Design of human granzyme B variants resistant to serpin B9

Losasso, Valeria; Schiffer, Sonja; Barth, Stefan; Carloni, Paolo

doi:10.1002/prot.24133

Cited by 26 publications

(34 citation statements)

References 51 publications

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“…Gb was produced in HEK293 cells with an N-terminal protective peptide fused to an enterokinase cleavage site (EGb) to suppress the enzymatic activity in the host cells, as previously described (11). Activity was restored by the enzymatic removal of this peptide using 0.02 U of recombinant enterokinase (Novagen; Merck) per g of protein (12). The restored enzymatic activity was confirmed using a colorimetric activity assay (13).…”

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confidence: 99%

“…Reimann, R. Fischer, S. Barth, and R. Fendel, unpublished data), by splicing by overlap extension (SOE)-PCR using a glycine-serine linker peptide and fusing it to the SerpinB9-resistant EGb R201K mutant (12). This was expressed in HEK293-6E cells using a vector based on pTT5 (25) modified with an expression cassette designed for EGb-scFv fusion proteins (26,27).…”

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confidence: 99%

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Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

Kapelski

Almeida

Fischer

et al. 2015

Antimicrob Agents Chemother

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We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC 50 ], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC 50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B. Malaria is caused by parasites of the genus Plasmodium and remains one of the most widespread and dangerous infectious diseases, causing ϳ627,000 deaths worldwide per year (1). Among the six species that infect humans, Plasmodium falciparum causes the severest form of the disease (2). There is no effective vaccine against these parasites (3, 4), and resistances are emerging against a wide variety of antimalarial drugs (5). It was recently shown in vitro that natural killer (NK) cells can eliminate erythrocytes infected with P. falciparum (6) and that this is associated with the production of the serine protease granzyme B (Gb) (7). In vivo, NK cells are essential for protection against plasmodial infections in mice (8), and increased levels of circulating Gb are found in naturally infected humans (9).We therefore investigated the antimalarial effect of recombinant Gb on P. falciparum (strain 3D7A) in a standardized 72-h drug susceptibility assay starting with synchronized ring-stage parasites (10). Gb was produced in HEK293 cells with an N-terminal protective peptide fused to an enterokinase cleavage site (EGb) to suppress the enzymatic activity in the host cells, as previously described (11). Activity was restored by the enzymatic removal of this peptide using 0.02 U of recombinant enterokinase (Novagen; Merck) per g of protein (12). The restored enzymatic activity was confirmed using a colorimetric activity assay (13). Parasite growth was specifically inhibited by activated Gb, with a half-maximal inhibitory concentration (IC 50 ) of 1,590 nM (95% confidence interval [95% CI], 1,197 to 2,112 nM, calculated using the Hill equation in GraphPad Prism version 5). Undigested (inactive) EGb showed no inhibition ( Fig. 1 and Table 1). To our knowledge, this is the first time that the antimalarial activity of Gb has been directly confirmed in vitro.We developed a strategy to target Gb to the parasite and thus reduce the required dose. Targeted toxin delivery via the parasite transferrin receptor has already been reported (14,15). Although some authors claim to have identified and characterized this receptor (16, 17), others argue that iron uptake by the parasite is nonspecific and that the P. falciparum transferrin receptor remains elusive (18). Promising alternative targets include the merozoite surface proteins (MSPs), especially MSP1, MSP2, MSP4, and MSP8, which bear glycosylphosphatidylinositol (GPI) anchors and th...

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confidence: 99%

Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

Kapelski

Almeida

Fischer

et al. 2015

Antimicrob Agents Chemother

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“…[27][28][29][30] We have addressed this challenge by developing a PI9-independent variant of granzyme B. 31 We previously showed that human granzyme B fused to the humanized single chain antibody H22 (which binds specifically and with high affinity to CD64) induces apoptosis in AML cells.…”

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Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Schiffer

Rosinke

Jost

et al. 2014

Intl Journal of Cancer

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CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.The clonal hematopoietic stem cell disorder CMML (chronic myelomonocytic leukemia), is a highly aggressive and resistant form of leukemia. It is characterized by persistent monocytosis in the peripheral blood, at least one dysplasia component in the bone marrow, and <20% promonocytes and blasts in peripheral blood and bone marrow. 1 CMML can be classified further according to blast numbers, that is, CMML1 (<5% blasts in peripheral blood and <10% blasts in bone marrow) and CMML2 (<20% blasts in peripheral blood and 10-19% blasts in bone marrow).2,3 The disease occurs mainly in elderly people with a median survival of 15-20 months and leukemic transformation rates of 15-30%, both factors depending on the disease subtype. 4 Other diagnostic criteria include immunophenotyping based on the overlapping antigen expression patterns in CMML and acute myelomonocytic leukemia (AMML). The expression of CD56 combined with reductions in the levels of myeloid markers such as CD15 and CD13 is a unique signature of CMML monocytes.5 Furthermore, CD14 is expressed at higher levels on bone marrow monocytes in CMML compared with reactive monocytosis and normal marrow samples. 5 High levels of CD33 have also been reported. 6 Clonal cytogenetic abnormalities are found in 20-40% of CMML patients, but these do not appear to be specific. [7][8][9] The close relationship between CMML and other myelodysplastic (MDS)/MPNs makes correct diagnosis and treatment a significant challenge. Novel molecular markers described more recently include specific alleles of TET2 and CBL, but these

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“…Based on in silico analysis carried out by the German Research School for Simulation Sciences in Jülich, we recently identified the molecular contacts between granzyme B and PI-9 by molecular modeling [144]. The inhibition of granzyme B by PI-9 involves the generation of a reversible Michaelis complex followed by a covalent stoichiometric 1:1 interaction, resulting in the irreversible inactivation of both the enzyme and the inhibitor [81,145,146].…”

Section: Design Of Granzyme B Variants That Are Insensitive Towards Pi-9mentioning

confidence: 99%

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

Cremer

Hehmann-Titt²,

Schiffer

et al. 2015

Resistance to Targeted Anti-Cancer Therapeutics

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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

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Design of human granzyme B variants resistant to serpin B9

Cited by 26 publications

References 51 publications

Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

Antimalarial Activity of Granzyme B and Its Targeted Delivery by a Granzyme B–Single-Chain Fv Fusion Protein

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

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

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