The major limitation of second and third generation ITs reflects the long-term nature of cancer treatment and the need for repeated doses, which means any component of nonhuman origin can induce neutralizing antibodies in patients with a functional immune system, thereby reducing therapeutic efficiency [36,37,38,39]. These problems can be caused either by the tumor-selective ligand or the cytotoxic component so that the complete humanization of ITs is preferable when repeated dose schedules are required, to improve safety and to ensure a positive clinical outcome.

If the tumor-selective ligand is an antibody or antibody fragment of murine origin, it can induce a human anti-mouse antibody (HAMA) response in some patients [40,41]. HAMA responses not only reduce treatment efficiency, but also have been shown to cause allergic reactions, in extreme cases leading to life-threatening complications such as renal failure.

Several strategies can be used to humanize the tumor-selective ligand including the fusion of the effector moiety to human cytokines and growth factors that target tumor receptors, e.g., IL-2, IL-4, EGF (epidermal growth factor), TGFα (tumor growth factor alpha) or the CD30 ligand [29,34,42]. Recombinant DNA technology can also be used to engineer antibodies for reduced immunogenicity [43]. The generation of chimeric antibodies comprising a murine Fab fragment and a human Fc region can reduce HAMA responses because the constant region makes the most significant contribution to immunogenicity [44,45]. Another strategy is the generation of humanized antibodies by grafting murine complementarity determining regions onto human V-region frameworks, an approach known as CDR replacement [46,47,48]. Complete humanization is also possible by selecting the V-regions from human antibody libraries or using transgenic knock-out mice or rats in which the gene loci for the light and heavy antibody chains are replaced by their human counterparts [49,50]. Single chain fragment variable (scFv) antibodies used for the production of fourth-generation ITs can be derived from humanized or human monoclonal antibodies or selected directly from a human scFv library.

The conventional use of plant or bacterial toxins can induce an undesirable immune response and therefore several strategies have been investigated to reduce the immunogenicity of the cytotoxic component. These include the identification and removal (by mutation) of major immunogenic B-cell and T-cell epitopes within the toxins [51,52] as well as the derivatization of ITs using polyethylene glycol [53] or the co-application of immunosuppressive drugs [54,55].

A more favorable strategy for complete humanization is the use of endogenous human cytolytic enzymes, such as enzymes that induce cell death by a variety of mechanisms. Pro-apoptotic human proteins that are potentially useful for targeted cancer therapy include proteases and RNases such as angiogenin as well as death receptor ligands such as sTRAIL and FASL and pro-apoptotic members of the Bcl-2 family [32]. In addition to pro-apoptotic proteins, enzymes with tumor suppressive function such as DAPK2 (death-associated protein kinase 2) allow the targeted restoration of tumor suppressor activity in cancer cells [33,42].

The efficacy of ITs/hCFPs depends on the sensitivity of tumor cells to pro-apoptotic signals. One of the major challenges in both targeted cancer therapy and standard chemotherapy approaches that induce programmed cell death is the tendency for tumor cells to develop resistance to pro-apoptotic signals allowing the invasion of host immunosurveillance [56]. Tumor cells can evolve multiple intrinsic and extrinsic escape mechanisms to overcome innate and adaptive immune responses. These include impaired tumor antigen presentation by the downregulation of MHC class I molecules, and the expression of factors that either directly kill or inhibit effector cells or recruit regulatory T-cells and thereby induce immunotolerance [57].

Tumor cells can regulate apoptosis by overexpressing different classes of anti-apoptotic proteins. For example, FLICE-inhibitory proteins (FLIPs) interfere with death receptor signal transduction pathways [58], whereas anti-apoptotic proteins of the Bcl-2 family regulate apoptosis at the mitochondrial level [59,60] and IAPs (inhibitors of apoptosis proteins) inhibit caspase activity [61]. Apoptosis-related escape mechanisms in tumor cells can reduce the efficiency of targeted therapy using human pro-apoptotic enzymes, but GrB is unusual because it utilizes multiple apoptosis induction mechanisms (see below) and is therefore more likely to overcome resistance mechanisms that evolve in tumors. The inherent pro-apoptotic activity of recombinant GrB in vitro emphasizes its potential in targeted cancer therapy (Table 1).

Granzyme B, a pro-apoptotic serine protease produced by effector cells of the innate and adaptive immune system, is involved in both non-specific and specific host rejection of tumors and cells infected with viruses [62,63]. GrB belongs to a family of serine proteases stored in the cytotoxic granules of natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) [64]. Five different human granzymes have been identified (A, B, H, K and M), each with unique expression profiles and substrate specificities [65]. The most intensively studied enzymes are GrB and GrA, both of which are considered to be major inducers of granule-mediated apoptosis [66]. Active GrB is stored as a complex on a scaffold comprising the chondroitin sulfate proteoglycan serglycin, the pore-forming protein perforin (PFN) and other members of the granzyme family [67,68]. During NK cell-mediated immunosurveillance, secretory granules are deposited into an immunological synapse that forms following the specific recognition of abnormal cells through an elaborate repertoire of cell surface receptors [69]. The precise mechanism by which GrB is taken up into cells and released from the endosome into the cytosol is still controversial. Two potential models for delivery into target cells have been reviewed in detail [70]. Following the recognition of the target cell, granules release their cytolytic contents into the immunological synapse and this directs GrB, probably with the assistance of perforin, into the cytosol of the target cell. Once released, GrB activates different signal transduction pathways to induce cell death.

The classical induction of apoptosis by GrB is comparable to the Fas mediated pathway, and involves the cleavage of several executive and downstream caspases mediated by both the caspase-pathway and the BID-pathway, the latter associated with the BAX/BAK-dependent release of cytochrome c from the mitochondria into the cytosol [71,72,73]. Direct activation of the effector caspase 3 is controlled by the inhibitor of apoptosis protein (IAP), and this must be inactivated, e.g., by HtrA2/OMI, a pro-apoptotic effector released from disrupted mitochondria (see below). Successful processing of pro-caspase-3 leads to the cleavage of several downstream death substrates including the inhibitor of caspase-activated DNase (ICAD), poly(ADP ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), the nuclear mitotic apparatus protein (NuMA) and the nuclear-envelope intermediate-filament protein lamin B. This results in the typical morphological changes associated with apoptosis, i.e., DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage and the formation of apoptotic bodies [71,74,75,76,77].

The activation of the BID pathway (either directly by GrB or indirectly via caspase-8) leads to the cleavage of mitochondrial BID (BH3-interacting domain death agonist) involving BAX (BCL-2-associated X protein) and BAK (BCL-2 antagonist). The translocation of tBID (truncated BID) to the mitochondria results in the permeabilization of the outer mitochondrial membrane and thus mitochondrial depolarization and the subsequent release of cytochrome c (cyt c) and other pro-apoptotic molecules such as endonuclease G and HtrA2/OMI [90,91]. Cytochrome c must be present in the cytosol for the apoptosome to form, which requires the assembly of apoptotic protease activation factor APAF-1 which in turn leads to the autocatalytic activation of pro-caspase-9 and the subsequent processing of the effector caspase-3. In addition to the BID-mediated loss of mitochondrial transmembrane potential, GrB can directly induce mitochondrial depolarization without the assistance of cytosolic mediators, but the exact mechanisms remain unclear [59,92,93,94,95]. The natural apoptotic pathways induced by GrB have been comprehensively reviewed [96,97,98].

GrB cleaves downstream of aspartic or glutamic acid residues and therefore has a substrate specificity similar to that of caspases. GrB can therefore also directly target and activate the downstream caspase substrates ICAD, PARP, DNA-PK, NuMA and lamin B [99,100]. Taken together, these data show that GrB is not only a highly potent inducer of apoptosis due to its status as the major mediator of cellular immunity, but also because of its ability to induce apoptosis at multiple levels using distinct pathways.

Production according to good manufacturing practice (GMP) guidelines is a prerequisite before biopharmaceuticals can enter clinical studies. The production of GrB (and CFPs derived therefrom) in various heterologous expression systems is relatively straightforward, and success has been achieved in Escherichia coli, Pichia pastoris, insect cells and mammalian cells such as HEK 293T, HeLa, Jurkat and COS [78,82,85,87,88,89,101,102,103]. GrB requires a free N-terminus for enzymatic activity, therefore native GrB is expressed as an inactive zymogen containing an N-terminal pre-pro leader sequence. The signal peptide is cleaved by a signal peptidase in the endoplasmic reticulum to produce an inactive proenzyme with an N-terminal pro-peptide, protecting the cell from the induction of apoptosis, thus the pro-peptide must be removed to generate an active protease [104]. In the Golgi apparatus, a mannose-6-phosphate tag is attached which directs GrB to the cytotoxic granules [105]. The pro-peptide is removed by the dipeptidyl peptidase I (DPPI) (also known as cathepsin C) [106].

Several strategies have been developed to accomplish the correct in vitro or in vivo processing and activation of recombinant GrB, including the insertion of an enterokinase cleavage site (ECS) upstream of the sequence of the mature GrB polypeptide, to allow activation after purification by in vitro processing [78,80,81,86,89]. Secretory expression in the methylotropic yeast P. Pastoris has been achieved by the direct N-terminal fusion of the mature polypeptide to the Saccharomyces cerevisiae mating factor α prepro-leader sequence, allowing the straightforward purification of active GrB from the culture supernatant based on local Kex2-protease activity in the trans-Golgi compartment [82,107]. Secretion and functional in vivo activation by a host cell signal peptidase has been achieved in insect cells and COS cells using the native sequence of the GrB precursor protein with the propeptide deleted, or the genetic fusion of a furin recognition motif allowing in vivo processing by endogenous P. pastoris furin-like proteases [102,103,108]. In addition, in vitro and in vivo activation has been achieved by either coexpression of rat DPPI in COS cells or subsequent incubation with bovine spleen DPPI [109].

Despite encouraging data confirming the targeted cytotoxicity of GrB in the context of hCFPs, the involvement of GrB in natural cellular defense is a mixed blessing in targeted cancer therapy. On one hand, the induction of multiple apoptosis pathways allows GrB to evade typical apoptosis inhibitors and it is easy to express in heterologous systems. On the other hand however, two structural features reduce its therapeutic potential.

First, the endogenous GrB inhibitor PI-9 protects cytotoxic lymphocytes from misdirected GrB during an immune response, but the same molecule can be overexpressed by tumor cells allowing them to escape from the impact of GrB secreted by NK cells and CTLs (compare Table 2 and Table 3). Second, GrB has a significant positive surface charge which allows it to bind non-specifically to the negatively-charged cell surface and cause off-target effects. CFPs based on human enzymes have shown promising results in vitro and in preclinical evaluations, but are less efficacious than the more potent bacterial ITs. This probably reflects the lack of an endogenous translocation domain typically present in bacterial toxins, which is required for efficient delivery to the cytosol where the apoptotic machinery is located. The endosomal trapping of human pro-apoptotic enzymes is a major bottleneck in the development of highly effective humanized ITs.

Recombinant GrB-based CFPs have been shown to bind non-specifically to non-target cells which has the potential to cause adverse effects [151]. Different receptors have been shown to facilitate the uptake of GrB after its release from cytotoxic granules, which could lead to the non-specific delivery of recombinant GrB after systemic administration. GrB may bind non-specifically to the mannose-6-phosphate receptor [152,153,154,155] but this can be prevented by deglycosylation following secretory expression in mammalian cells, or by manufacturing the protein in Escherichia coli, which does not glycosylate proteins, or the yeast Pichia pastoris, which does not support mannose-6-phosphate modification [156]. Glycosylation has no impact on the cytotoxic activity of GrB [80,85,86] and might improve the clinical potential of GrB-based ITs. Other structures that may interact with GrB include the CD44 receptor [157] and cell surface-bound heat shock protein 70 (Hsp70) [158], which should be considered when designing GrB-based ITs to prevent off-target effects.

The high isoelectric point (pI) of GrB is a more challenging issue because the net positive charge on the protein surface facilitates electrostatic interactions with the negatively-charged heparan sulfate proteoglycans found on the surface of almost all cells [154,159,160]. The non-specific binding of GrB to the cell-surface does not generally increase off-target cytotoxic activity because the enzyme is not taken up into the cytosol so that it does not interact with its intracellular substrates, but this nevertheless reduces the therapeutic efficacy of the enzyme because it becomes trapped on non-target tissues [151]. Endogenous GrB is stored as a complex with perforin with the negatively-charged chondroitin sulfate proteoglycan serglycin, helping to shield the positive charges [64,161]. The pI of recombinant GrB can be reduced in the context of CFPs by engineering the antibody component with a low pI so that the overall charge is lower but the cytotoxic efficacy is unaffected [162]. Additionally, extracellular proteolytic GrB activity is important for tissue remodeling during an immune response [63], during which the most prominent proteins cleaved by GrB are vitronectin, fibronectin and laminin [163]. Excessive extracellular GrB activity can cause pathophysiological tissue destruction, resulting in disorders such as rheumatoid arthritis [164,165,166], atherosclerosis or abdominal aortic aneurysm [166,167,168].

The amino acids that confer a positive surface charge and thereby facilitate the interaction with glucosaminoglycans on the cell surface are mainly found within the two major heparin-binding motifs RKAKRTRA (residues 96–103 of mature GrB) and KKTMKR (residues 221–226 of mature GrB). The basic amino acid residues involved in heparan sulfate binding were substituted for alanine to reduce cell binding and endocytosis [159]. The substitution of cationic sequences has no impact on the enzymatic and pro-apoptotic activity of GrB [159,169] so that a recombinant version of GrB modified in this matter is evaluated in the context of a hCFP targeting EFGR-positive cancer cells, by exchanging the following amino acids for alanine by site-directed mutagenesis: R96, R100, R102, K221, K222, K225 and R226 [151]. The mutant version of GrB was then fused to the EGFR ligand TGFα, expressed in P. Pastoris and purified from the culture supernatant [82].

Comparative studies with wild-type GrB involving flow cytometric binding analysis on breast cancer cell lines revealed that the modified GrB (GrBcs) showed much weaker binding to EGFR-negative MDA-MB453 cells. Furthermore, cytotoxicity assays using mixed EGFR-positive and EGFR-negative cells showed no reduction in GrBcs cytotoxicity due to trapping by non-target cells, which has been demonstrated for wild-type GrB. The mutations did not interfere with ligand binding or enzyme activity and reduced the negative impact of extracellular matrix effects. Therefore, the specific cytotoxicity of GrB against the EGFR-positive MDA-MB468 cell line could be increased by using the GrBcs variant [151].

We therefore suggest using the PI-9 insensitive and surface-charged modified GrB variant GrBR201Kcs for immunotherapeutic applications. A GrB-based CFP including both modifications is shown in Figure 2.