Michael Huhn

Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions

ABSTRACT The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coliunder osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.

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.

Macrophage-Targeted Therapy: CD64-Based Immunotoxins for Treatment of Chronic Inflammatory Diseases

Diseases caused by chronic inflammation (e.g., arthritis, multiple sclerosis and diabetic ulcers) are multicausal, thus making treatment difficult and inefficient. Due to the age-associated nature of most of these disorders and the demographic transition towards an overall older population, efficient therapeutic intervention strategies will need to be developed in the near future. Over the past decades, elimination of activated macrophages using CD64-targeting immunotoxins has proven to be a promising way of resolving inflammation in animal models. More recent data have shown that the M1-polarized population of activated macrophages in particular is critically involved in the chronic phase. We recapitulate the latest progress in the development of IT. These have advanced from full-length antibodies, chemically coupled to bacterial toxins, into single chain variants of antibodies, genetically fused with fully human enzymes. These improvements have increased the range of possible target diseases, which now include chronic inflammatory diseases. At present there are no therapeutic strategies focusing on macrophages to treat chronic disorders. In this review, we focus on the role of different polarized macrophages and the potential of CD64-based IT to intervene in the process of chronic inflammation.

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

Immunotoxins are promising targeted therapeutic agents comprising an antibody-based ligand that specifically binds to diseased cells, and a pro-apoptotic protein. Toxic components from bacteria or plants can trigger a neutralizing immune response, so that human effector molecules are more suitable. In this context, the protease granzyme B has been successfully tested in cytotoxicity assays against different cancer cells in vitro and in vivo. Our aim here was to introduce granzyme M as an alternative and novel component of human cytolytic fusion proteins. We fused it to the humanized single-chain antibody fragment (scFv) H22 which specifically binds to CD64, an FcγRI receptor overexpressed on activated myeloid cells and leukemic cells. We show that the humanized cytolytic fusion protein Gm-H22(scFv) specifically targets the acute myeloid leukemia cell line HL60 in vitro and is cytotoxic with an IC50 between 1.2 and 6.4 nM. These findings were confirmed ex vivo using leukemic primary cells from patients, which were killed by granzyme M despite the presence of the granzyme B inhibitor serpin B9. In conclusion, granzyme M is a promising new cell-death inducing component for hCFPs because it specifically and efficiently kills target cells when fused to a targeting component.

A Human Recombinant Autoantibody-Based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth In Vitro and in a Murine Transplantation Model

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) γ-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

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′).

Nickel complexes with heterofunctionalized phosphine ligands. Catalytic oligomerization of ethylene with [Ni(C5Ph5){Ph2PCHC(O)Ph}]

Reaction of [Ni(C5Ph5)Br(CO)] with Ph2PCH2C(O)Ph in the presence of 1,8-bis(dimethylamino)naphthalene (proton sponge) gives the chelate complex [Ni(C5Ph5){Ph2PCHC(O)Ph}]1. The complexes [Ni(C5Ph5){Ph2PCHC(O)(C5H4)Fe(C5H5)}]2, [Ni(C5Ph5){Ph2PCHC(O)(1,3-C6H4)C(O)HCPPh2}Ni(C5Ph5)]3, [Ni(C5H5){Ph2PCHC(O)Ph}]4 and [Ni(C5H5){Ph2PCHC(O)(C5H4)Fe(C5H5)}]5 have been synthesised using the appropriate phosphine ligand and applying a procedure similar to that for 1. Complex 1, activated with NaBH4, exhibits catalytic activity in the low-pressure oligomerization of ethylene. At 130 °C, under 38 bar of ethylene, selectivities of up to 98% towards linear α-olefins were achieved. Protonation of the bimetallic complex 2 with HBF4 gave the cationic complex [Ni(C5Ph5){Ph2PCH2C(O)(C5H4)Fe(C5H5)}]BF46. Crystais of 6 belong to the orthorhombic space group Pbca with a= 19.427(6), b= 38.595(11), c= 13.001(4)A and Z= 8. The structure was refined to R= 0.049 (R′= 0.067). The bonding of C5Ph5 contrasts with the bonding modes usually found for cyclopentadienyl ligands. The ring contains two short [1.384(8) and 1.389(8)], two medium [1.444(8) and 1.443(8)], and one long [1.474(8)A] C–C bonds so that the C5Ph5 may be viewed as an (alkyl, diene) ligand. This unusual bonding is likely to result from the ‘chemical’ dissymmetry of the P,O ligand and the low trans influence of the oxygen atom. The extremely low ν, (CO) frequency of the co-ordinated ketone (1525 cm–1) reflects the strong electron-withdrawing effect of the Ni(C5Ph5)+ moiety.

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

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.

Reaktionen von Pentaphenylcyclopentadienylnickel-Komplexen mit Thiuramen und Dithiocarbamat: Nickel in den Oxidationsstufen I, II and III

Abstract Tetramethylthiuram monosulfide and tetramethylthiuram disulfide react with [(C5Ph5)Ni(NCCH3)2]BF4 (2) to give the black pentaphenylcyclopentadienyl nickel complex [(C5Ph5)Ni{S2CN(CH3)2}]BF4 (7). Its crystal structure has been determined. It is the first 17-electron cyclopentadienyl nickel(III) compound to be structurally characterized. The formation reaction corresponds formally to an oxidative addition of tetramethylthiuram monosulfide and tetramethylthiuram disulfide to the nickel(II) complex 2. The solid-state magnetic moment of the paramagnetic nickel(III) complex 7 is μeff = 1,77 B.M. The reaction of sodium dimethyldithiocarbamate with [(C4Ph5)Ni(CO)Br] yields the dark-red pentaphenylcyclopentadienyl nickel(II) complex [(C5Ph5)Ni{S2CN(CH3)}] (6). Cyclic voltammetry reveals that the dithiocarbamate complex 6 can be oxidised electrochemically and chemically reversibly at +0.265 V vs. SCE to give the cation 7. The reduction at −1.43 V leads to the nickel(I) complex [(C5Ph5Ni{S2CN(CH3)2}]−. The nickel(III) complex 7 can also be prepared on a preparative scale by oxidation of 6 with ferrocene(1 + )tetrafluoroborate in dichloromethane. The reduction of 7 to give 6 is possible in dichloromethane with tetrakis(dimethylamino)ethylene as the reducing agent.

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.