Cancer immunotherapy aims to selectively target and kill tumor cells whilst limiting the damage to healthy tissues. Controlled delivery of plant, bacterial and human toxins or enzymes has been shown to promote the induction of apoptosis in cancerous cells. The 4th generation of targeted effectors are being designed to be as humanized as possible—a solution to the problem of immunogenicity encountered with existing generations. Granzymes are serine proteases which naturally function in humans as integral cytolytic effectors during the programmed cell death of cancerous and pathogen-infected cells. Secreted predominantly by cytotoxic T lymphocytes and natural killer cells, granzymes function mechanistically by caspase-dependent or caspase-independent pathways. These natural characteristics make granzymes one of the most promising human enzymes for use in the development of fusion protein-based targeted therapeutic strategies for various cancers. In this review, we explore research involving the use of granzymes as cytolytic effectors fused to antibody fragments as selective binding domains.

Immunotherapies are disease management strategies that target or manipulate components of the immune system. Infectious diseases pose a significant threat to human health as evidenced by countries continuing to grapple with several emerging and re-emerging diseases, the most recent global health threat being the SARS-CoV2 pandemic. As such, various immunotherapeutic approaches are increasingly being investigated as alternative therapies for infectious diseases, resulting in significant advances towards the uncovering of pathogen-host immunity interactions. Novel and innovative therapeutic strategies are necessary to overcome the challenges typically faced by existing infectious disease prevention and control methods such as lack of adequate efficacy, drug toxicity and the emergence of drug resistance. As evidenced by recent developments and success of pharmaceuticals such as monoclonal antibodies, immunotherapies already show abundant promise to overcome such limitations while also advancing the frontiers of medicine. In this review we summarize some of the most notable inroads made to combat infectious disease, over mainly the last 5 years, through the use of immunotherapies such as vaccines, monoclonal antibody-based therapies, T-cell-based therapies, manipulation of cytokine levels and checkpoint inhibition. Whilst its most general applications are founded in cancer treatment, advances made towards the curative treatment of HIV, tuberculosis, malaria, zika virus and, most recently COVID-19, reinforce the role of immunotherapeutic strategies in the broader field of disease control. Ultimately, the comprehensive specificity, safety and cost of immunotherapeutics will impact its widespread implementation.

Patient-specific targeted therapy represents the holy grail of anti-cancer therapeutics, allowing potent tumor depletion without detrimental off-target toxicities. Disease-specific monoclonal antibodies have been employed to bind to oncogenic cell-surface receptors, representing the earliest form of immunotherapy. Targeted drug delivery was first achieved by means of antibody-drug conjugates, which exploit the differential expression of tumor-associated antigens as a guiding mechanism for the specific delivery of chemically-conjugated chemotherapeutic agents to diseased target cells. Biotechnological advances have expanded the repertoire of immunology-based tumor-targeting strategies, also paving the way for the next intuitive step in targeted drug delivery: the construction of recombinant protein drugs consisting of an antibody-based targeting domain genetically fused with a cytotoxic peptide, known as an immunotoxin. However, the most potent protein toxins have typically been derived from bacterial or plant virulence factors and commonly feature both off-target toxicity and immunogenicity in human patients. Further refinement of immunotoxin technology thus led to the replacement of monoclonal antibodies with humanized antibody derivatives, including the substitution of non-human toxic peptides with human cytolytic proteins. Preclinically tested human cytolytic fusion proteins (hCFPs) have proven promising as non-immunogenic combinatory anti-cancer agents, however they still require further enhancement to achieve convincing candidacy as a single-mode therapeutic. To date, a portfolio of highly potent human toxins has been established; ranging from microtubule-associated protein tau (MAP tau), RNases, granzyme B (GrB) and death-associated protein kinase (DAPk). In this review, we discuss the most recent findings on the use of these apoptosis-inducing hCFPs for the treatment of various cancers.

Chondroitin-sulfate proteoglycan 4 (CSPG4) is a transmembrane glycoprotein overexpressed on malignant cells in several cancer types with only limited expression on normal cells. CSPG4 is implicated in several signaling pathways believed to drive cancer progression, particularly proliferation, motility and metastatic spread. Expression may serve as a prognostic marker for survival and risk of relapse in treatment-resistant malignancies including melanoma, triple negative breast cancer, rhabdomyosarcoma and acute lymphoblastic leukemia. This tumor-associated overexpression of CSPG4 points towards a highly promising therapeutic target for antibody-guided cancer therapy. Monoclonal αCSPG4 antibodies have been shown to inhibit cancer progression by blocking ligand access to the CSPG4 extracellular binding sites. Moreover, CSPG4-directed antibody conjugates have been shown to be selectively internalized by CSPG4-expressing cancer cells via endocytosis. CSPG4-directed immunotherapy may be approached in several ways, including: (1) antibody-based fusion proteins for the selective delivery of a pro-apoptotic factors such as tumor necrosis factor-related apoptosis-inducing ligand to agonistic death receptors 4 and 5 on the cell surface; and (2) CSPG4-specific immunotoxins which bind selectively to diseased cells expressing CSPG4, are internalized by them and induce arrest of biosynthesis, closely followed by initiation of apoptotic signaling. Here we review various methods of exploiting tumor-associated CSPG4 expression to improve targeted cancer therapy.

The epidermal growth factor receptor (EGFR) has been recognized as an important therapeutic target in oncology. It is commonly overexpressed in a variety of solid tumors and is critically involved in cell survival, proliferation, metastasis, and angiogenesis. This multi-dimensional role of EGFR in the progression and aggressiveness of cancer, has evolved from conventional to more targeted therapeutic approaches. With the advent of hybridoma technology and phage display techniques, the first anti-EGFR monoclonal antibodies (mAbs) (Cetuximab and Panitumumab) were developed. Due to major limitations including host immune reactions and poor tumor penetration, these antibodies were modified and used as guiding mechanisms for the specific delivery of readily available chemotherapeutic agents or plants/bacterial toxins, giving rise to antibody-drug conjugates (ADCs) and immunotoxins (ITs), respectively. Continued refinement of ITs led to deimmunization strategies based on depletion of B and T-cell epitopes or substitution of non-human toxins leading to a growing repertoire of human enzymes capable of inducing cell death. Similarly, the modification of classical ADCs has resulted in the first, fully recombinant versions. In this review, we discuss significant advancements in EGFR-targeting immunoconjugates, including ITs and recombinant photoactivable ADCs, which serve as a blueprint for further developments in the evolving domain of cancer immunotherapy.