Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

Abstract

The use of therapies based on antibody fusion proteins for the selective elimination of tumor cells has increased markedly over the last two decades because the severe side effects associated with conventional chemotherapy and radiotherapy are reduced or even eliminated. However, the initial development of immunotoxins suffered from a number of drawbacks such as nonspecific cytotoxicity and the induction of immune responses because the components were non-human in origin. The most recent iteration of this approach is a new class of targeted human cytolytic fusion proteins (hCFPs) comprising a tumor-specific targeting component such as a human antibody fragment fused to a human effector domain with pro-apoptotic activity. Certain tumors resist the activity of hCFPs by upregulating the intracellular expression of native inhibitors, which rapidly bind and inactivate the human effector domains. Higher doses of the hCFPs are, therefore, required to improve therapeutic efficacy. To circumvent these inhibitory processes, novel isoforms of the enzymes granzyme B and angiogenin have been designed to increase their intrinsic activity and reduce their interactions with native inhibitors resulting in more potent hCFPs that can be applied at lower doses. This chapter summarizes the basic scientific knowledge that can facilitate the rational development of human enzymes with novel and beneficial characteristics, including the ability to avoid neutralization by native inhibitors

CD64: An Attractive Immunotherapeutic Target for M1-type Macrophage Mediated Chronic Inflammatory Diseases

“…Hence, these mutants can be used at low doses. In addition, the introduction of a sophisticated adapter sequence containing a membrane transfer peptide (MTD) in between an endosomal and cytosolic cleavable domains has also allowed an increase in the intracellular pool of Ang with a significant increase in cytotoxic activity to levels comparable to ETA’ based bacterial immunotoxins [ 80 ]. The potential combination of H22(scFv) and these Ang variants is expected to offer a powerful benefit for the treatment of M1 macrophage related chronic inflammatory conditions.…”

CSPG4: A Target for Selective Delivery of Human Cytolytic Fusion Proteins and TRAIL

“…Enzymatic activity may be influenced via various methods involving modification of the cytolytic effector peptide in order to reduce its sensitivity to endogenous inhibitors and/or boost its affinity for catalytic substrates [ 73 , 74 ]. Granzyme B (R201K) [ 44 , 57 ] and angiogenin (G85R/G86R and Q117G) [ 39 , 40 ] are examples pro-apoptotic proteins which have undergone such modifications to enhance their cytolytic activity. Further modifications of angiogenin are currently undergoing cell model screening in fusion with αCSPG4 (scFv) for improved cytolytic efficacy against several cancers.…”

“…However, these same characteristics of ETA are also the source of tumor specificity problems, as non-specific cell binding leads to off-target toxicity. This problem was largely overcome by employing a truncated form of ETA (ETA′) which lacks its cell binding Domain Ia [ 44 ], such as used in an ETA′-based IT targeting MSCP (CSPG4) recently generated by Brehm and co-workers [ 21 ] using a novel bacterial expression system [ 45 ].…”

“…Ang, which induces apoptosis by degrading tRNA, has also been shown to exhibit cytotoxicity upon delivery via a disease-specific scFv, while producing no immunogenicity or off-target toxicity [ 38 , 43 , 58 ]. Both these cytotoxic effectors are subject to endogenous inhibition as a tumor survival mechanism and the most recent stages of proofing have involved modifications to reduce sensitivity to inhibition [ 44 ].…”

Immunotoxins: Targeted Toxin Delivery for Cancer Therapy

“…[38][39][40] Granzyme B GrB is a serine protease found in cytoplasmic granules of NK cells and cytotoxic T cells. 86 Due to cytotoxicity with GrB, it was usually expressed as an inactive zymogen form, and the activity was recovered by lysosomal dipeptide protease to remove two propeptide amino acids, it is expressed as an inactive zymogen form, and can recover activity by lysosomal dipeptide protease to remove two propeptide amino acids. GrB can enter the cytoplasm of target cells through the pores in the cell membrane, and cause apoptosis by cutting various structural and functional proteins containing Aps-X, Glu-X sites, etc., such as a variety of caspases, BH3 proapoptotic protein.…”