Glioblastoma CD105+ cells define a SOX2- cancer stem cell-like subpopulation in the pre-invasive niche.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. Glioma stem like cells (GSC) represent the highest cellular hierarchy in GBM and have a determining role in tumor growth, recurrence and patient prognosis. However, a better definition of GSC subpopulations, especially at the surgical resection margin, is warranted for improved oncological treatment options. The present study interrogated cells expressing CD105 (CD105+) specifically within the tumor front and the pre-invasive niche as a potential GSC subpopulation. GBM primary cell lines were generated from patients (n = 18) and CD105+ cells were isolated and assessed for stem-like characteristics. In vitro, CD105+ cells proliferated and enriched in serum-containing medium but not in serum-free conditions. CD105+ cells were characterized by Nestin+, Vimentin+ and SOX2-, clearly distinguishing them from SOX2+ GCS. GBM CD105+ cells differentiated into osteocytes and adipocytes but not chondrocytes. Exome sequencing revealed that GBM CD105+ cells matched 83% of somatic mutations in the Cancer cell line encyclopedia, indicating a malignant phenotype and in vivo xenotransplantation assays verified their tumorigenic potential. Cytokine assays showed that immunosuppressive and protumorigenic cytokines such as IL6, IL8, CCL2, CXCL-1 were produced by CD105+ cells. Finally, screening for 88 clinical drugs revealed that GBM CD105+ cells are resistant to most chemotherapeutics except Doxorubicin, Idarubicin, Fludarabine and ABT-751. Our study provides a rationale for targeting tumoral CD105+ cells in order to reshape the tumor microenvironment and block GBM progression.

Human endoglin-CD3 bispecific T cell engager antibody induces anti-tumor effect in vivo.

Rationale: Endoglin, also known as CD105, is a homo-dimeric membrane glycoprotein required for angiogenesis and serves as a marker for cancer vasculature. In this study, we constructed a bispecific T-cell engager (BiTE) antibody that targets human endoglin and CD3 (hEND-CD3/BiTE). We examined BiTE binding to endoglin-expressing cells and its effects on the cytolytic activity of T cells and cancer development. Methods: The in vitro effects of hEND-CD3/BiTE, including binding to target cells, T-cell activation, proliferation, and cytotoxicity, were examined in endoglin-expressing 293T cells, human umbilical vascular endothelial cells, tumor-derived endothelial cells, and CD3+ T cells. An in vivo xenograft tumor model was established using A549 human lung cancer cells. The therapeutic efficacy of hEND-CD3/BiTE was assessed by monitoring tumor growth, angiogenesis, and mouse survival. Results: hEND-CD3/BiTE specifically bound to endoglin-expressing cells and CD3+ T cells in vitro and stimulated T-cell activation, proliferation, and Th1 cytokine secretion, and promoted T-cell-mediated cytolysis of endoglin-expressing cells. The hEND-CD3/BiTE in vivo caused minimal toxicity to major organs, reduced tumor neoangiogenesis, inhibited tumor growth, and significantly improved mouse survival. Conclusions: Our study demonstrated the therapeutic potential of hEND-CD3/BiTE and provided a novel approach to clinical cancer treatment.

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy.

Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105+ target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105+ target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105+ tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.

Renal carcinoma CD105-/CD44- cells display stem-like properties in vitro and form aggressive tumors in vivo.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. Prognosis for ccRCC is generally poor since it is largely resistant to chemo- and radiotherapy. Many studies suggested that cancer stem cells/tumor initiating cells (CSCs/TICs) are responsible for development of tumor, disease progression, aggressiveness, metastasis and drug resistance. However, tumorigenic potential of CSCs/TICs isolated from established RCC cell lines - basic ccRCC research model - has never been investigated in vivo. CD105+, CD105-, CD44+ and CD44- as well as CD44-/CD105- CD44+/CD105+ and CD44-/CD105+ cells were isolated from Caki-1 RCC cell line, confirming coexistence of multiple subpopulations of stem-related phenotype in stable cell line. Sorted cells were injected subcutaneously into NOD SCID mice and tumor growth was monitored with MRI and PET/CT. Tumor growth was observed after implantation of CD105+, CD44+, CD44-, CD44-/CD105+ and CD44-/CD105- but not CD105- or CD44+/CD105+. Implantation of CD44-/CD105- cells induced tumors that were characterized by longer T1 and distinct metabolic pattern than other tumors. All the tumors were characterized by low uptake of [18F]FDG. CD105+ and CD44- tumors expresses Nanog and Oct-4, while CD44- tumors additionally expressed endothelial cell marker - CD31.

PEGylated immunoliposome-loaded endoglin single-chain antibody enhances anti-tumor capacity of porcine α1,3GT gene.

Tumor could not be completely removed due to the absence of immune storm against tumor. The porcine α1,3 galactosyltransferase (α1,3 GT) induce the hyperacute rejection by synthesizing Galα1-3Galß1-(3)4GlcNAc-R (αGal) on the surface of graft endothelial cells (ECs) during xeno-transplantation. This study aimed to develop anti-endoglin single-chain Fv fragments (ENG-scFv) conjugated PEGylated immunoliposomes (iLPs) to induce immune storm against tumor. Immune fluorescence was performed to detect the binding of ENG-scFv to human ENG, the endosomal/lysosomal escape of ENG-scFv-iLPs/α1,3 GT, and αGal expression in hENG-HEK293 cells. In vitro MTT assay was performed to measure ENG-scFv-iLPs/α1,3 GT cytotoxicity. NOD/SCID mouse born A549 tumor model was used to evaluate the therapeutic potency of ENG-scFv-iLPs/α1,3 GT. ENG-scFv-iLPs enabled efficient targeting delivery of α1,3 GT plasmid to ENG + tumors neovascular endothelial cells (TnECs), promoted endosomal/lysosomal escape due to the pH-sensitive ability, then synthesized carbohydrate epitope αGal on the surface of these cells to achieve the purpose of destroying the tumor. The mechanism of uptake for nanoparticles was energy driven, the clathrin-mediated endocytosis was the main endocytic pathway of the ENG-mAb-iLPs/α1,3 GT and lipid-raft-mediated of the ENG-scFv-iLPs/α1,3 GT, and macropinocytosis was also involved in intracellular entry. The inhibition of tumor angiogenesis and proliferation by ENG-scFv-iLPs/α1,3 GT was closely related to down-regulation of VEGF. Our findings establish an alternative therapeutic paradigm for scFv-conjugated nanoparticles to induce tumor cell apoptosis and inhibit tumor growth early. Such iLPs nanocarrier could efficiently release α1,3 GT to their distinct sites of action, where the endoglin + tumor neovascular endothelial cells (ENG + TnECs) exist, in a site-specific manner. Therefore, we believe that these scFv-targeted core-shell immunocomplexes are an important potential α1,3 GT delivery system for various solid tumor-targeted therapy.

Anti-CD105 Antibody Eliminates Tumor Microenvironment Cells and Enhances Anti-GD2 Antibody Immunotherapy of Neuroblastoma with Activated Natural Killer Cells.

PURPOSE: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells. EXPERIMENTAL DESIGN: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft. RESULTS: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes, and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs, monocytes, and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added, which depleted human MSCs and murine endothelial cells and macrophages from the TME. CONCLUSIONS: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME, but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells.

Immunohistochemical Grading of Epidural Fibrosis with CD105 Antibody.

OBJECTIVE: Grading of epidural fibrosis (EF) is usually performed by histopathologic staining in experimental studies. Immunohistochemical methods for grading are not available in routine practice yet. In our study, the effect of tranexamic acid (TXA), a commonly used hemostatic agent in surgical interventions, was evaluated for use against the development of EF with classical histopathologic methods and immunohistochemistry using the CD105 antibody, a marker of angiogenesis. METHODS: Sixteen rats were used. The rats were assigned to 2 groups, control and TXA. Laminectomy was performed on the control group. In the treatment group, laminectomy + topical TXA was applied. After sacrificing the rats in the sixth week, histopathologic and immunohistochemical examinations and grading of the EF tissue were performed. RESULTS: Conventional histopathologic parameters of fibroblast count, intensity of fibrosis density, and inflammatory cell density, as well as immunohistochemical evaluation with CD105, showed that the grading of EF was comparable between groups I and II (P < 0.001). DISCUSSION: The results of our study have demonstrated that CD105 is compatible with the conventional histopathologic grading methods and can be used as a marker to determine the grades of angiogenesis and fibrosis in experimental studies. The results of our study have also shown that TXA, administered locally for hemostasis, reduces the grade of EF in rats following laminectomy. TXA has been observed to cause no toxic effects on neural tissue as it is already commonly used in clinical practice.

PROGNOSTIC ROLE OF CD44 EXPRESSION AND NEOVASCULARIZATION DETERMINED BY ENDOGLIN (CD105) IN GLIOBLASTOMA PATIENTS.

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans. Clinically useful molecular markers that help predict response to therapy and prognosis are still rare. The research was conducted in 55 patients with GBM, 26 (47.3%) women and 29 (52.7%) men, mean age 62.58 years. On immunohistochemical analysis, primary antibody to CD44 (dilution 1:50) and primary antibody to endoglin (CD105) (dilution 1:250) were used to evaluate neovascularization. Statistical analysis showed negative correlation between CD44 and survival (p=0.023) (higher expression of CD44 was correlated with shorter survival), but there was no correlation between neovascularization determined by CD105 in GBM and patient survival. Thus, significant individual predictors of longer survival were lower expression of CD44 (p=0.004), higher Karnofsky score (p=0.045), and female gender (p=0.017). The results obtained suggested the possible role of CD44 in the progression and tumor neovascularization of GBM.

A Combination Therapy Targeting Endoglin and VEGF-A Prevents Subretinal Fibro-Neovascularization Caused by Induced Müller Cell Disruption.

Purpose: Subretinal fibroneovascularization is one of the most common causes of vision loss in neovascular AMD (nAMD). Anti-VEGF therapy effectively inhibits vascular leak and neovascularization but has little effect on fibrosis. This study aimed to identify a combination therapy to concurrently inhibit subretinal neovascularization and prevent fibrosis. Methods: We generated transgenic mice in which induced disruption of Müller cells leads to subretinal neovascularization, which is reliably accompanied by subretinal fibrosis. We conducted Western blots and immunohistochemistry to study changes in transforming growth factor-ß (TGFß) signaling including endoglin, a coreceptor essential for TGFß signaling, and then tested the effects of monthly intravitreal injection of anti-VEGF-A and anti-endoglin, either alone or in combination, on the development of subretinal fibroneovascularization in our transgenic mice. Results: Müller cell disruption increased expression of TGFß1, TGFß type 1 receptor, and phosphorylated-Smad3. Endoglin was strongly expressed in subretinal fibroneovascular tissue. Fluorescein angiography and measurements of retinal vascular permeability indicated that intravitreal anti-VEGF-A in combination with anti-endoglin treatment more efficiently inhibited vascular leak compared with either monotherapy. Immunostaining of retinal wholemounts with antibodies against glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 indicated that the combination therapy also effectively prevented subretinal fibrosis and inhibited microglial activation. Luminex cytokine assays indicated that intravitreal anti-VEGF-A and anti-endoglin treatment, either alone or in combination, reduced the production of IL33 and macrophage inflammatory protein-3α. Conclusions: Our findings offer a potentially novel combination approach to concurrently managing subretinal neovascularization and fibrosis in nAMD.

New highly sensitive sandwich ELISA system for soluble endoglin quantification in different biological fluids.

Soluble endoglin (sEng) is a fragment of a membrane-associated receptor (CD105) expressed on endothelial cells, mesenchymal stem cells and trophoblast cells. It is considered as a regulatory factor involved in the development of preeclampsia (PE) and cancer-associated neo-angiogenesis. The purpose of this study was to describe a new sandwich ELISA for sEng quantification. In contrast to three commercial kits, the new ELISA was able to quantify sEng not only in blood plasma and cell culture supernatants, but also in urine and cerebrospinal fluid. The assay detected up to two orders of magnitude higher antigen levels than did the commercial kits. Using Western blot assay followed by SDS-PAGE or Blue Native PAGE, we demonstrated a heterogeneous nature of sEng molecules. Antigen heterogeneity is considered as a factor contributing to the pronounced differences in its content estimations by different ELISAs. We obtained evidence indicating that the assay was capable of detecting heterogenious sEng molecules. The new ELISA was validated as a quick, specific and accurate method for sEng quantification. Despite the differences in antigen content estimates, the assay had similar diagnostic performance as widely used commercial kit for the detection of severe PE in pregnant women based on plasma sEng contents. Moreover, the new assay was able to delineate diseased patients based on antigen levels in urine. Therefore, the new ELISA is a potentially valuable tool for in vitro and clinical studies.

Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes.

BACKGROUND: Endoglin (CD105) is overexpressed on tumor cells and tumor vasculatures, making it a potential target for diagnostic imaging and therapy of different neoplasms. Therefore, studies on nanocarrier systems designed for endoglin-directed diagnostic and drug delivery purposes would expose the feasibility of targeting endoglin with therapeutics. METHODS: Liposomes carrying high concentrations of a near-infrared fluorescent dye in the aqueous interior were prepared by the lipid film hydration and extrusion procedure, then conjugated to single chain antibody fragments either selective for murine endoglin (termed mEnd-IL) or directed towards human endoglin (termed hEnd-IL). A combination of Dynamic Light Scattering, electron microscopy, cell binding and uptake assays, confocal microscopy and in vivo fluorescence imaging of mice bearing xenografted human breast cancer and human fibrosarcoma models were implemented to elucidate the potentials of the liposomes. RESULTS: The mEnd-IL and hEnd-IL were highly selective for the respective murine- and human endoglin expressing cells in vitro and in vivo. Hence, the hEnd-IL bound distinctly to the tumor cells and enabled suitable fluorescence imaging of the tumors, whereas the mEnd-IL bound the tumor vasculature, but also to the liver, kidney and lung vasculature of mice. CONCLUSIONS: The work highlights key differences between targeting vascular (murine) and neoplastic (human) endoglin in animal studies, and suggests that the hEnd-IL can serve as a delivery system that targets human endoglin overexpressed in pathological conditions. GENERAL SIGNIFICANCE: The endoglin-targeting liposomes presented herewith represent strategic tools for the future implementation of endoglin-directed neoplastic and anti-angiogenic therapies.

Targeting angiogenesis for radioimmunotherapy with a 177Lu-labeled antibody.

PURPOSE: Increased angiogenesis is a marker of aggressiveness in many cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic. CD105, or endoglin, has a primary role in angiogenesis in a number of cancers, making this a widely applicable target for targeted radioimmunotherapy. METHODS: The anti-CD105 antibody, TRC105 (TRACON Pharmaceuticals), was conjugated with DTPA for radiolabeling with 177Lu (t 1/2 6.65 days). Balb/c mice were implanted with 4T1 mammary carcinoma cells, and five study groups were used: 177Lu only, TRC105 only, 177Lu-DTPA-IgG (a nonspecific antibody), 177Lu-DTPA-TRC105 low-dose, and 177Lu-DTPA-TRC105 high-dose. Toxicity of the agent was monitored by body weight measurements and analysis of blood markers. Biodistribution studies of 177Lu-DTPA-TRC105 were also performed at 1 and 7 days after injection. Ex vivo histology studies of various tissues were conducted at 1, 7, and 30 days after injection of high-dose 177Lu-DTPA-TRC105. RESULTS: Biodistribution studies indicated steady uptake of 177Lu-DTPA-TRC105 in 4T1 tumors between 1 and 7 days after injection (14.3 ± 2.3%ID/g and 11.6 ± 6.1%ID/g, respectively; n = 3) and gradual clearance from other organs. Significant inhibition of tumor growth was observed in the high-dose group, with a corresponding significant increase in survival (p < 0.001, all groups). In most study groups (all except the nonspecific IgG group), the body weights of the mice did not decrease by more than 10%, indicating the safety of the injected agents. Serum alanine transaminase levels remained nearly constant indicating no damage to the liver (a primary clearance organ of the agent), and this was confirmed by ex vivo histological analyses. CONCLUSION: 177Lu-DTPA-TRC105, when administered at a sufficient dose, is able to curtail tumor growth and provide a significant survival benefit without off-target toxicity. Thus, this targeted agent could be used in combination with other treatment options to slow tumor growth allowing the other agents to be more effective.

Durable remission for a woman with refractory choriocarcinoma treated with anti-endoglin monoclonal antibody and bevacizumab: A case from the New England Trophoblastic Disease Center, Brigham and Women's Hospital and Dana-Farber Cancer Institute.

A 36-year-old with metastatic and refractory choriocarcinoma following single- and multi-agent chemotherapy and surgical metastectomy experienced a durable remission after receiving therapy with an anti-endoglin monoclonal antibody and bevacizumab. Treatment options and scientific advances in this disease are highlighted.

CD105+-mesenchymal stem cells migrate into osteoarthritis joint: An animal model.

Mesenchymal stem cells are being the focus of connective tissue technology and regenerative medicine, presenting a good choice cell source for improving old and well recognized techniques of cartilage defect repair. For instance, the autologous chondrocyte transplantation using new concepts of regenerative medicine. The present study investigated the risk of xenogenicity of human synovial membrane-derived MSCs, injected into the monkeys using intravenous and intra-articular administration. The animal models used were adult monkeys Rhesus which had been injured into the left knee to create an Osteoarthritis (OA) animal model. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were euthanized one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. We concluded that a characterized MSCs subset could be a safe alternative for cell therapy in clearly localized pathologies.

Dendritic-cell-based immunotherapy evokes potent anti-tumor immune responses in CD105+ human renal cancer stem cells.

Cancer stem cells (CSCs) are responsible for tumor initiation, progression, and resistance to therapeutic agents; they are usually less sensitive to conventional cancer therapies, and could cause tumor relapse. An ideal therapeutic strategy would therefore be to selectively target and destroy CSCs, thereby preventing tumor relapse. The aim of the present study was to evaluate the effectiveness of dendritic cells (DCs) pulsed with antigen derived from CD105+ human renal cell carcinoma (RCC) CSCs against renal cancer cells in vitro and in vivo. We identified "stem-like" characteristics of CD105+ cells in two human RCC cell lines: A498 and SK-RC-39. Loading with cell lysates did not change the characteristics of the DCs. However, DCs loaded with lysates derived from CD105+ CSCs induced more functionally specific active T cells and specific antibodies against CSCs, and clearly depressed the tumor growth in mice. Our results could form the basis for a novel strategy to improve the efficacy of DC-based immunotherapy for human RCC.

Intracoronary allogeneic cardiosphere-derived stem cells are safe for use in dogs with dilated cardiomyopathy.

Cardiosphere-derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non-ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c-kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells.

Association of Stromal Factors With the Histologic Risk Assessment Model in Oral Squamous Cell Carcinoma.

The aim of the present study was to evaluate angiogenesis, lymphangiogenesis, and mast cell density in association with the histologic risk assessment (HRA) model in oral squamous cell carcinoma. One hundred oral squamous cell carcinomas were graded according to the HRA system and immunostained with antibodies against D2-40, CD34, and CD105 to determine lymphvessel density (LVD) and microvessel density (MVD). Mast cells were detected by toluidine blue and counted in all samples. Assessments were made between the evaluated factors and the histologic variables of HRA. Kruskal-Wallis and Mann-Whitney U test were used for statistical analysis and P<0.05 was considered significant. There were 32, 26, and 42 cases of low, intermediate, and high-grade neoplasms, respectively. Only LVD (P=0.05) and CD34MVD (P=0.03) showed significant associations with lymphocytic infiltration and were both higher in score 0 cases compared with score 3 tumors (P=0.05 and <0.001, respectively). None of the other variables showed significant relationships with the HRA risk scores or subcategories (P>0.05). According to our findings, it appears that the role of lymphangiogenesis and angiogenesis is limited in the HRA system. The significant relationship of lymphocytic infiltration with LVD and CD34MVD, but not CD105MVD, might indicate that "inflammatory lymphangiogenesis/angiogenesis" may differ from that induced by noninflamed neoplastic tissues. It also seems that the vasculature in inflamed tumor tissues is not entirely newly formed.

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Endoglin (CD105) is an accessory component of the TGF-ß receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

Biodistribution of (125)I-labeled anti-endoglin antibody using SPECT/CT imaging: Impact of in vivo deiodination on tumor accumulation in mice.

INTRODUCTION: Radiolabeled antibodies directed against endoglin (CD105) are promising tools for imaging and antiangiogenic cancer therapy. To validate iodinated antibodies as reliable tracers, we investigated the influence of the radiolabeling method (direct or indirect) on their in vivo stability. METHODS: Anti-CD105 mAbs were radioiodinated directly using chloramine-T ((125)I-anti-CD105-mAbs) or indirectly using D-KRYRR peptide as a linker ((125)I-KRYRR-anti-CD105-mAbs). The biodistribution was studied in B16 tumor-bearing mice via SPECT/CT imaging. RESULTS: Radioiodinated mAbs were stable in vitro. In vivo, thyroid showed the most important increase of uptake after 24h for (125)I-anti-CD105-mAbs (91.9±4.0%ID/ml) versus(125)I-KRYRR-anti-CD105-mAbs (4.4±0.6%ID/ml). Tumor uptake of (125)I-anti-CD105-mAbs (0.9±0.3%ID/ml) was significantly lower than that of (125)I-KRYRR-anti-CD105-mAbs (4.7±0.2%ID/ml). CONCLUSIONS: An accurate characterization of the in vivo stability of radioiodinated mAbs and the choice of an appropriate method for the radioiodination are required, especially for novel targets. The indirect radioiodination of internalizing anti-CD105 mAbs leads to more stable tracer by decreasing in vivo deiodination and improves the tumor retention of radioiodinated mAbs. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: To date, the only antiangiogenic antibody approved for clinical indications is bevacizumab. There is a need to develop more antibodies that have targets highly expressed on tumor endothelium. CD105 represents a promising marker of angiogenesis, but its therapeutic relevance in cancer needs to be further investigated. In this context, this study suggests the potential use of indirectly iodinated anti-CD105 mAbs for tumor imaging and for therapeutic purposes.

Identification of a distinct subpopulation of fibroblasts from murine dermis: CD73(-) CD105(+) as potential marker of dermal fibroblasts subset with multipotency.

Skin dermis includes various types of multipotent stromal cells (MSCs) and a subpopulation of dermal fibroblasts that exhibit the ability to differentiate. However, characterization of this dermal fibroblast subtype remains less understood. In this study, we isolated dermal cells from the skin of newborn C57/B6 mice and investigated their characteristics. Isolated murine dermal cells exhibited a fibroblast phenotype as judged by accepted criteria including a lack of MSC-related antigens and the differentiation potential of MSCs, and the positive expression of fibroblast markers. A comparative analysis demonstrated that CD73(-) CD105(+) but not CD73(-) CD105(-) dermal fibroblasts exhibited some of the functional properties of MSCs. Furthermore, the multipotent phenotype of CD73(-) CD105(+) cells was diminished by treatment of CD105 siRNA and shRNA, indicating that CD105 expression was critical for the retention of differentiation potential of those cells. Overall, these results suggest that CD73(-) CD105(+) cells are a distinct subset of dermal fibroblasts with multipotency and that their surface antigens could help to classify this subpopulation. These cells may contribute to the regeneration of damaged tissue.