Mehmet Kemal Tur

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

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcγRI), the high-affinity 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 (IC50) 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− 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]

Targeted restoration of down-regulated DAPK2 tumor suppressor activity induces apoptosis in Hodgkin lymphoma cells.

Death-associated protein kinase 2 (DAPK2) is a calcium/calmodulin-regulated proapoptotic serine/threonine kinase that acts as a tumor suppressor. Here we show that DAPK2 is down-regulated in Hodgkin lymphoma-derived tumor cell lines and that promoter-region hypermethylation is one mechanism for DAPK2 inactivation. To determine whether selective reconstitution of DAPK2 catalytic activity in these cells could induce apoptosis, we created a fusion protein comprising a human CD30 ligand conjugated to a human DAPK2 calmodulin-deletion mutant. Thus, recombinant immunokinase DAPK2′-CD30L has a constitutive kinase activity with enhanced proapoptotic function. We show that this immunokinase fusion protein inhibits cell proliferation and induces apoptotic cell death specifically in CD30+/DAPK2-negative tumor cell lines. This proof-of–concept study provides the first demonstration of therapeutic strategies based on the restoration of a defective, tumor-suppressing kinase activity by a novel class of recombinant immunotherapeutics.

SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells.

Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.

In vivo efficacy of the recombinant anti-CD64 immunotoxin H22(scFv)-ETA' in a human acute myeloid leukemia xenograft tumor model.

Target‐specific acute myeloid leukemia (AML) immunotherapy requires selective cell‐surface antigens on AML blast cells. CD64 is a promising candidate antigen because it is abundantly expressed on monocytoid differentiated AML subtypes. In previous studies, a chemically linked full‐length anti‐CD64 immunotoxin based on ricin A showed promising results in several animal models, but further development has been hindered by its substantial, dose‐limiting off‐target effects. We recently constructed the recombinant immunotoxin H22(scFv)‐ETA′, comprising a truncated Pseudomonas exotoxin A (PE) and a humanized scFv antibody against CD64. This molecule was shown to kill CD64+ AML‐derived tumor cell lines and primary patient‐derived AML cells specifically, both in vitro and ex vivo. Here we describe the in vivo efficiency of H22(scFv)‐ETA′ in the U937/SCID mouse xenograft model for human AML, by providing immunohistochemical evidence for the elimination of human CD64+ tumor cells in mouse organs. H22(scFv)‐ETA′ showed potent antitumor activity against myeloid tumor cells and significantly prolonged the overall survival of AML xenograft animals. In conclusion, H22(scFv)‐ETA′ is efficacious against AML with monocytoid differentiation in vitro and in animal models in vivo, providing the basis for a novel therapeutic strategy for the treatment of AML patients.

Recombinant, ETA′-based CD64 immunotoxins: improved efficacy by increased valency, both in vitro and in vivo in a chronic cutaneous inflammation model in human CD64 transgenic mice

Background  Dysregulated, activated macrophages play a pivotal role in chronic inflammatory diseases such as arthritis and atopic dermatitis. These cells display increased expression of the high‐affinity Fcγ receptor (CD64), making them ideal targets for CD64‐specific immunotoxins. We previously showed that a chemically linked immunotoxin, the monoclonal H22‐RicinA, specifically eliminated infiltrating activated macrophages and resolved chronic cutaneous inflammation. However, several disadvantages are associated with classic immunotoxins, and we therefore followed a fusion protein strategy to express the antigen‐binding site alone (scFv H22) fused to a derivative of Pseudomonas exotoxin A (ETA′).

Targeted Delivery of Short Interfering RNAs - Strategies for In Vivo Delivery

RNA interference (RNAi) is a powerful endogenous process initiated by short double stranded RNAs, which results in sequence-specific posttranscriptional gene silencing. The ability to block the expression of any disease-causing or disease-related protein emphasizes the huge therapeutic potential of this technology. In a clinical setting, however, the use of RNAi-based therapeutics is limited by their short serum half lives and poor uptake into cells. In this review, we provide an overview of recent patents in the field of short interfering RNA (siRNA) delivery and discuss recent progress in the development of efficient siRNA delivery vehicles enhancing the pharmacokinetic properties of RNAi-based therapeutics and promoting cellular uptake.

An Aptamer–siRNA Chimera Silences the Eukaryotic Elongation Factor 2 Gene and Induces Apoptosis in Cancers Expressing αvβ3 Integrin

Small interfering RNAs (siRNAs) silence gene expression by triggering the sequence-specific degradation of mRNAs, but the targeted delivery of such reagents remains challenging and a significant obstacle to therapeutic applications. One promising approach is the use of RNA aptamers that bind tumor-associated antigens to achieve the delivery of siRNAs to tumor cells displaying specific antigens. Wholly RNA-based constructs are advantageous because they are inexpensive to synthesize and their immunogenicity is low. We therefore joined an aptamer-recognizing alpha V and integrin beta 3 (αvβ3) integrin to a siRNA that targets eukaryotic elongation factor 2 and achieved for the first time the targeted delivery of a siRNA to tumor cells expressing αvβ3 integrin, causing the inhibition of cell proliferation and the induction of apoptosis specifically in tumor cells. The impact of our results on the development of therapeutic aptamer-siRNA constructs is discussed.

Fcγ Receptor 1 (CD64), a Target Beyond Cancer

Immunotoxins are powerful tools to specifically eliminate deviated cells. Due to the side effects of the original immunotoxins, they were only considered for the treatment of cancer as in these cases, the potential favourable effect outweighed the unwanted toxic side effects. Over time, many improvements in the construction of immunotoxins have been implemented that circumvent, or at least strongly diminish, the side effects. In consequence this opens the way to employ these immunotoxins for the treatment of non-life threatening diseases. One such category of disease could be the many chronic inflammatory disorders in which an uncontrolled interaction between inflammatory cells leads to chronicity. In several of these chronic conditions, activated macrophages, which are characterised by an increased expression of CD64, are known to play a key role. In this review we discus the data presently available on elimination of activated macrophages through CD64 immunotoxins in several animal models for chronic disease. A chemically linked complete antibody with the plant toxin Ricin-A, proved very effective and provided proof of concept. Subsequently, the development towards genetically engineered, fully human, multivalent single chain based immunotoxins that have diminished immunogenicity, is discussed. The data show that the specific elimination of activated macrophages through CD64 is indeed beneficial for the course of disease. As opposed to other methods used to inactivate or eliminate macrophages, with the CD64 based immunotoxins only the activated population is killed. This may open the way to apply these immunotoxins as therapeutics in chronic inflammatory disease.

Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins

BackgroundConventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs) are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP).ResultsAs an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv) Ki-4(scFv) and the anti-MucI single-chain fragment variable M12(scFv). During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct.ConclusionsOur low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.

Metalloproteinase inhibition augments antitumor efficacy of the anti-CD30 immunotoxin Ki-3(scFv)-ETA' against human lymphomas in vivo.

There is increasing evidence that the shedding of extracellular antigen domains impedes selective immunotherapy. One example is CD30, which is overexpressed on the surface of malignant lymphoma cells and has been identified as a promising target for antibody‐based immunotherapy. However, CD30 is cleaved from the surface of target cells and the resulting soluble ectodomain (sCD30) is protecting the cells from antibody binding. Shedding can be inhibited by hydroxamate inhibitors of metalloproteinases such as BB‐3644. We thus evaluated the influence of BB‐3644 on the efficacy of the anti‐CD30 single‐chain immunotoxin Ki‐3(scFv)‐ETA′. In vitro, the addition of BB‐3644 augmented the antitumor effect of Ki‐3(scFv)‐ETA′ against Hodgkin‐derived L540Cy cells by a factor of 2.75. Severe combined immunodeficiency (SCID) mice challenged with CD30‐positive L540Cy cells were treated with the immunotoxin. One single nontoxic dose of BB‐3644 increased the mean survival time of animals treated concomitantly with Ki‐3(scFv)‐ETA′ to 93 days as compared with 35 days in the control (p = 0.0017). When BB‐3644 was continuously delivered using subcutaneously implanted pumps, this effect was even more pronounced with no observed tumor growth in the animals within 200 days. Thus, concomitant application of metalloproteinase inhibitors might become clinically relevant in antibody‐based immunotherapy against targets known to be shed from tumor cells.