Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells

Stöcker, Michael; Tur, Mehmet Kemal; Sasse, Stephanie; Krüssmann, Anne; Barth, Stefan; Engert, Andreas

doi:10.1016/s1046-5928(02)00709-x

Cited by 58 publications

(59 citation statements)

References 33 publications

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“…The sequence encoding mature granzyme B was amplified from human blood-derived RNA and cloned into a pMS vector (32,33) to obtain pMS-LKGb-II, a plasmid encoding Ki4(scFv)-granzyme B, as described elsewhere (32).…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…After centrifugation, cleared supernatants were blended with 4Â incubation buffer (pH 7.4) as described previously (33), filter sterilized, and applied to an XK16 column (Amersham/GE Healthcare) containing 5 mL Talon Superflow resin (Clontech/Takara) pre-equilibrated with 1Â incubation buffer (pH 7.4). Washing and elution were also done with the buffers described by Stocker et al (33) modified to pH 7.4. Eluted protein was rebuffered in PBS containing 20 mmol/L Tris using a HiPrep 26/10 desalting column (Amersham/ GE Healthcare) and concentrated by centrifugation at 3,800 Â g with a Vivaspin 6 ultrafiltration spin column 5 kDa MWCO (Sartorius).…”

Section: Expression Purification and Activation Of Gb-h22(scfv)mentioning

confidence: 99%

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Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Stahnke

Thepen

Stöcker³

et al. 2008

Molecular Cancer Therapeutics

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Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (Fc;RI), the highaffinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8 + T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64 + U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC 50 ) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64 + AML cells, whereas CD64 À AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells. [Mol Cancer Ther 2008;7(9):2924 -32]

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Section: Construction Of Plasmidsmentioning

confidence: 99%

Section: Expression Purification and Activation Of Gb-h22(scfv)mentioning

confidence: 99%

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Stahnke

Thepen

Stöcker³

et al. 2008

Molecular Cancer Therapeutics

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“…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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“…Expression vector pL (previously designated as pMS) has been described previously. 20,50 In brief, pL is a derivative of the pSecTag2 plasmid (Invitrogen) that contains the IVS/IRES-EGFP sequence of the pIRES-EGFP plasmid (Clontech). Further, both aCD19 and aCD33 scFvs were PCR amplified to replace SfiI by NotI restriction site at the 5 0 end to generate respective scFv flanked by NotI sites.…”

Section: Methodsmentioning

confidence: 99%

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activityin vitroandin vivoagainst chronic lymphocytic leukemia

et al. 2016

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Chronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor in situ effector functions. NKG2D is an activating receptor found on NK and CD8 C T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono-and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant in vivo activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept.

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Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells

Cited by 58 publications

References 33 publications

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes

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

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activityin vitroandin vivoagainst chronic lymphocytic leukemia

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