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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.

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell's metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents., Competing Interests: The authors declare no conflict of interest.

To date, no curative therapy is available for the treatment of most chronic inflammatory diseases such as atopic dermatitis, rheumatoid arthritis, or autoimmune disorders. Current treatments require a lifetime supply for patients to alleviate clinical symptoms and are unable to stop the course of disease. In contrast, a new series of immunotherapeutic agents targeting the Fc γ receptor I (CD64) have emerged and demonstrated significant clinical potential to actually resolving chronic inflammation driven by M1-type dysregulated macrophages. This subpopulation plays a key role in the initiation and maintenance of a series of chronic diseases. The novel recombinant M1-specific immunotherapeutics offer the prospect of highly effective treatment strategies as they have been shown to selectively eliminate the disease-causing macrophage subpopulations. In this review, we provide a detailed summary of the data generated, together with the advantages and the clinical potential of CD64-based targeted therapies for the treatment of chronic inflammatory diseases., Competing Interests: Theo Thepen and Stefan Barth are co-inventors on CD64-related patents and patent applications, which are assigned to Fraunhofer or UCT. The authors otherwise declare no conflicts of interest.

Some of the most promising small molecule toxins used to generate antibody drug conjugates (ADCs) include anti-mitotic agents (e.g., auristatin and its derivatives) which are designed to attack cancerous cells at their most vulnerable state during mitosis. We were interested in identifying a human cystostatic protein eventually showing comparable activities and allowing the generation of corresponding targeted fully human cytolytic fusion proteins. Recently, we identified the human microtubule associated protein tau (MAP tau), which binds specifically to tubulin and modulates the stability of microtubules, thereby blocking mitosis and presumably vesicular transport. By binding and stabilizing polymerized microtubule filaments, MAP tau-based fusion proteins skew microtubule dynamics towards cell cycle arrest and apoptosis. This biological activity makes rapidly proliferating cells (e.g., cancer and inflammatory cells) an excellent target for MAP tau-based targeted treatments. Their superior selectivity for proliferating cells confers additional selectivity towards upregulated tumor-associated antigens at their surface, thereby preventing off-target related toxicity against normal cells bearing tumor-associated antigens at physiologically normal to low levels. In this review, we highlight recent findings on MAP tau-based targeted cytolytic fusion proteins reported in preclinical immunotherapeutic studies., Competing Interests: Dmitrij Hristodorov, Radoslav Mladenov, and Stefan Barth are inventors on a European Patent Application on MAP based targeted human cytolytic fusion proteins assigned to Fraunhofer. No potential conflicts of interest are disclosed by the other authors.