A Bispecific Antibody That Targets the Membrane-Proximal Region of Mesothelin and Retains High Anticancer Activity in the Presence of Shed Mesothelin.

Mesothelin (MSLN) is a cell-surface protein that is expressed in many cancers, which makes it a popular target for Ab-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patients' fluids and tumors and can block Ab-based MSLN-targeting drugs from killing cancer cells. A previously established mAb, 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. Moreover, 15B6 variable fragment (Fv)-derived chimeric antigen receptor T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived chimeric antigen receptor T cells, which bind an epitope retained in shed MSLN. In this study, we have established 15B6 Fv-derived MSLN × CD3 bispecific antibodies (BsAb) that target MSLN-expressing cancers. We identified our lead candidate BsAb 5 after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

A bispecific antibody that targets the membrane-proximal region of mesothelin and retains high anticancer activity in the presence of shed mesothelin.

Mesothelin (MSLN) is a cell-surface protein that is expressed on many cancers, which makes it a popular target for antibody-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patient fluids and tumors and can block antibody-based MSLN-targeting drugs from killing cancer cells. A previously established monoclonal antibody (mAb), 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. 15B6 variable fragment (Fv)-derived chimeric antigen receptor (CAR) T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived CAR T cells, which bind an epitope retained in shed MSLN. Here, we have established 15B6 Fv-derived MSLN x CD3 bispecific antibodies (BsAbs) that target MSLN-expressing cancers. We identified our lead candidate, BsAb 5, after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

Highly active CAR T cells that bind to a juxtamembrane region of mesothelin and are not blocked by shed mesothelin.

SignificanceMesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.

Immunotherapy-based targeting of MSLN+ activated portal fibroblasts is a strategy for treatment of cholestatic liver fibrosis.

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln-/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1-/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN+ aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)-injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.

Anti-BCMA Immunotoxins: Design, Production, and Preclinical Evaluation.

Multiple myeloma (MM) is a B-cell malignancy that is incurable for a majority of patients. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed in multiple myeloma cells but not in other normal tissues except normal plasma B cells. Due to the restricted expression and being a cell surface membrane protein, BCMA is an ideal target for immunotherapy approaches in MM. Recombinant immunotoxins (RITs) are a novel class of protein therapeutics that are composed of the Fv or Fab portion of an antibody fused to a cytotoxic agent. RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in E. coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays using BCMA expressing cell lines and on cells isolated from MM patients. The in vivo efficacy of RITs was tested in a xenograft mouse model using BCMA expressing multiple myeloma cell lines. Anti-BCMA recombinant immunotoxins are very effective in killing myeloma cell lines and cells isolated from myeloma patients expressing BCMA. Two mouse models of myeloma showed that the anti-BCMA immunotoxins can produce a long-term complete response and warrant further preclinical development.

Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and Antitumor Activity.

Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using ß-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl)phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10- to 30-fold increase in half-life that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.

Anti-Mesothelin Recombinant Immunotoxin Therapy for Colorectal Cancer.

BACKGROUND: Mesothelin (MSLN) is a cell surface glycoprotein expressed at a high level on many malignancies, including pancreatic adenocarcinoma, serous ovarian cancer, and epithelioid mesothelioma. MSLN-targeted recombinant immunotoxins (RITs) consist of an anti-MSLN Fv fused to the catalytic domain of Pseudomonas exotoxin A. Recent data has also shown that MSLN is expressed at clinically relevant levels on the surface of colorectal cancer (CRC). In this study, CRC cell lines were tested for MSLN expression and susceptibility to MSLN-targeted RITs. MATERIALS AND METHODS: CRC cell lines were tested for membranous MSLN expression via flow cytometry. Cell lines expressing MSLN were tested by WST-8 cell viability assay for sensitivity to various RITs and chemotherapeutic agents. CRC cell line SW-48 was tested in a mouse model for response to RIT as a single agent or in combination with actinomycin D and oxaliplatin. RESULTS: CRC cell lines were susceptible to anti-MSLN RITs at half maximal inhibitory concentration levels comparable with those previously described in pancreatic cancer cell lines. In a nude mouse model, MSLN-targeted RIT treatment of SW48 CRC tumors resulted in a significant decrease in tumor volume. Although combination therapy with standard of care chemotherapeutic oxaliplatin did not improve tumor regressions, combination therapy with actinomycin D resulted in > 90% tumor volume reduction with 50% complete regressions. CONCLUSIONS: These data support the development of anti-MSLN RITs as well as other MSLN-targeted therapies for CRC.

Generation of a Transgenic BALB/c Mouse Line With Selective Expression of Human Mesothelin in Thyroid Gland: Application in Mesothelin-targeted Immunotherapy.

Despite encouraging clinical results with immune checkpoint inhibitors and other types of immunotherapies, the rate of failure is still very high. The development of proper animal models which could be applied to the screening of effective preclinical antitumor drugs targeting human tumor antigens, such as mesothelin (MSLN), is a great need. MSLN is a 40 kDa cell-surface glycoprotein which is highly expressed in a variety of human cancers, and has great value as a target for antibody-based therapies. The present study reports the establishment of an immunocompetent transgenic mouse expressing human MSLN (hMSLN) only in thyroid gland by utilizing an expression vector containing a thyroid peroxidase (TPO) promoter. These mice do not reject genetically modified tumor cells expressing hMSLN on the cell membrane, and tolerate high doses of hMSLN-targeted immunotoxin. Employing this TPO-MSLN mouse model, we find that the combination treatment of LMB-100 and anti-CTLA-4 induces complete tumor regression in 91% of the mice burdened with 66C14-M tumor cells. The combination therapy provides a significant survival benefit compared with both LMB-100 and anti-CTLA-4 monotherapy. In addition, the cured mice reject tumor cells when rechallenged, indicating the development of long-term antitumor immunity. This novel TPO-MSLN mouse model can serve as an important animal tool to better predict tumor responses to any immunomodulatory therapies that target MSLN.

Anti-BCMA immunotoxins produce durable complete remissions in two mouse myeloma models.

Multiple myeloma (MM) is a B cell malignancy for which new treatments are urgently needed. The B cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed on myeloma. Recombinant immunotoxins (RITs) are proteins composed of the Fv or Fab portion of an antibody fused to a bacterial toxin. We previously treated H929 myeloma s.c. tumors with anti-BCMA immunotoxins, very active on killing cultured cells, and observed tumor growth inhibition but not complete tumor responses. To determine if immunotoxins were more active against cells growing in the bone marrow (BM), the normal location of myeloma cells, we developed a BM mouse model that is more relevant to human disease. H929 cells were transfected with luciferase and GFP, enriched by flow, recycled through the BM of a mouse, and injected IV into nonobese diabetic scid γ mice (NSG) mice. A second myeloma mouse model used the MM.1S-GFP-luc cell line. Mice were treated IV with immunotoxins, and the tumor burden was assessed using bioluminescence imaging. We achieved complete durable remissions when treating mice with H929-GFP-luc cells with anti-BCMA RITs both leptomycin B-75 (LMB-75) [anti-BCMA-disulfide-stabilized (ds)-Fv-PE24] (where PE represents Pseudomonas exotoxin A) or LMB-70 (anti-BCMA-Fab-PE24) given every other day for 5-d (QOD×5) doses beginning on day 4 or day 8. Mice were disease free at 3 months; untreated mice became moribund around day 40. We also achieved long-term responses using the MM.1S-GFP-luc myeloma cell line. Treatment with an 1.5 mg/kg LMB-75 QOD×5 anti-BCMA RIT beginning on day 4 caused the complete disappearance of tumors for 80 days. To summarize, LMB-75 and LMB-70, our anti-BCMA RITs, induced complete durable responses in two myeloma models.

Preclinical development of anti-BCMA immunotoxins targeting multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy that is incurable for the majority of patients. New treatments are urgently needed. Recombinant immunotoxins (RITs) are chimeric proteins that are composed of the Fv or Fab portion of an antibody fused to a bacterial toxin. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein and an ideal target for antibody-based treatments for MM. METHODS: RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in Escherichia coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays. We also measured their binding to human and mouse serum albumins and to BCMA and measured their serum half-life in mice. RESULTS: Using Fvs from different anti-BCMA antibodies, we produced RITs that specifically kill BCMA-expressing MM cells in vitro. To increase the serum half-life in vivo, we generated RITs that are fused with albumin-binding domains (ABDs). All RITs with ABDs have some decreased activity compared to the parent RIT, which is not due to decreased binding to BCMA. CONCLUSIONS: Various new anti-BCMA immunotoxins were produced and evaluated. None of these were better than LMB-75 (anti-BCMA BM306-disulfide-stabilized Fv-LRggs) supporting the further preclinical development of LMB-75.

Domain II of Pseudomonas Exotoxin Is Critical for Efficacy of Bolus Doses in a Xenograft Model of Acute Lymphoblastic Leukemia.

Moxetumomab pasudotox is a fusion protein of a CD22-targeting antibody and Pseudomonas exotoxin. Minutes of exposure to Moxetumomab achieves similar cell killing than hours of exposure to a novel deimmunized variant against some acute lymphoblastic leukemia (ALL). Because blood levels fall quickly, Moxetumomab is more than 1000-fold more active than the deimmunized variant in vivo. We aimed to identify which part of Moxetumomab increases in vivo efficacy and generated five immunotoxins, tested time-dependent activity, and determined the efficacy in a KOPN-8 xenograft model. Full domain II shortened the time cells had to be exposed to die to only a few minutes for some ALL; deimmunized domain III consistently extended the time. Against KOPN-8, full domain II accelerated time to arrest protein synthesis by three-fold and tripled PARP-cleavage. In vivo efficacy was increased by more than 10-fold by domain II and increasing size, and therefore half-life enhanced efficacy two- to four-fold. In summary, in vivo efficacy is determined by the time cells have to be exposed to immunotoxin to die and serum half-life. Thus, domain II is most critical for activity against some ALL treated with bolus doses; however, immunotoxins lacking all but the furin-cleavage site of domain II may be advantageous when treating continuously.

Recombinant immunotoxins with albumin-binding domains have long half-lives and high antitumor activity.

Recombinant immunotoxins (RITs) are chimeric proteins consisting of a Fv that binds to a cancer cell and a portion of a protein toxin. One of these, Moxetumomab pasudotox, was shown to be effective in treating patients with some leukemias, where the cells are readily accessible to the RIT. However, their short half-life limits their efficacy in solid tumors, because penetration into the tumors is slow. Albumin and agents bound to albumin have a long half-life in the circulation. To increase the time tumor cells are exposed to RITs, we have produced and evaluated variants that contain either an albumin-binding domain (ABD) from Streptococcus or single-domain antibodies from Llama. We have inserted these ABDs into RITs targeting mesothelin, between the Fv and the furin cleavage site. We find that these proteins can be produced in large amounts, are very cytotoxic to mesothelin-expressing cancer cell lines, and have a high affinity for human or mouse serum albumin. In mice, the RIT containing an ABD from Streptococcus has a longer half-life and higher antitumor activity than the other two. Its half-life in the circulation of mice ranges from 113 to 194 min compared with 13 min for an RIT with no ABD. Cell uptake studies show the RIT enters the target cell bound to serum albumin. We conclude that RITs with improved half-lives and antitumor activity should be evaluated for the treatment of cancer in humans.

Combining Local Immunotoxins Targeting Mesothelin with CTLA-4 Blockade Synergistically Eradicates Murine Cancer by Promoting Anticancer Immunity.

Immune checkpoint blockade using antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) benefits a limited number of cancer patients. SS1P and LMB-100 are immunotoxins that target mesothelin. We observed delayed responses to SS1P in patients with mesothelioma suggesting that antitumor immunity was induced. Our goal was to stimulate antitumor immunity by combining SS1P or LMB-100 with anti-CTLA-4. We constructed a BALB/c breast cancer cell line expressing human mesothelin (66C14-M), which was implanted in one or two locations. SS1P or LMB-100 was injected directly into established tumors and anti-CTLA-4 administered i.p. In mice with two tumors, one tumor was injected with immunotoxin and the other was not. The complete regression rate was 86% for the injected tumors and 53% for the uninjetced tumors. No complete regressions occurred when drugs were given separately. In regressing tumors, dying and dead tumor cells were intermingled with PMNs and surrounded by a collar of admixed eosinophils and mononuclear cells. Tumor regression was associated with increased numbers of tumor-infiltrating CD8+ cells and blocked by administration of antibodies to CD8. Surviving mice were protected from tumor rechallenge by 66C14 cells not expressing mesothelin, indicating the development of antitumor immunity. The antitumor effect was abolished when a mutant noncytotoxic variant was used instead of LMB-100, showing that the antitumor response is not mediated by recognition of a foreign bacterial protein. Our findings support developing a therapy composed of immunotoxins and checkpoint inhibitors for patients. Cancer Immunol Res; 5(8); 685-94. ©2017 AACR.

Reduced Shedding of Surface Mesothelin Improves Efficacy of Mesothelin-Targeting Recombinant Immunotoxins.

Mesothelin (MSLN) is a differentiation antigen that is highly expressed in many epithelial cancers. MSLN is an important therapeutic target due to its high expression in cancers and limited expression in normal human tissues. Although it has been assumed that shed antigen is a barrier to immunotoxin action, a modeling study predicted that shed MSLN may enhance the action of MSLN-targeting recombinant immunotoxins such as SS1P and similar therapeutics by facilitating their redistribution within tumors. We aimed to determine whether shed MSLN enhances or reduces the antitumor effect of MSLN-targeting immunotoxins SS1P and RG7787. We engineered a cell line, A431/G9 (TACE mutant) that expresses a mutant form of MSLN in which the TNF-converting enzyme protease site is replaced with GGGS. We compared the response of the TACE-mutant cells with immunotoxins SS1P and RG7787 with that of the parental A431/H9 cell line. We show that TACE-mutant cells shed 80% less MSLN than A431/H9 cells, that TACE-mutant cells show a 2- to 3-fold increase in MSLN-targeted immunotoxin uptake, and that they are about 5-fold more sensitive to SS1P killing in cell culture. Tumors with reduced shedding respond significantly better to treatment with SS1P and RG7787. Our data show that MSLN shedding is an impediment to the antitumor activity of SS1P and RG7787. Approaches that decrease MSLN shedding could enhance the efficacy of immunotoxins and immunoconjugates targeting MSLN-expressing tumors. Mol Cancer Ther; 15(7); 1648-55. ©2016 AACR.

An improved recombinant Fab-immunotoxin targeting CD22 expressing malignancies.

Moxetumomab pasudotox (HA22) is a recombinant immunotoxin, now in clinical trials, that combines an anti-CD22-Fv with a 38-kDa fragment of Pseudomonas exotoxin A. To produce a less immunogenic molecule without reducing the half-life in circulation, we constructed LMB11 combining an anti-CD22 Fab with a less immunogenic version of PE38. We found that LMB11 retains full activity toward CD22-expressing cells. In mice, the half-life of LMB11 is 29 min and the antitumor activity of LMB11 is better than that of HA22. Because it can be safely given at much higher doses, LMB11 produced complete tumor remissions in 7/7 mice.

Megakaryocytic potentiating factor and mature mesothelin stimulate the growth of a lung cancer cell line in the peritoneal cavity of mice.

The mesothelin (MSLN) gene encodes a 71 kilodalton (kDa) precursor protein that is processed into megakaryocytic potentiating factor (MPF), a 31 kDa protein that is secreted from the cell, and mature mesothelin (mMSLN), a 40 kDa cell surface protein. The mMSLN binds to CA125, an interaction that has been implicated in the intra-cavitary spread of mesothelioma and ovarian cancer. To better define the role of MPF and mMSLN, growth of the lung cancer cell line A549 was evaluated in immuno-deficient mice with inactivation of the Msln gene. We observed that Msln-/- mice xenografted with intraperitoneal A549 tumors survive significantly long than tumor-bearing Msln+/+ mice. When tumor-bearing Msln-/- mice are supplemented with recombinant MPF (and to a lesser extent mMSLN), most of this survival advantage is lost. These studies demonstrate that MPF and mMSLN have an important role in the growth of lung cancer cells in vivo and raise the possibility that inactivation of MPF may be a useful treatment for lung and other MSLN expressing cancers.

Methylation of the DPH1 promoter causes immunotoxin resistance in acute lymphoblastic leukemia cell line KOPN-8.

Moxetumomab pasudotox (HA22) is an immunotoxin with an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A that kills CD22 expressing ALL cells. HA22 produced significant responses in some cases of ALL. To understand how to increase response rate, we isolated HA22-resistant KOPN-8 cells and found that HA22 cannot inactivate elongation factor-2 (EF2) due to low levels of DPH1 RNA and protein. Resistance was associated with methylation of the CpG island in the DPH1 promoter. 5-Azacytidine prevented resistance and methylation of the CpG residues and merits evaluation to determine if it can increase the efficacy of HA22 in ALL.

Identification and enhancement of HLA-A2.1-restricted CTL epitopes in a new human cancer antigen-POTE.

Identification of CD8(+) T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA) substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272-280 and POTE 323-331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9), POTE 553-1Y (with tyrosine at position 1) and POTE 323-3F (with phenylalanine at position 3) conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F) induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2(+) human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.

A modified form of diphthamide causes immunotoxin resistance in a lymphoma cell line with a deletion of the WDR85 gene.

HA22 is a recombinant immunotoxin that kills CD22-expressing cells by ADP-ribosylating and inactivating elongation factor-2 (EF2). HA22 is composed of an Fv that binds to CD22 fused to a portion of Pseudomonas exotoxin A. HA22 is very active in drug-resistant hairy cell leukemia but is less active in children with acute lymphoblastic leukemia. To understand why some patients do not respond to HA22, we isolated an HA22-resistant lymphoma cell line and showed that resistance was due to the inability of HA22 to ADP-ribosylate and inactivate EF2. We analyzed the diphthamide synthesis genes and found that the WDR85 gene was deleted. We show that WDR85 knockdown conferred HA22 resistance to sensitive cells and that sensitivity was restored by introduction of a WDR85 cDNA into resistant cells. Analysis of EF2 in the mutant cells revealed a novel form of diphthamide with an additional methyl group that prevented ADP-ribosylation and inactivation of EF2. The abnormal methylation appeared to be catalyzed by DPH5. Inactivation of the WDR85 gene could be a mechanism of immunotoxin resistance in patients undergoing immunotoxin therapy.

ANKRD26 and its interacting partners TRIO, GPS2, HMMR and DIPA regulate adipogenesis in 3T3-L1 cells.

Partial inactivation of the Ankyrin repeat domain 26 (Ankrd26) gene causes obesity and diabetes in mice and increases spontaneous and induced adipogenesis in mouse embryonic fibroblasts. However, it is not yet known how the Ankrd26 protein carries out its biological functions. We identified by yeast two-hybrid and immunoprecipitation assays the triple functional domain protein (TRIO), the G protein pathway suppressor 2 (GPS2), the delta-interacting protein A (DIPA) and the hyaluronan-mediated motility receptor (HMMR) as ANKRD26 interacting partners. Adipogenesis of 3T3-L1 cells was increased by selective down-regulation of Ankrd26, Trio, Gps2, Hmmr and Dipa. Furthermore, GPS2 and DIPA, which are normally located in the nucleus, were translocated to the cytoplasm, when the C-terminus of ANKRD26 was introduced into these cells. These findings provide biochemical evidence that ANKRD26, TRIO, GPS2 and HMMR are novel and important regulators of adipogenesis and identify new targets for the modulation of adipogenesis.