Granzyme M as a novel effector molecule for human cytolytic fusion proteins: CD64-specific cytotoxicity of Gm-H22(scFv) against leukemic cells

Schiffer, Sonja; Letzian, Soriba; Jost, Edgar; Mladenov, Radoslav; Hristodorov, Dmitrij; Hühn, Michael; Fischer, Rainer; Barth, Stefan; Thepen, Theo

doi:10.1016/j.canlet.2013.08.005

Cited by 22 publications

(23 citation statements)

References 46 publications

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“…Several hCFPs have demonstrated specific activity and efficiency in vitro and in preclinical studies. 22,23 We show here for the first time that targeting CD64 on both murine (transgenic for hCD64) and human macrophages results in selective elimination of M1, but not M2 cells, although both subtypes express CD64. Compared to M2, M1 macrophages have reduced endosomal protease activity, which limits the proteolytic degradation of the immunotoxin after internalization through CD64.…”

Section: Introductionmentioning

confidence: 82%

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

Hristodorov¹,

Mladenov²,

Felbert³

et al. 2015

mAbs

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(2015) Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies, mAbs, 7:5, 853-862, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 (classical M1 or alternative M2), that play a differential role in immune regulation. In general, the M1 contribute to onset of inflammation, whereas the M2 orchestrate resolution and repair, whereby failure to switch from predominantly M1 to M2 reinforces a pro-inflammatory environment and chronic inflammation. Here, we show selective elimination of M1 macrophages in vitro by a range of CD64-targeted immunotoxins, including H22 (scFv)-ETA'. After re-polarization of already polarized macrophages, still only M1 polarization showed sensitivity toward CD64-directed immunotoxins. The selectivity for M1 was found linked to reduced endosomal protease activity in M1 macrophages as demonstrated by inhibition of endosomal proteases. Using the H22(scFv)-ETA' in a transgenic mouse model for chronic cutaneous inflammation, the M1 specificity was confirmed in vivo and a beneficial effect on inflammation demonstrated. Also ex vivo on skin biopsies from atopic dermatitis and diabetes type II patients with chronically-inflamed skin, a clear M1 specific effect was found. This indicates the potential relevance for human application. Our data show that targeting M1 macrophages through CD64 can be instrumental in developing novel intervention strategies for chronic inflammatory conditions.

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Section: Introductionmentioning

confidence: 82%

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

Hristodorov¹,

Mladenov²,

Felbert³

et al. 2015

mAbs

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“…Incubation of the primary cells with the inactive variant GbS184A-H22(scFv) 38 and the nonbinding control GbR201K-Ki4(scFv) 39 had no impact on cell viability (data shown in Schiffer et al 37 and Supporting Information Fig. S1, respectively).…”

Section: Cancer Therapymentioning

confidence: 97%

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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“…The concept of recombinant immunotoxins has been extensively investigated in the field of targeted tumor therapy with various bacterial and human effector domains (30)(31)(32), including Gb (11,13,27,33). After binding to a disease-specific cell surface antigen, these immunotoxins are internalized, e.g., by receptor-mediated endocytosis, released from endosomal compartments into the cytoplasm, and efficiently kill the malignant cell by their catalytic activity.…”

Section: Figmentioning

confidence: 99%

“…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). Two unrelated EGbscFv fusion constructs named EGb-H22 (targeting human CD64) (11) and EGb-Ki4 (targeting human CD30) (13) were used as negative controls.…”

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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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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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Granzyme M as a novel effector molecule for human cytolytic fusion proteins: CD64-specific cytotoxicity of Gm-H22(scFv) against leukemic cells

Cited by 22 publications

References 46 publications

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies

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

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

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