Murine Monoclonal IgG1 Research Articles

IgG against the Membrane-Proximal Portion of the Desmoglein 3 Ectodomain Induces Loss of Keratinocyte Adhesion, a Hallmark in Pemphigus Vulgaris

Pemphigus vulgaris is a severe autoimmune blistering disease characterized by IgG autoantibodies (auto-abs) against the desmosomal adhesion molecules desmoglein (DSG) 3 and DSG1. Underlying mechanisms leading to blister formation upon binding of DSG-specific IgG auto-abs are not fully understood. Numerous studies showed the pathogenicity of IgG auto-ab binding to the aminoterminal region 1 (EC1) of the DSG3 ectodomain. However, auto-abs in pemphigus vulgaris are polyclonal, including IgG against both aminoterminal- and membrane-proximal epitopes of the DSG3 ectodomain. In this study, the pathogenicity of a previously uncharacterized murine monoclonal IgG antibody, 2G4, directed against the membrane-proximal region (EC5) of the DSG3 ectodomain was characterized and tested in various specificity and functionality assays. The results clearly show that 2G4 is capable of inhibiting intercellular keratinocyte adhesion and of inducing cellular DSG3 redistribution by activation of the p38MAPK signal transduction pathway. In this study, we provide evidence that an IgG auto-abs directed against the membrane-proximal region EC5 of DSG3 induces acantholysis, the hallmark in pemphigus vulgaris. These findings challenge the current concept that IgG auto-abs targeting the NH2-terminal portion of the DSG3 ectodomain are pathogenic only. Our study provides further aspects for a deeper understanding of desmosomal keratinocyte adhesion and improves our insight into the complex auto-ab‒induced blister formation in pemphigus vulgaris.

In vitro immunization approach to generate specific murine monoclonal IgG antibodies

Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophilain vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species.

Hinge influences in murine IgG binding to Cryptococcus neoformans capsule.

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C.neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.

Antibodies against apoB100 peptide 210 inhibit atherosclerosis in apoE-/- mice

Atherosclerotic plaques are characterized by an accumulation and subsequent oxidation of LDL, resulting in adaptive immune responses against formed or exposed neoepitopes of the LDL particle. Autoantibodies against native p210, the 3136–3155 amino acid sequence of the LDL protein apolipoprotein B-100 (apoB100) are common in humans and have been associated with less severe atherosclerosis and decreased risk for cardiovascular events in clinical studies. However, whether apoB100 native p210 autoantibodies play a functional role in atherosclerosis is not known. In the present study we immunized apoE-/- mice with p210-PADRE peptide to induce an antibody response against native p210. We also injected mice with murine monoclonal IgG against native p210. Control groups were immunized with PADRE peptide alone or with control murine monoclonal IgG. Immunization with p210-PADRE induced an IgG1 antibody response against p210 that was associated with reduced atherosclerotic plaque formation in the aorta and reduced MDA-LDL content in the lesions. Treatment with monoclonal p210 IgG produced a similar reduction in atherosclerosis as immunization with p210-PADRE. Our findings support an atheroprotective role of antibodies against the apoB100 native p210 and suggest that vaccines that induce the expression of native p210 IgG represent a potential therapeutic strategy for lowering cardiovascular risk.

The expression of urokinase-type plasminogen activator system in upper tract urothelial carcinoma and its prognostic value after radical nephroureterectomy

BackgroundTo evaluate the expression pattern and prognostic role of the urokinase-type plasminogen activator (uPA) system in patients who underwent radical nephroureterectomy (RNU) for nonmetastatic upper tract urothelial carcinoma (UTUC). MethodsA total of 732 patients who were treated with RNU for clinically nonmetastatic UTUC comprised our analytical cohort. Immunohistochemical staining of uPA, uPA receptor (uPAR) and uPA inhibitor (PAI-1) was performed using Murine IgG1 monoclonal antibodies. Outcomes of interest were recurrence-free survival, cancer-specific survival, and overall survival. ResultsThe median age of the patients was 69.8 years and 56.6% of them were males. Overall, overexpression of uPA, uPAR, and PAI-1 was observed in 292 (39.9%), 346 (47.3%), and 345 (47.1%) patients, respectively. The uPA system components showed a statistically significant association with adverse clinicopathologic features such as lymphovascular invasion, multifocality, sessile tumors, and advanced pathologic stage (P < 0.01). On multivariable models, higher pathologic tumor stage, multifocality, and lymph node involvement were associated with RFS, OS, and CSS, but not the overexpression of uPA, uPAR, or PAR-1. In patients with organ-confined disease (≤pT2N0), however, uPA was significantly associated with RFS (hazard ratio [HR]: 2.04, 95% confidence interval [CI]: 1.21–3.43), OS (HR: 1.59, 95% CI:1.08–2.24) and CSS (HR: 2.55, 95% CI:1.44–4.52). uPA improved the predictive accuracy of a standard post-RNU model for all 3 endpoints, in organ-confined disease, by a prognostically significant margin. ConclusionsOverexpression of uPA system components was associated with adverse clinicopathologic characteristics and survival outcomes on the univariable, but not multivariable analyses. uPA expression was an independent predictor of survival outcomes in patients with organ-confined disease. While the clinical value of the uPA system remains limited in this cohort, further studies are needed to identify a marker or constellation of markers of high predictive value to help in counseling and treatment planning of UTUC patients.

Abstract CT006: Intraperitoneal radioimmunotherapy for desmoplastic small round cell tumor: Results of a phase I study (NCT01099644)

Abstract Background / Objectives: Desmoplastic small round cell tumor (DSRCT), a rare sarcoma of adolescents and young adults, has a long-term survival of &amp;lt;20% despite aggressive multimodality therapy, warranting a search for novel treatments. The murine monoclonal IgG1 antibody 8H9 recognizes cell surface antigen 4Ig-B7H3 and binds to 96% of DSRCTs with restricted normal tissue reactivity. DSRCT recurrences often present as multifocal peritoneal implants. We hypothesized that intraperitoneal (IP) radioimmunotherapy (RIT) by virtue of prolonged residence time and slow transfer to the circulation, may selectively target IP DSRCT. Design / Methods: We conducted a phase I study of radioiodinated 8H9 to evaluate toxicity, pharmacokinetics, biodistribution and efficacy. Cohorts of 3-6 patients were treated with escalated doses of IP 131I-8H9. A prior dose of 2mCi 124I-8H9 IP was used to acquire PET images and biodistribution data. Toxicity was monitored clinically and biochemically. Pharmacokinetics was studied using serial blood draws. Results: Forty-one DSRCT patients were treated at doses of 30-90mCi/m2. Maximum tolerated dose was not reached; there were no dose-limiting toxicities. Major adverse events (n=1 each) were transient: grade 3 transaminitis, neutropenia, and thrombocytopenia. Blood and peritoneal half-times were 32.5h and 14.6h respectively. Mean projected absorbed doses to blood, kidney, liver, lung and spleen were 0.7,1.72,1.92,0.64 and 1.03 rad/mCi 131I-8H9 respectively (n=12 patients analyzed). Dehalogenation was insignificant: &amp;gt;80% blood 131I remained protein-bound 66h post-RIT. Recommended phase II dose was 80mCi/m2. External beam intensity-modulated whole-abdominopelvic radiotherapy (WAP-IMRT) to 3000cGy was administered after protocol therapy to 25 patients without unexpected adverse events. Median progression-free survival (PFS) from RIT for first remission patients (n=21) undergoing gross total resection followed by RIT and whole-abdominal radiotherapy was 17.9±1.7 months. Of these, 19 and 13 patients are PF and overall survivors respectively at a median of 13 months post-RIT. In contrast patients with gross residual disease pre-RIT or those receiving RIT in ≥2nd remission (n=20) had median PFS of 8.1±3.5 months (p&amp;lt;0.05 for PFS and OS). Conclusions: 124I-8H9-directed radioimmuno-PET successfully determined biodistribution, whole-body and organ exposure. 131I-8H9 IP RIT had a satisfactory safety profile and appears to have activity against micro-metastatic DSRCT. A phase II trial combining IP RIT and WAP-IMRT will commence shortly. Citation Format: Shakeel Modak, Jorge Carrasquillo, Michael LaQuaglia, Zanzonico Pat, Todd Heaton, Nai-Kong Cheung, Neeta Pandit-Taskar. Intraperitoneal radioimmunotherapy for desmoplastic small round cell tumor: Results of a phase I study (NCT01099644) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT006.

A Cherry Seed-Derived Spice, Mahleb, is Recognized by Anti-Almond Antibodies Including Almond-Allergic Patient IgE.

There are a number of examples of immunologic cross-reactivity elicited by pollens, fruits, seeds, and nuts of closely related plant species. Such cross-reactivity is of particular concern for patients with food allergies. In this report, we investigated a spice (mahleb) that is prepared from the kernel of the St. Lucie cherry, Prunus mahaleb, for cross-reactivity with almond (Prunus dulcis), using enzyme-linked immunosorbent assay (ELISA) and Western blot. Almond and mahleb are members of the same genus. Cross-reactivity between the mahleb and almond was demonstrated by reaction of cherry and almond kernel protein extracts with antibodies raised against almond proteins. Almond-specific murine monoclonal IgG, rabbit polyclonal IgG, and almond-allergic serum IgE each exhibited cross-reactivity with cherry kernel protein. Because of the demonstrated cross-reactivity between almond and mahleb, these findings should be of special concern to almond-allergic patients and attending medical personnel.

A curative approach to central nervous system metastases of neuroblastoma.

10545 Background: Neuroblastoma metastatic to the central nervous system (CNS NB) is associated with significant mortality (median survival &lt; 6 months, &lt; 10% survival at 36 months). Intraventricular compartmental radioimmunotherapy (cRIT) with radio-iodinated murine IgG1 monoclonal antibody 131I-8H9 targeting tumor cell-surface glycoprotein B7-H3 offers a therapeutic strategy. We analyzed overall survival of patients with CNS NB treated with intraventricular 131I-8H9 cRIT at Memorial Sloan Kettering Cancer Center (MSK) since 2003. Methods: After radiographic and/or pathologic confirmation of CNS NB, and assessment of adequate CSF flow, cRIT eligible patients underwent treatment on an IRB-approved protocol with either temozolomide/irinotecan-based CNS salvage regimen incorporating craniospinal radiation therapy, 131I-8H9 cRIT plus systemic immunotherapy (group 1), or non-regimen therapies with 131I-8H9 cRIT (group 2). cRIT administration involved a 2mCi tracer of 124I- or 131I-8H9 with nuclear imaging and CSF sampling for dosimetry followed by 1 or 2 therapeutic injections up to 70 mCi 131I-8H9. Disease surveillance included serial MR brain/spine, MIBG, CT, and bone marrow evaluation. Data are presented as overall survival after detection of CNS metastasis. Results: 105 patients with CNS NB were evaluated;80 patients (76%) were treated (57 group 1, 23 group 2). Of the 25 patients who were not eligible for cRIT, survival averaged 8.6 months. Of 19 patients with radiographic evidence of disease at the time of cRIT, 7 (36%) demonstrated post cRIT radiographic improvement. At analysis, 45/80 (56%) patients were alive 4.8–152 months (median 58 months) after CNS metastasis, including 36 (45%) at 36 months and 23 (29%) &gt; 60 months. Subgroup analyses of 131I-8H9–treated patients identified age at NB diagnosis (≤18 months), relapse restricted to CNS and group 1 status as factors positively correlated with survival. Conclusions: 76% of patients with CNS NB treated at MSK received 131I-8H9 cRIT, and approximately half completed multimodality CNS salvage regimen with 131I-8H9 cRIT. Despite advanced CNS involvement, over 50% of patients treated with 131I-8H9 cRIT are still alive and nearly 50% have survived at least 36 months. Clinical trial information: NCT00089245.

MILGa-01: A first-in-human study assessing the safety and tolerability of chMIL-38 in metastatic prostate, bladder, and pancreatic cancers.

e565 Background: Metastatic or recurrent cancer continues to be a challenge in patients with urogenital and pancreatic cancers despite the development of newer anti-androgen therapies. MIL-38 is an IgG1 murine monoclonal antibody directed against Glypican-1 (GPC-1). The proteoglycan GPC-1 is upregulated in prostate, pancreatic and bladder cancer cell lines. Targeting of tumor xenografts and lack of adverse events in pre-clinical models suggest chimeric MIL-38 (chMIL-38) is a good candidate for both radioimmunotherapy and antibody-drug conjugate development. Methods: Patients with known metastatic prostate and pancreatic cancer were injected with a single dose of MILGa drug (1mg, 250MBq dose) and imaged with whole body gamma camera scans and SPECT/CT at 30 minutes, 6 h, 24 h, 48 h, 72 h (if required) and 144h post infusion. Blood and urine samples for pharmacokinetic, biomarker studies, cancer markers and hematology and biochemistry analysis were collected at each time point. Dosimetry analysis of tumour images was performed to determine the relative accumulation of MILGa in different organs. Results: To date one pancreatic cancer patient and one prostate cancer patient with metastatic disease have been infused with the MILGa drug. The drug was safe and well tolerated in both patients with no infusion reactions or drug-related adverse events. Tracer uptake was observed in the liver, with mild tracer uptake in the spleen, skeletal system and kidneys. Significant MILGa targeting was seen in Patient 2 24 hours post infusion onwards with uptake in the right foot (right calcaneum and cuneiform bones). SPECT/CT at 24 hours confirmed uptake was in the bone, not in the joint space or soft tissue. Conclusions: MILGa has shown a promising safety profile to date with indication of tumour targeting in metastatic prostate cancer. Additional data from this study may permit the selection and evaluation of potentially sensitive tumor types to be studied for further therapeutic and diagnostic studies. Clinical trial information: ACTRN12616000787482.

Evaluation of CD146 as Target for Radioimmunotherapy against Osteosarcoma.

BackgroundOsteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo.Methods and ResultsA murine monoclonal anti-CD146 IgG1 isotype antibody, named OI-3, was developed along with recombinant chimeric versions with human IgG1 or human IgG3 Fc sequences. Using flow cytometry, selective binding of OI-3 to human osteosarcoma cell lines OHS, KPDX and Saos-2 was confirmed. The results confirm a higher expression level of CD146 on human osteosarcoma cells than HER2 and EGFR; antigens targeted by commercially available therapeutic antibodies. The biodistribution of 125I-labeled OI-3 antibody variants was compared with 125I-labeled chimeric anti-EGFR antibody cetuximab in nude mice with subcutaneous OHS osteosarcoma xenografts. OI-3 was able to target CD146 expressing tumors in vivo and showed improved tumor to tissue targeting ratios compared with cetuximab. Subsequently, the three OI-3 variants were conjugated with p-SCN-Bn-DOTA and labeled with a more therapeutically relevant radionuclide, 177Lu, and their biodistributions were studied in the nude mouse model. The 177Lu-labeled OI-3 variants were stable and had therapeutically relevant biodistribution profiles. Dosimetry estimates showed higher absorbed radiation dose to tumor than all other tissues after administration of the chimeric IgG1 OI-3 variant.ConclusionOur results indicate that CD146 can be targeted in vivo by the radiolabeled OI-3 antibodies.

Abstract 2303: The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients

Abstract Background: Chimeric antigen receptor (CAR)-engineered T cells have demonstrated potent clinical efficacy in patients with B cell lymphoma. However, the application of CAR-T cell therapy targeting other solid tumors has been limited. The GD2 gangliosides are sialic acid-containing glycosphingolipids that play a role in signal transduction and cell-cell recognition. 1st generation gangliosides GD2 chimeric antigen receptor T cells in some patients with refractory neuroblastoma already studied in a phase I clinical trials, indicate that T cells expressing 1st generation anti-GD2 chimeric antigen receptors were safe and mediated modest antitumor activity. In a phase I study, the murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, was administered intravenously to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma. Antitumor responses occurred in 7 of 17 patients. However, the expression of the gangliosides GD2 in Asian melanoma populations has not been reported. Methods: This study involved tumor samples from 228 melanoma patients (129 acral melanomas, 65 mucosal melanomas, 13 melanomas on skin with chronic sun-induced damage, 21 melanomas on skin without chronic sun-induced damage) in Peking University Cancer Hospital &amp; Institute. Clinical data were collected. Immunohistochemistry assays using antibodies against gangliosides GD2 were performed on formalin-fixed, paraffin-embedded specimens. The ability of gangliosides GD2 chimeric antigen receptor T cells to kill gangliosides GD2+ melanoma cell was evaluated by MTT in vitro. Results: Among the 228 samples,40.3% of the cases(92/228) demonstrated positive staining with gangliosides GD2. Expression of gangliosides GD2 were relatively more frequent in acral (48.1%) and mucosal (36.9%) melanomas than in CSD (15.4%) and Non-CSD (19.0%) melanomas. The median survival time for patients with expression of gangliosides GD2 was significantly shorter than patients with absence of gangliosides GD2 expression (21.4 vs 57.6 months, P&amp;lt;0.05). Statistical differences were found between primary melanomas with metastatic melanomas (34.9% vs 53.0%, P&amp;lt;0.05). In addition, the gangliosides GD2 chimeric antigen receptor T cells exhibited specific lysis of melanoma cell with gangliosides GD2 expression in vitro experiment. Conclusion: These data demonstrate the expression of ganglioside GD2 are more frequent in acral and mucosal melanomas than in cutaneous melanomas in Chinese patients. The higher expression of gangliosides GD2 on metastatic melanomas compared to primary melanomas was in conformity with the viewpoint that GD2 expression was related to increased metastatic potential. Gangliosides GD2 chimeric antigen receptor T cells represents a clinically appealing treatment strategy for melanoma patients with ganglioside GD2 expression. Citation Format: Yan Kong, Jiayi Yu, Lu Si, Zhihong Chi, Chuanliang Cui, Xinan Sheng, Jun Guo. The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2303.

A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors.

c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers.

Generation of recombinant antibody fragments with toxin-neutralizing potential in loxoscelism

Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32–35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms.

Immunodetection of the “brown” spider (Loxosceles intermedia) dermonecrotoxin with an scFv-alkaline phosphatase fusion protein

Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32–35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39–20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.

Abstract 640: Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs

Abstract Ion channels are important portals that facilitate ionic passage across membranes in all organisms. KCNK9, a member of the family of two-pore domain potassium K+ channels, mediates “leak” currents critical to maintaining cell resting membrane potentials. Since its identification, KCNK9 has been implicated in various human disorders including cancer. KCNK9 overexpression has been observed in melanoma, colorectal, breast and lung tumors and KCNK9 has been found to promote tumor survival and growth, by increasing resistance to hypoxia and apoptosis. However, KCNK9's underlying pathophysiologic mechanisms have been elusive due to the absence of specific inhibitors. Here, we describe the development of novel monoclonal antibodies directed against the KCNK9 extracellular domain and their molecular/cellular effects on KCNK9-expressing cells. Murine monoclonal IgG1 antibodies were raised against recombinant KCNK9 extracellular domains expressed in a mammalian system. 40 monoclonal antibodies were generated and demonstrated channel subtype-specific recognition of human KCNK9. Among them, 1 antibody designated Y4-mAb displayed sub-nanomolar binding affinity and inhibited KCNK9 channel conductance by up to 30% when tested in a fluorescent ion-influx assay. Through confocal microscopic analysis, channel inhibition was found to result from antibody-induced internalization and endocytosis of cell-surface channels. Y4-mAb induced up to 30∼40% reduction in cell viability and increased cell death in BEN non-small cell lung carcinoma cells and BT-549 and 410.4 breast cancer cells. In addition to “direct cell killing”, this antibody activated complement, resulting in complement-dependent cancer cell cytotoxicity in vitro. Initial experiments in vivo showed that systemic Y4-mAb inhibited the capacity of BEN non-small cell lung carcinoma cells to propagate subcutaneous tumor xenografts in nude mice (p&amp;lt;0.05). In conclusion, we have generated a high affinity KCNK9-specific monoclonal antibody that perturbs KCNK9 function and inhibits tumor cell survival and xenograft formation. Anti-KCNK9 antibodies have the potential to be valuable research tools and may lead to novel therapeutics. Moreover, our strategies should be generally applicable to studying the functions of other ion channels in cancer. Citation Format: Han Sun, Liqun Luo, Bachchu Lal, John Laterra, Min Li. Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2015-640

Preclinical imaging of kallikrein-related peptidase 2 (hK2) in prostate cancer with a (111)In-radiolabelled monoclonal antibody, 11B6.

BackgroundProstate cancer is a leading cause of death in the male population of the western world. Human kallikrein-related peptidase 2 (hK2) is abundantly expressed in malignant prostatic tissue, and its gene, KLK2, is regulated by the androgen receptor. 11B6 is a murine IgG1 monoclonal antibody directed against free human hK2. In this study, we performed a preclinical evaluation of 111In-labelled 11B6 in mouse xenografts to investigate its potential in the clinical staging and assessment of metastatic prostate cancer.Methods11B6 was radiolabelled with 111In through CHX-A″-DTPA chelation. In vivo biodistribution and uptake of 111In-DTPA-11B6 were measured until 168 h post-injection in NMRI nude mice bearing subcutaneous LNCaP xenografts. The binding specificity to hK2 was evaluated by both in vivo competitive binding assays with excess non-labelled 11B6 and hK2-negative DU145 xenografts. SPECT/CT imaging of subcutaneous and intra-tibial LNCaP xenografts was used to visualize the tumours.ResultsTumour uptake of 111In-DTPA-11B6 in LNCaP xenografts was 19% ± 0.78%IA/g at 48 h, giving a tumour-to-blood ratio of 1.6, which increases to 2.4 at 1 week post-injection. Accumulation was low in other organs except for the salivary glands, which is probably the result of cross-reactivity with mouse kallikreins. Significantly lower tumour accumulation was observed in competitive assays and DU145 xenografts. SPECT/CT imaging could clearly visualize the subcutaneous and intra-tibial LNCaP xenografts.ConclusionsOur study demonstrates the potential of 111In-DTPA-11B6 for the detection of metastatic prostate cancer and monitoring anti-androgen therapy, as it exhibits an increased uptake and accumulation in viable tumour when compared to normal tissue. A humanised version of the 11B6 monoclonal antibody is currently under evaluation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-014-0051-5) contains supplementary material, which is available to authorized users.

P17.13 * ABSCESUALIZED GLIOMAS: PREDICTIVE VALUE OF 99MTC-LEUKOSCAN

Abscess formation within brain tumor is uncommon. Intrasellar or parasellar tumors are among the most common neoplasms that developed such complications as the results of direct extension of microbial flora from contiguous infected sinuses. Cerebral gliomas with abscess are extremely rare. The major challenge is detection of a correct diagnosis. The diagnosis of brain tumor associated with abscess is particularly difficult by conventional neuroradiological studies. Often, computed tomography (ct) and magnetic resonance imaging (mri) can reveal a sudden increase in size of the mass and significant perilesional edema. However, these studies did not consent a correct early diagnosis and only surgical treatment with histological examination allow the exact diagnosis. 99mtc-labeled sulesomab (leukoscan®) can be useful in the diagnosis of brain tumors with intratumoral abscess. 99mtc-labeled sulesomab (leukoscan®) consists of monoclonal murine igg antibody fab' fragment, bound with technetium-99m with affinity for a 42 kda glycoprotein present on the surface of activated granulocytes (nca-90 surface antigen). It is used in different clinical settings as a potential diagnostic tool for the infections of various tissues. We presented three rare cases of cerebral glioma showing intratumoral abscess formation. All patients had been submitted to 99mtc-leukoscan study. Labeled leukocytes scintigraphy seems to be able to differentiate infectious from non-infectious brain lesions. In our patients mri and magnetic resonance spectroscopy (mrs) were unconvincing while the results of the leukocyte scintigraphy, were successively confirmed by histopathological examination, which also showed the presence of a brain abscess. Thus in our cases the sensitivity and the positive predictive value (ppv) of the granulocyte scintigraphy were both 100%. To the best of our knowledge no data have been reported in the literature on the role of nuclear medicine investigation for the recognition of brain abscesses so far. To date this represents the first description of the role of leukoscan in cerebral gliomas with abscess formation.

A novel IgE antibody targeting the prostate-specific antigen as a potential prostate cancer therapy

BackgroundProstate cancer (PCa) is the second leading cause of cancer deaths in men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 and CD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal was to take advantage of the unique properties of IgE in order to trigger immune activation against PCa.MethodsBinding characteristics of the antibody were determined by ELISA and flow cytometry. In vitro degranulation was determined by the release of β-hexosaminidase from effector cells. In vivo degranulation was monitored in human FcεRIα transgenic mice using the passive cutaneous anaphylaxis assay. These mice were also used for a vaccination study to determine the in vivo anti-cancer effects of this antibody. Significant differences in survival were determined using the Log Rank test. In vitro T-cell activation was studied using human dendritic cells and autologous T cells.ResultsThe anti-PSA IgE, expressed in murine myeloma cells, is properly assembled and secreted, and binds the antigen and FcεRI. In addition, this antibody is capable of triggering effector cell degranulation in vitro and in vivo when artificially cross-linked, but not in the presence of the natural soluble antigen, suggesting that such an interaction will not trigger systemic anaphylaxis. Importantly, the anti-PSA IgE combined with PSA also triggers immune activation in vitro and in vivo and significantly prolongs the survival of human FcεRIα transgenic mice challenged with PSA-expressing tumors in a prophylactic vaccination setting.ConclusionsThe anti-PSA IgE exhibits the expected biological properties and is capable of triggering immune activation and anti-tumor protection. Further studies on this antibody as a potential PCa therapy are warranted.

Abstract 2848: IgE-mediated immune activation targeting the prostate specific antigen: a potential prostate cancer therapy.

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths among men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 and CD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal is to take advantage of the unique properties of the IgE molecule in order to trigger immune activation against PCa. The properly assembled and secreted anti-PSA IgE binds the antigen and the Fc epsilon RI (FcεRI) as demonstrated by ELISA and flow cytometry, respectively. This novel IgE antibody is capable of triggering effector cell degranulation in vitro as determined by the release of β-hexosaminidase from rat basophil leukemic effector cells that express human FcεRI (RBL SX-38). Furthermore, the IgE triggered degranulation in vivo in human FcεRIα transgenic mice using the passive cutaneous anaphylaxis assay. Degranulation was only observed when the IgE was artificially cross-linked, but not in the presence of the natural soluble antigen, suggesting that such an interaction will not trigger systemic anaphylaxis in patients. Importantly, when incubated in vitro with human dendritic cells the anti-PSA IgE complexed with the antigen (PSA) triggered autologous CD4 and CD8 T-cell activation, suggesting enhancement of antigen presentation. Immune activation was also observed in vivo where a prophylactic vaccination with the anti-PSA IgE complexed to PSA significantly prolonged the survival of human FcεRIα transgenic mice challenged with PSA-expressing tumors, under conditions in which its chimeric mouse/human anti-PSA IgG1 counterpart failed to confer protection. Our results provide initial proof-of principle that the anti-PSA IgE can stimulate immune activation against tumor cells expressing PSA, and further studies on the anti-tumor activity of this antibody are warranted. These studies belong to the new field of AllergoOncology that aims to reveal the function of IgE-mediated immune responses against cancer and develop novel IgE-based cancer therapies. Citation Format: Tracy R. Daniels-Wells, Gustavo Helguera, Richard K. Leuchter, Rafaela Quintero, Maggie Kozman, Jose A. Rodríguez, Elizabeth Ortiz-Sánchez, Otoniel Martínez-Maza, Birgit C. Schultes, Christopher F. Nicodemus, Manuel L. Penichet. IgE-mediated immune activation targeting the prostate specific antigen: a potential prostate cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2848. doi:10.1158/1538-7445.AM2013-2848

Reactivity of Human IgM Binding Murine Monoclonal 6B6C1 (IgG2a) with Other Murine Monoclonal IgG Antibodies

Approximately 6% of sera positive in a dengue virus IgM-capture enzyme immunoassay (EIA) represent false-positives due to interaction between IgM and horseradish peroxidase (HRP)-labeled monoclonal antibody (MAb) 6B6C1 (IgG2a). To better understand this interaction, we assessed the reactivity of captured IgM from these sera with other HRP-labeled MAbs. Fifty dengue IgM false-positive sera (recognizing 6B6C1) were evaluated for IgM reactivity with the HRP-labeled MAbs H3A4 (IgG2a), 53.8 (IgG2b), and IL-A2 (IgG1). The sera were also tested in an EIA for human anti-mouse antibody (HAMA). Forty-three sera (86%) reacted with IgG2a MAb (H3A4). Most (31/43 = 72%) of these sera recognizing 6B6C1 and H3A4 also recognized the IgG2 MAb and/or the IgG1 MAb. In contrast, HAMA was increased in only 9 of 50 (18%) sera reacting with 6B6C1. IgM from most sera-binding IgG2a MAb 6B6C1 also binds another IgG2a MAb, suggesting that IgM-6B6C1 reactivity is not idiotype specific. In many cases, IgM recognizing 6B6C1 also binds MAbs of other IgG subclasses, but is negative in a HAMA assay. These findings indicate that samples positive in IgM-capture EIAs utilizing conjugated MAbs should always be retested in the absence of antigen to identify false-positive reactivity caused by direct IgM-MAb interaction.