to the same survivin peptide targeted tumor cells but did not cause fratricidal toxicity (119). Molecular modeling of TCR–peptide–HLA complexes and alanine scanning of the survivin peptide demonstrated that maximal peptide recognition was critical for TCR selectivity for tumor cells. Thus, the specificity of the peptide–MHC binder could be as critical as the choice of target peptide.

Future Directions for TCRm Antibodies

T-cell receptor mimic antibodies have not yet entered the clinic, although Novartis have partnered with Eureka Therapeutics and Memorial Sloan Kettering Cancer Center to develop their ESK1 TCRm targeting WT1. Several key factors have the potential to improve the development of TCRm antibodies further with the prospect of undertaking clinical studies and ultimately establishing them as cancer therapeutics. These include epitope expression, production methodology, specificity validation and mechanism of action. It is also important to consider that TCRm antibodies may represent theranostics, combining diagnostic utility to determine target epitope presentation and therapeutic activity within a single agent.

The low epitope density of peptide–MHC complexes on the cell surface poses some limitations for TCRm antibody-based therapy. This might be addressed by choosing target epitopes that do not have low cell surface expression, by increasing MHC-I expression in tumors, by making TCRm antibodies more sensitive to low density epitopes or by choosing effector mechanisms that do not require high epitope density for cytotoxicity.

High-affinity, peptide-specific TCRm antibodies have proven difficult to produce in large numbers by either traditional phage or hybridoma approaches. Enhanced display technologies, particularly those capable of isolating fully human antibodies within a short period of time, offer some exciting opportunities to accelerate future TCRm antibody discovery. Having a wider array of antibodies for characterization will improve the chances of identifying those with the necessary affinity and specificity for further development.

It is crucial to ensure that the TCRm antibody does not recognize the MHC-I alone, as this molecule is found on most nucleated cells. Therefore, the TCRm antibody must be specific for the peptide–MHC complex, which also highlights that it should not cross-react with other processed peptides. As the TCRm antibody recognizes only few amino acid residues in the peptide, it will be crucial to assess which other processed peptides possess the same amino acids at those positions and whether there would be any risk of cross-reactivity. The importance of this is exemplified in a clinical trial of an affinity-enhanced TCR, which targeted a MAGE-A3 epitope (120). Following administration of the therapy, it was discovered that the TCR also recognized an epitope on the unrelated protein titin that is expressed in cardiac tissue. The cardiac toxicity led to two patient deaths. This cross-reactivity was not observed in normal tissue screening and the titin peptide was not conserved in mice. However, a limitation of in silico screening by amino acid substitution is that it may identify a wider variety of potentially cross-reactive peptides than can be functionally evaluated. Furthermore, even potentially cross-reactive peptides shown to bind MHC-I in T2 presentation assays may not be processed endogenously or presented on the cell surface in normal tissues.

One of the key limitations of TCRm antibody therapy is the MHC-restricted nature of the therapy—although this is crucial to enable the recognition of intracellular proteins. Most studies to date focus on the HLA-A*0201 haplotype, which is prevalent in up to 40% of Caucasians, and in up to 20% of populations of different ethnicities, which covers a large proportion of the world’s population. There are other dominant HLA alleles worldwide, including HLA-A*2402 and HLA-A*1101 in Oriental populations. Although TCRm antibodies are HLA-restricted, it has been proposed that antibodies to three HLA alleles for a particular target antigen would cover >96% of the world’s population (53). Structural analysis of the TCRm antibody ESK1 shows that it binds multiple HLA-A*02 variants and not only the HLA-A*0201 subtype, which it is designed to target (121). This is due to the fact that ESK1 binds a portion of the MHC molecule that is conserved among the various HLA-A2 subtypes, thereby suggesting that the certain TCRm could target a larger population of patients with a variety of HLA subtypes. In addition, designing TCRm antibodies that target different antigens or different epitopes on the same antigen and using a combination of these as a therapeutic regimen could increase the chances of successful tumor eradication and minimize escape variants.

The manufacture and regulatory approval pathways for TCRm antibodies are likely to have similarities to that for classical monoclonal antibodies and share commonalities with TCR-based therapies. The latter being the lack of availability of suitable animal models to study agents targeting a dual epitope where potentially neither the MHC-I or target peptide is conserved. One opportunity potentially available for TCRm antibodies would be to use in vivo imaging studies to study the biodistribution of a subtherapeutic dose of the TCRm antibody in early clinical safety studies.

Conclusion

The generation of antibodies that can target intracellular antigens offers an unparalleled opportunity to expand the repertoire of therapeutic antibodies that are available to treat human disease. When coupled with advances in genomic sequencing technologies, proteomic investigations and the increasing numbers of antibodies being made available to the research community, new disease-related proteins and their variants (post translational modifications, splice variants, mutations, etc.) that are suitable for antibody targeting will continue to be identified. Further developments in the production technology, delivery, and regulatory approval pathways for antibodies targeting intracellular antigens should also contribute to the introduction of many new exciting antibodies into the clinic in the future.

Author Contributions

All authors contributed to drafting, revising, and approving the final article.

Conflict of Interest Statement

AB and DL are inventors, and IT is a contributor, on a patent application describing the production and characterization of TCR mimic antibodies targeting p53:HLA-A2.

Funding

DL is supported by a Cancer Research UK project grant awarded to AB, DL, and Prof. Adrian L. Harris (C10396/A21667). IT is supported by a University of Oxford Medical Sciences Graduate School Studentship funded by the Medical Research Council, UK.

References

1. Abramson RG. Overview of Targeted Therapies for Cancer. My Cancer Genome (2017). Available from: https://www.mycancergenome.org/content/molecular-medicine/overview-of-targeted-therapies-for-cancer/

Google Scholar

2. Guo K, Li J, Tang JP, Tan CPB, Hong CW, Al-Aidaroos AQO, et al. Targeting intracellular oncoproteins with antibody therapy or vaccination. Sci Transl Med (2011) 3:99ra85. doi:10.1126/scitranslmed.3002296

PubMed Abstract | CrossRef Full Text | Google Scholar

3. Thura M, Al-Aidaroos AQ, Yong WP, Kono K, Gupta A, Lin YB, et al. PRL3-zumab, a first-in-class humanized antibody for cancer therapy. JCI Insight (2016) 1:1–15. doi:10.1172/jci.insight.87607

PubMed Abstract | CrossRef Full Text | Google Scholar

4. Saha S, Bertilsson H, Abdollahi P, Størkersen Ø, Våtsveen TK, Rye MB, et al. A phosphatase associated with metastasis of colorectal cancer. Science (2001) 294:1343–6. doi:10.1126/science.1065817

CrossRef Full Text | Google Scholar

5. Bessette DC, Qiu D, Pallen CJ. PRL PTPs: mediators and markers of cancer progression. Cancer Metastasis Rev (2008) 27:231–52. doi:10.1007/s10555-008-9121-3

PubMed Abstract | CrossRef Full Text | Google Scholar

6. Takechi Y, Hara I, Naftzger C, Xu Y, Houghton AN. A melanosomal membrane protein is a cell surface target for melanoma therapy. Clin Cancer Res (1996) 2:1837–42.

PubMed Abstract | Google Scholar

7. Weidle UH, Maisel D, Klostermann S, Schiller C, Weiss EH. Intracellular proteins displayed on the surface of tumor cells as targets for therapeutic intervention with antibody-related agents. Cancer Genomics Proteomics (2011) 8:49–64.

PubMed Abstract | Google Scholar

8. Weidle UH, Maisel D, Brinkmann U, Tiefenthaler G. The translational potential for target validation and therapy using intracellular antibodies in oncology. Cancer Genomics Proteomics (2013) 10:239–50.

PubMed Abstract | Google Scholar

9. Rock KL, Kono H. The inflammatory response to cell death. Annu Rev Pathol Dis (2008) 3:99–126. doi:10.1146/annurev.pathmechdis.3.121806.151456

CrossRef Full Text | Google Scholar

10. Nickel W, Rabouille C. Mechanisms of regulated unconventional protein secretion. Nat Rev Mol Cell Biol (2008) 10:148–55. doi:10.1038/nrm2645

CrossRef Full Text | Google Scholar

11. Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol (2008) 8:34–47. doi:10.1038/nri2206

PubMed Abstract | CrossRef Full Text | Google Scholar

12. Weiskopf K, Weissman IL. Macrophages are critical effectors of antibodies therapies for cancer. MAbs (2015) 2:303–10. doi:10.1080/19420862.2015.1011450

CrossRef Full Text | Google Scholar

13. Guo K, Tang JP, Jie L, Al-Aidaroos AQO, Hong CW, Tan CPB, et al. Engineering the first chimeric antibody in targeting intracellular PRL-3 oncoprotein for cancer therapy in mice. Oncotarget (2012) 3:158–71. doi:10.18632/oncotarget.442

PubMed Abstract | CrossRef Full Text | Google Scholar

14. Wörn A, Plückthun A. Stability engineering of antibody single-chain Fv fragments. J Mol Biol (2001) 305:989–1010. doi:10.1006/jmbi.2000.4265

PubMed Abstract | CrossRef Full Text | Google Scholar

15. Bird RE, Hardman KD, Jacobson JW, Johnson S, Bennett M, Lee S, et al. Single-chain antigen-binding proteins. Science (1988) 242:423–6. doi:10.1126/science.3140379

CrossRef Full Text | Google Scholar

16. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotný J, Margolies MN, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A (1988) 85:5879–83. doi:10.1073/pnas.85.16.5879

CrossRef Full Text | Google Scholar

17. Tanaka T, Lobato MN, Rabbitts TH. Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. J Mol Biol (2003) 331:1109–20. doi:10.1016/S0022-2836(03)00836-2

PubMed Abstract | CrossRef Full Text | Google Scholar

18. Harmsen MM, De Haard HJ. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol (2007) 77:13–22. doi:10.1007/s00253-007-1142-2

PubMed Abstract | CrossRef Full Text | Google Scholar

19. Streltsov V, Nuttall S. Do sharks have a new antibody lineage? Immunol Lett (2005) 97:159–60. doi:10.1016/j.imlet.2004.09.018

CrossRef Full Text | Google Scholar

20. Tanaka T, Rabbitts TH. Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds. J Mol Biol (2008) 376:749–57. doi:10.1016/j.jmb.2007.11.085

CrossRef Full Text | Google Scholar

21. Melchionna T, Cattaneo A. A protein silencing switch by ligand-induced proteasome-targeting intrabodies. J Mol Biol (2007) 374:641–54. doi:10.1016/j.jmb.2007.09.053

PubMed Abstract | CrossRef Full Text | Google Scholar

22. Tse E, Rabbitts TH. Intracellular antibody-caspase-mediated cell killing: an approach for application in cancer therapy. Proc Natl Acad Sci U S A (2000) 97:12266–71. doi:10.1073/pnas.97.22.12266

PubMed Abstract | CrossRef Full Text | Google Scholar

23. Ivanov AA, Khuri FR, Fu H. Targeting protein-protein interactions as an anticancer strategy. Trends Pharmacol Sci (2013) 34:393–400. doi:10.1016/j.tips.2013.04.007

PubMed Abstract | CrossRef Full Text | Google Scholar

24. Wells JA, McClendon CL. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature (2007) 450:1001–9. doi:10.1038/nature06526

PubMed Abstract | CrossRef Full Text | Google Scholar

25. Tanaka T, Williams RL, Rabbitts TH. Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J (2007) 26:3250–9. doi:10.1038/sj.emboj.7601744

PubMed Abstract | CrossRef Full Text | Google Scholar

26. Tanaka T, Rabbitts TH. Interfering with RAS–effector protein interactions prevent RAS-dependent tumour initiation and causes stop–start control of cancer growth. Oncogene (2010) 29:6064–70. doi:10.1038/onc.2010.346

CrossRef Full Text | Google Scholar

27. Marschall AL, Dübel S, Böldicke T. Specific in vivo knockdown of protein function by intrabodies. MAbs (2015) 7:1010–35. doi:10.1080/19420862.2015.1076601

PubMed Abstract | CrossRef Full Text | Google Scholar

28. Jendreyko N, Popkov M, Rader C, Barbas CF. Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesis in vivo. Proc Natl Acad Sci U S A (2005) 102:8293–8. doi:10.1073/pnas.0503168102

PubMed Abstract | CrossRef Full Text | Google Scholar

29. Marschall ALJ, Dübel S. Antibodies inside of a cell can change its outside: can intrabodies provide a new therapeutic paradigm? Comput Struct Biotechnol J (2016) 14:304–8. doi:10.1016/j.csbj.2016.07.003

PubMed Abstract | CrossRef Full Text | Google Scholar

30. Ruiz-Argüelles A, Alarcón-Segovia D. Penetration of autoantibodies into living cells. Isr Med Assoc J (2001) 3:121–6. doi:10.2174/1381612033454379

CrossRef Full Text | Google Scholar

31. Sun KH, Tang SJ, Lin ML, Wang YS, Sun GH, Liu WT. Monoclonal antibodies against human ribosomal P proteins penetrate into living cells and cause apoptosis of Jurkat T cells in culture. Rheumatology (Oxford) (2001) 40:750–6. doi:10.1093/rheumatology/40.7.750

PubMed Abstract | CrossRef Full Text | Google Scholar

32. Douglas JN, Gardner LA, Levin MC. Antibodies to an intracellular antigen penetrate neuronal cells and cause deleterious effects. J Clin Cell Immunol (2013) 04:1–7. doi:10.4172/2155-9899.1000134

CrossRef Full Text | Google Scholar

33. Pérez-Martínez D, Tanaka T, Rabbitts TH. Intracellular antibodies and cancer: new technologies offer therapeutic opportunities. Bioessays (2010) 32:589–98. doi:10.1002/bies.201000009

PubMed Abstract | CrossRef Full Text | Google Scholar

34. Verma IM, Weitzman MD. Gene therapy: twenty-first century medicine. Annu Rev Biochem (2005) 74:711–38. doi:10.1146/annurev.biochem.74.050304.091637

CrossRef Full Text | Google Scholar

35. Chatin B, Mével M, Devallière J, Dallet L, Haudebourg T, Peuziat P, et al. Liposome-based formulation for intracellular delivery of functional proteins. Mol Ther Nucleic Acids (2015) 4:e244. doi:10.1038/mtna.2015.17

PubMed Abstract | CrossRef Full Text | Google Scholar

36. Liu X, Huang G. Formation strategies, mechanism of intracellular delivery and potential clinical applications of pH-sensitive liposomes. Asian J Pharm Sci (2013) 8:319–28. doi:10.1016/j.ajps.2013.11.002

CrossRef Full Text | Google Scholar

37. Zelphati O, Wang Y, Kitada S, Reed JC, Felgner PL, Corbeil J. Intracellular delivery of proteins with a new lipid-mediated delivery system. J Biol Chem (2001) 276:35103–10. doi:10.1074/jbc.M104920200

PubMed Abstract | CrossRef Full Text | Google Scholar

38. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol (2007) 2:751–60. doi:10.1038/nnano.2007.387

PubMed Abstract | CrossRef Full Text | Google Scholar

39. Noble GT, Stefanick JF, Ashley JD, Kiziltepe T, Bilgicer B. Ligand-targeted liposome design: challenges and fundamental considerations. Trends Biotechnol (2014) 32:32–45. doi:10.1016/j.tibtech.2013.09.007

PubMed Abstract | CrossRef Full Text | Google Scholar

40. Chen J, Guo Z, Tian H, Chen X. Production and clinical development of nanoparticles for gene delivery. Mol Ther Methods Clin Dev (2016) 3:16023. doi:10.1038/mtm.2016.23

PubMed Abstract | CrossRef Full Text | Google Scholar

41. Cui Y, Cui P, Chen B, Li S, Guan H. Monoclonal antibodies: formulations of marketed products and recent advances in novel delivery system. Drug Dev Ind Pharm (2017) 9045:1–39. doi:10.1080/03639045.2017.1278768

PubMed Abstract | CrossRef Full Text | Google Scholar

42. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release (2012) 161:505–22. doi:10.1016/j.jconrel.2012.01.043

PubMed Abstract | CrossRef Full Text | Google Scholar

43. Son S, Lee WR, Joung YK, Kwon MH, Kim YS, Park KD. Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles. Int J Pharm (2009) 368:178–85. doi:10.1016/j.ijpharm.2008.09.061

PubMed Abstract | CrossRef Full Text | Google Scholar

44. Uherek C, Wels W. DNA-carrier proteins for targeted gene delivery. Adv Drug Deliv Rev (2000) 44:153–66. doi:10.1016/S0169-409X(00)00092-2

PubMed Abstract | CrossRef Full Text | Google Scholar

45. Gillies ER, Frechet JMJ. Dendrimers and dendritic polymers in drug delivery. Drug Discov Today (2005) 10:35–43. doi:10.1016/S1359-6446(04)03276-3

PubMed Abstract | CrossRef Full Text | Google Scholar

46. Muller S, Zhao Y, Brown TL, Morgan AC, Kohler H. TransMabs: cell-penetrating antibodies, the next generation. Expert Opin Biol Ther (2005) 5:237–41. doi:10.1517/14712598.5.2.237

PubMed Abstract | CrossRef Full Text | Google Scholar

47. Zhao Y, Brown TL, Kohler H, Müller S. MTS-conjugated-antiactive caspase 3 antibodies inhibit actinomycin D-induced apoptosis. Apoptosis (2003) 8:631–7. doi:10.1023/A:1026139627930

PubMed Abstract | CrossRef Full Text | Google Scholar

48. Fonseca SB, Pereira MP, Kelley SO. Recent advances in the use of cell-penetrating peptides for medical and biological applications. Adv Drug Deliv Rev (2009) 61:953–64. doi:10.1016/j.addr.2009.06.001

PubMed Abstract | CrossRef Full Text | Google Scholar

49. El-Sayed A, Futaki S, Harashima H. Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment. AAPS J (2009) 11:13–22. doi:10.1208/s12248-008-9071-2

PubMed Abstract | CrossRef Full Text | Google Scholar

50. Shin S-M, Choi D-K, Jung K, Bae J, Kim J, Park S, et al. Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration. Nat Commun (2017) 8:15090. doi:10.1038/ncomms15090

PubMed Abstract | CrossRef Full Text | Google Scholar

51. Rock KL, York IA, Goldberg AL. Post-proteasomal antigen processing for major histocompatibility complex class I presentation. Nat Immunol (2004) 5:670–7. doi:10.1038/ni1089

PubMed Abstract | CrossRef Full Text | Google Scholar

52. Weidanz JA, Hawkins O, Verma B, Hildebrand WH. TCR-like biomolecules target peptide/MHC class I complexes on the surface of infected and cancerous cells. Int Rev Immunol (2011) 30:328–40. doi:10.3109/08830185.2011.604880

PubMed Abstract | CrossRef Full Text | Google Scholar

53. Cohen M, Reiter Y. T-cell receptor-like antibodies: targeting the intracellular proteome therapeutic potential and clinical applications. Antibodies (2013) 2:517–34. doi:10.3390/antib2030517

CrossRef Full Text | Google Scholar

54. Dahan R, Reiter Y. T-cell-receptor-like antibodies – generation, function and applications. Expert Rev Mol Med (2012) 14:e6. doi:10.1017/erm.2012.2

CrossRef Full Text | Google Scholar

55. Verma B, Jain R, Caseltine S, Rennels A, Bhattacharya R, Markiewski MM, et al. TCR mimic monoclonal antibodies induce apoptosis of tumor cells via immune effector-independent mechanisms. J Immunol (2011) 186:3265–76. doi:10.4049/jimmunol.1002376

PubMed Abstract | CrossRef Full Text | Google Scholar

56. Li D, Bentley C, Anderson A, Wiblin S, Cleary KLS, Koustoulidou S, et al. Development of a T cell receptor mimic antibody against wild-type p53 for cancer immunotherapy. Cancer Res (2017) 77(10):2699–711. doi:10.1158/0008-5472.CAN-16-3247

CrossRef Full Text | Google Scholar

57. Denkberg G, Lev A, Eisenbach L, Benhar I, Reiter Y. Selective targeting of melanoma and APCs using a recombinant antibody with TCR-like specificity directed toward a melanoma differentiation antigen. J Immunol (2003) 171:2197–207. doi:10.4049/jimmunol.171.5.2197

PubMed Abstract | CrossRef Full Text | Google Scholar

58. Klechevsky E, Gallegos M, Denkberg G, Palucka K, Banchereau J, Cohen C, et al. Antitumor activity of immunotoxins with T-cell receptor-like specificity against human melanoma xenografts. Cancer Res (2008) 68:6360–7. doi:10.1158/0008-5472.CAN-08-0928

PubMed Abstract | CrossRef Full Text | Google Scholar

59. Oren R, Hod-Marco M, Haus-Cohen M, Thomas S, Blat D, Duvshani N, et al. Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody–based chimeric antigen receptors indicates affinity/avidity thresholds. J Immunol (2014) 193:5733–43. doi:10.4049/jimmunol.1301769

CrossRef Full Text | Google Scholar

60. Zhang G, Wang L, Cui H, Wang X, Zhang G, Ma J, et al. Anti-melanoma activity of T cells redirected with a TCR-like chimeric antigen receptor. Sci Rep (2014) 4:3571. doi:10.1038/srep03571

PubMed Abstract | CrossRef Full Text | Google Scholar

61. Polakova K, Plaksin D, Chung DH, Belyakov M, Berzofsky JA, Margulies DH. Antibodies directed against the MHC-I molecule H-2D d complexed with an antigenic peptide: similarities to a T cell receptor with the same specificity. J Immunol (2017) 165(10):5703–12. doi:10.4049/jimmunol.165.10.5703

CrossRef Full Text | Google Scholar

62. Sastry SR, Too CT, Kaur K, Gehring AJ, Low L, Javiad A, et al. Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody. J Virol (2011) 85:1935–42. doi:10.1128/JVI.01990-10

PubMed Abstract | CrossRef Full Text | Google Scholar

63. Chames P, Hufton SE, Coulie PG, Uchanska-Ziegler B, Hoogenboom HR. Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library. Proc Natl Acad Sci U S A (2000) 97:7969–74. doi:10.1073/pnas.97.14.7969

PubMed Abstract | CrossRef Full Text | Google Scholar

64. Chames P, Willemsen RA, Rojas G, Dieckmann D, Rem L, Schuler G, et al. TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity. J Immunol (2002) 169:1110–8. doi:10.4049/jimmunol.169.2.1110

PubMed Abstract | CrossRef Full Text | Google Scholar

65. Denkberg G, Klechevsky E, Reiter Y. Modification of a tumor-derived peptide at an HLA-A2 anchor residue can alter the conformation of the MHC-peptide complex: probing with TCR-like recombinant antibodies. J Immunol (2002) 169:4399–407. doi:10.4049/jimmunol.169.8.4399

PubMed Abstract | CrossRef Full Text | Google Scholar

66. Hoogenboom HR, Reiter Y. MHC-Peptide Complex Binding Ligands (2003). Available from: https://www.google.com/patents/US20030223994

Google Scholar

67. Lev A, Denkberg G, Cohen CJ, Reiter Y, Tzukerman M, Skorecki KL, et al. Isolation and characterization of human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells directed toward the widely expressed tumor T-cell epitopes of the telomerase catalytic sub. Cancer Res (2002) 62:3184–94.

Google Scholar

68. Cohen CJ, Hoffmann N, Farago M, Hoogenboom HR, Eisenbach L, Reiter Y. Direct detection and quantitation of a distinct T-cell epitope derived from tumor-specific epithelial cell-associated mucin using human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T. Cancer Res (2002) 62:5835–44.

Google Scholar

69. Held G, Matsuo M, Epel M, Gnjatic S, Ritter G, Lee SY, et al. Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments. Eur J Immunol (2004) 34:2919–29. doi:10.1002/eji.200425297

PubMed Abstract | CrossRef Full Text | Google Scholar

70. Bernardeau K, Gouard S, David G, Ruellan AL, Devys A, Barbet J, et al. Assessment of CD8 involvement in T cell clone avidity by direct measurement of HLA-A2/Mage3 complex density using a high-affinity TCR like monoclonal antibody. Eur J Immunol (2005) 35:2864–75. doi:10.1002/eji.200526307

PubMed Abstract | CrossRef Full Text | Google Scholar

71. Wittman VP, Woodburn D, Nguyen T, Neethling FA, Wright S, Weidanz JA. Antibody targeting to a class I MHC-peptide epitope promotes tumor cell death. J Immunol (2006) 177:4187–95. doi:10.4049/jimmunol.177.6.4187

PubMed Abstract | CrossRef Full Text | Google Scholar

72. Neethling FA, Ramakrishna V, Keler T, Buchli R, Woodburn T, Weidanz JA. Assessing vaccine potency using TCRmimic antibodies. Vaccine (2008) 26:3092–102. doi:10.1016/j.vaccine.2008.02.025

PubMed Abstract | CrossRef Full Text | Google Scholar

73. Weidanz JA, Nguyen T, Woodburn T, Neethling FA, Chiriva-Internati M, Hildebrand WH, et al. Levels of specific peptide-HLA class I complex predicts tumor cell susceptibility to CTL killing. J Immunol (2006) 177:5088–97. doi:10.4049/jimmunol.177.8.5088

PubMed Abstract | CrossRef Full Text | Google Scholar

74. Held G, Wadle A, Dauth N, Stewart-Jones G, Sturm C, Thiel M, et al. MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing. Eur J Immunol (2007) 37:2008–17. doi:10.1002/eji.200636545

PubMed Abstract | CrossRef Full Text | Google Scholar

75. Epel M, Carmi I, Soueid-Baumgarten S. Targeting TARP, a novel breast and prostate tumor-associated antigen, with T-cell receptor-like human recombinant antibodies. Eur J Immunol (2008) 38:1706–20. doi:10.1038/jid.2014.371

CrossRef Full Text | Google Scholar

76. Weidanz JA, Hildebrand WH, Hawkins O. No Antibodies as T Cell Receptor Mimics, Methods of Production and Uses Thereof (2009). Available from: http://www.google.com/patents/US20090226474

Google Scholar

77. Li D, Bentley C, Yates J, Salimi M, Greig J, Wiblin S, et al. Engineering chimeric human and mouse major histocompatibility complex (MHC) class I tetramers for the production of T-cell receptor (TCR) mimic antibodies. PLoS One (2017) 12:e0176642. doi:10.1371/journal.pone.0176642

PubMed Abstract | CrossRef Full Text | Google Scholar

78. Michaeli Y, Denkberg G, Sinik K, Lantzy L, Chih-Sheng C, Beauverd C, et al. Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies. J Immunol (2009) 182:6328–41. doi:10.4049/jimmunol.0801898

PubMed Abstract | CrossRef Full Text | Google Scholar

79. Verma B, Hawkins OE, Neethling FA, Caseltine SL, Largo SR, Hildebrand WH, et al. Direct discovery and validation of a peptide/MHC epitope expressed in primary human breast cancer cells using a TCRm monoclonal antibody with profound antitumor properties. Cancer Immunol Immunother (2010) 59:563–73. doi:10.1007/s00262-009-0774-8

PubMed Abstract | CrossRef Full Text | Google Scholar

80. Hawkins O, Verma B, Lightfoot S, Jain R, Rawat A, McNair S, et al. An HLA-presented fragment of macrophage migration inhibitory factor is a therapeutic target for invasive breast cancer. J Immunol (2011) 186:6607–16. doi:10.4049/jimmunol.1003995

PubMed Abstract | CrossRef Full Text | Google Scholar

81. Sergeeva A, Alatrash G, He H, Ruisaard K, Lu S, Wygant J, et al. An anti-PR1/HLA-A2 T-cell receptor-like antibody mediates complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells. Blood (2011) 117:4262–72. doi:10.1182/blood-2010-07-299248

CrossRef Full Text | Google Scholar

82. Dao T, Yan S, Veomett N, Pankov D, Zhou L, Scott A, et al. Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med (2013) 5:1–22. doi:10.1126/scitranslmed.3005661.Targeting

PubMed Abstract | CrossRef Full Text | Google Scholar

83. Zhao Q, Ahmed M, Tassev D, Hasan A, Kuo TY, Guo HF, et al. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential. Leukemia (2015) 29:2238–47. doi:10.1038/leu.2015.125

PubMed Abstract | CrossRef Full Text | Google Scholar

84. Inaguma Y, Akahori Y, Murayama Y, Shiraishi K, Tsuzuki-Iba S, Endoh A, et al. Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H. Gene Ther (2014) 21:575–84. doi:10.1038/gt.2014.30

PubMed Abstract | CrossRef Full Text | Google Scholar

85. Chang AY, Dao T, Gejman RS, Jarvis CA, Scott A, Dubrovsky L, et al. A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens. J Clin Invest (2017) 127(7):2705–18. doi:10.1172/JCI92335

PubMed Abstract | CrossRef Full Text | Google Scholar

86. Garboczi DN, Hung DT, Wiley DC. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci U S A (1992) 89:3429–33. doi:10.1073/pnas.89.8.3429

PubMed Abstract | CrossRef Full Text | Google Scholar

87. Andersen PS, Stryhn A, Hansen BE, Fugger L, Engberg J, Buus S. A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells. Proc Natl Acad Sci U S A (1996) 93:1820–4. doi:10.1073/pnas.93.5.1820

CrossRef Full Text | Google Scholar

88. Denkberg G, Cohen CJ, Reiter Y. Critical role for CD8 in binding of MHC tetramers to TCR: CD8 antibodies block specific binding of human tumor-specific MHC-peptide tetramers to TCR. J Immunol (2001) 167:270–6. doi:10.4049/jimmunol.167.1.270

PubMed Abstract | CrossRef Full Text | Google Scholar

89. Dadaglio G, Nelson CA, Deck MB, Petzold SJ, Unanue ER. Characterization and quantitation of peptide-MHC complexes produced from hen egg lysozyme using a monoclonal antibody. Immunity (1997) 6:727–38. doi:10.1016/S1074-7613(00)80448-3

PubMed Abstract | CrossRef Full Text | Google Scholar

90. Porgador A, Yewdell JW, Deng Y, Bennink JR, Germain RN. Localization, quantitation, and in situ detection of specific peptide – MHC class I complexes using a monoclonal antibody. Immunity (1997) 6:715–26. doi:10.1016/S1074-7613(00)80447-1

CrossRef Full Text | Google Scholar

91. Rubin B, Malissen B, Jorgenssen PN, Zeuthen J. Recognition of MHC-class-II-expressing L929 cells by antibody and T cells. Res Immunol (1989) 140:67–74. doi:10.1016/0923-2494(89)90007-2

CrossRef Full Text | Google Scholar

92. Denkberg G, Cohen CJ, Lev A, Chames P, Hoogenboom HR, Reiter Y. Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC-restricted T cell receptor-like specificity. Proc Natl Acad Sci U S A (2002) 99:9421–6. doi:10.1073/pnas.132285699

PubMed Abstract | CrossRef Full Text | Google Scholar

93. Biddison WE, Turner RV, Gagnon SJ, Lev A, Cohen CJ, Reiter Y. Tax and M1 peptide/HLA-A2-specific Fabs and T cell receptors recognize nonidentical structural features on peptide/HLA-A2 complexes. J Immunol (2003) 171:3064–74. doi:10.4049/jimmunol.171.6.3064

CrossRef Full Text | Google Scholar

94. Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E. Rational development of high-affinity T-cell receptor-like antibodies. Proc Natl Acad Sci U S A (2009) 106:5784–8. doi:10.1073/pnas.0905399106

PubMed Abstract | CrossRef Full Text | Google Scholar

95. Makler O, Oved K, Netzer N, Wolf D, Reiter Y. Direct visualization of the dynamics of antigen presentation in human cells infected with cytomegalovirus revealed by antibodies mimicking TCR specificity. Eur J Immunol (2010) 40:1552–65. doi:10.1002/eji.200939875

PubMed Abstract | CrossRef Full Text | Google Scholar

96. Dubrovsky L, Dao T, Gejman R, Brea E, Chang A, Oh C, et al. T cell receptor mimic antibodies for cancer therapy. Oncoimmunology (2015) 5:e1049803. doi:10.1080/2162402X.2015.1049803

PubMed Abstract | CrossRef Full Text | Google Scholar

97. Worley BS, Van den Broeke LT, Goletz TJ, Pendleton CD, Daschbach EM, Thomas EK, et al. Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations. Cancer Res (2001) 61:6868–75.

PubMed Abstract | Google Scholar

98. Warren RL, Holt RA. A census of predicted mutational epitopes suitable for immunologic cancer control. Hum Immunol (2010) 71:245–54. doi:10.1016/j.humimm.2009.12.007

PubMed Abstract | CrossRef Full Text | Google Scholar

99. Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J. Generation of human cytotoxic T cells specific for human carcinoembryogenic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst (1995) 87:982–90. doi:10.1093/jnci/87.13.982

CrossRef Full Text | Google Scholar

100. Morgan RA, Chinnasamy N, Abate-daga DD, Gros A, Robbins F, Zheng Z, et al. Cancer regression and neurologic toxicity following anti-MAGEA3 TCR gene therapy. J Immunother (2014) 36:133–51. doi:10.1097/CJI.0b013e3182829903.Cancer

CrossRef Full Text | Google Scholar

101. Oleinika K, Nibbs RJ, Graham GJ, Fraser AR. Suppression, subversion and escape: the role of regulatory T cells in cancer progression. Clin Exp Immunol (2013) 171:36–45. doi:10.1111/j.1365-2249.2012.04657.x

PubMed Abstract | CrossRef Full Text | Google Scholar

102. Noy R, Pollard JW. Tumor-associated macrophages: from mechanisms to therapy. Immunity (2014) 41:49–61. doi:10.1016/j.immuni.2014.06.010

PubMed Abstract | CrossRef Full Text | Google Scholar

103. Dubrovsky L, Pankov D, Brea EJ, Dao T, Scott A, Yan S, et al. A TCR-mimic antibody to WT1 bypasses tyrosine kinase inhibitor resistance in human BCR-ABL 1 leukemias. Blood (2014) 123:3296–304. doi:10.1182/blood-2014-01-549022

CrossRef Full Text | Google Scholar

104. Veomett N, Dao T, Liu H, Xiang J, Pankov D, Dubrovsky L, et al. Therapeutic efficacy of an Fc-enhanced TCR-like antibody to the intracellular WT1 oncoprotein. Clin Cancer Res (2014) 20:4036–404. doi:10.1038/nature09421.Oxidative

PubMed Abstract | CrossRef Full Text | Google Scholar

105. Bassani-Sternberg M, Pletscher-Frankild S, Jensen LJ, Mann M. Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation. Mol Cell Proteomics (2015) 14:658–73. doi:10.1074/mcp.M114.042812

PubMed Abstract | CrossRef Full Text | Google Scholar

106. Khanna R. Tumour surveillance: missing peptides and MHC molecules. Immunol Cell Biol (1998) 76:20–6. doi:10.1046/j.1440-1711.1998.00717.x

PubMed Abstract | CrossRef Full Text | Google Scholar

107. Angell TE, Lechner MG, Jang JK, LoPresti JS, Epstein AL. MHC class I loss is a frequent mechanism of immune escape in papillary thyroid cancer that is reversed by interferon and selumetinib treatment in vitro. Clin Cancer Res (2014) 20:6034–44. doi:10.1158/1078-0432.CCR-14-0879

PubMed Abstract | CrossRef Full Text | Google Scholar

108. van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (1991) 254:1643–7. doi:10.1126/science.1840703

CrossRef Full Text | Google Scholar

109. Traversari C, van der Bruggen P, Luescher IF, Lurquin C, Chomez P, Van Pel A, et al. A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J Exp Med (1992) 176:1453–7. doi:10.1084/jem.176.5.1453

PubMed Abstract | CrossRef Full Text | Google Scholar

110. Kessler JH, Melief CJM. Identification of T-cell epitopes for cancer immunotherapy. Leukemia (2007) 21:1859–74. doi:10.1038/sj.leu.2404787

CrossRef Full Text | Google Scholar

111. Hoppes R, Ekkebus R, Schumacher TNM, Ovaa H. Technologies for MHC class I immunoproteomics. J Proteomics (2010) 73:1945–53. doi:10.1016/j.jprot.2010.05.009

CrossRef Full Text | Google Scholar

112. Backert L, Kowalewski DJ, Walz S, Berlin C, Neidert MC, Schemionek M, et al. A meta-analysis of HLA peptidome composition in different hematological entities: entity-specific dividing lines and “pan-leukemia” antigens. Oncotarget (2017) 8(27):43915–24. doi:10.18632/oncotarget.14918

CrossRef Full Text | Google Scholar

113. Matsui K, Boniface JJ, Reay PA, Schild H, Fazekas de St Groth B, Davis MM. Low affinity interaction of peptide-MHC complexes with T cell receptors. Science (1991) 254:1788–91.

Google Scholar

114. Mosquera LA, Card KF, Price-Schiavi SA, Belmont HJ, Liu B, Builes J, et al. In vitro and in vivo characterization of a novel antibody-like single-chain TCR human IgG1 fusion protein. J Immunol (2005) 174:4381–8. doi:10.4049/jimmunol.174.7.4381

PubMed Abstract | CrossRef Full Text | Google Scholar

115. Oates J, Hassan NJ, Jakobsen BK. ImmTACs for targeted cancer therapy: why, what, how, and which. Mol Immunol (2015) 67:67–74. doi:10.1016/j.molimm.2015.01.024

PubMed Abstract | CrossRef Full Text | Google Scholar

116. Ding Y-H, Smith KJ, Garboczi DN, Utz U, Biddison WE, Wiley DC. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity (1998) 8:403–11. doi:10.1016/S1074-7613(00)80546-4

PubMed Abstract | CrossRef Full Text | Google Scholar

117. Garcia KC, Degano M, Pease LR, Huang M, Peterson PA, Teyton L, et al. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science (1998) 279:1166–72. doi:10.1126/science.279.5354.1166

PubMed Abstract | CrossRef Full Text | Google Scholar

118. Mareeva T, Martinez-Hackert E, Sykulev Y. How a T cell receptor-like antibody recognizes major histocompatibility complex-bound peptide. J Biol Chem (2008) 283:29053–9. doi:10.1074/jbc.M804996200

PubMed Abstract | CrossRef Full Text | Google Scholar

119. Arber C, Feng X, Abhyankar H, Romero E, Wu MF, Heslop HE, et al. Survivin-specific T cell receptor targets tumor but not T cells. J Clin Invest (2015) 125:157–68. doi:10.1172/JCI75876

PubMed Abstract | CrossRef Full Text | Google Scholar

120. Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL, Emery L, et al. Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood (2013) 122:863–72. doi:10.1182/blood-2013-03-490565.G.P.L

PubMed Abstract | CrossRef Full Text | Google Scholar

121. Ataie N, Xiang J, Cheng N, Brea EJ, Lu W, Scheinberg DA, et al. Structure of a TCR-mimic antibody with target predicts pharmacogenetics. J Mol Biol (2016) 428:194–205. doi:10.1016/j.jmb.2015.12.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Keywords: T-cell receptor mimic antibody, intracellular antibody, intrabody, MHC class I presented peptide, T-cell epitope, cancer immunotherapy, therapeutic antibody, T-cell receptor-like antibody

Citation: Trenevska I, Li D and Banham AH (2017) Therapeutic Antibodies against Intracellular Tumor Antigens. Front. Immunol. 8:1001. doi: 10.3389/fimmu.2017.01001

Received: 30 June 2017; Accepted: 04 August 2017;
Published: 18 August 2017

Edited by:

Jose A. Garcia-Sanz, Consejo Superior de Investigaciones Científicas (CSIC), Spain

Reviewed by:

Daniel Olive, Institut national de la santé et de la recherche médicale, France
María Marcela Barrio, Fundación Cáncer FUCA, Argentina

Copyright: © 2017 Trenevska, Li and Banham. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Demin Li, demin.li@ndcls.ox.ac.uk;
Alison H. Banham, alison.banham@ndcls.ox.ac.uk