Optimized monoclonal antibody treatment against ELTD1 for GBM in a G55 xenograft mouse model

Glioblastoma is an aggressive brain tumour found in adults, and the therapeutic approaches available have not significantly increased patient survival. Recently, we discovered that ELTD1, an angiogenic biomarker, is highly expressed in human gliomas. Polyclonal anti‐ELTD1 treatments were effective in glioma pre‐clinical models, however, pAb binding is potentially promiscuous. Therefore, the aim of this study was to determine the effects of an optimized monoclonal anti‐ELTD1 treatment in G55 xenograft glioma models. MRI was used to assess the effects of the treatments on animal survival, tumour volumes, perfusion rates and binding specificity. Immunohistochemistry and histology were conducted to confirm and characterize microvessel density and Notch1 levels, and to locate the molecular probes. RNA‐sequencing was used to analyse the effects of the mAb treatment. Our monoclonal anti‐ELTD1 treatment significantly increased animal survival, reduced tumour volumes, normalized the vasculature and showed higher binding specificity within the tumour compared with both control‐ and polyclonal‐treated mice. Notch1 positivity staining and RNA‐seq results suggested that ELTD1 has the ability to interact with and interrupt Notch1 signalling. Although little is known about ELTD1, particularly about its ligand and pathways, our data suggest that our monoclonal anti‐ELTD1 antibody is a promising anti‐angiogenic therapeutic in glioblastomas.     

Dosimetry and pharmacokinetics of monoclonal antibody A6H with human renal cell carcinoma xenografts: Single dose study

Implantable miniature thermoluminescent dosimeters and conventional biodistribution analysis were used to determine the locally absorbed radiation dose delivered to three morphologically distinct human renal cell carcinoma xenografts (TK-39, TK-82 and TK-177C; N = 87) following a 50 μCi infusion of ¹³¹iodine-labeled monoclonal antibody A6H. Xenografts were clearly detected by radioimmunoscintigraphy. Pronounced differences were noted among the three xenografts in MAb pharmacokinetics and in the locally absorbed irradiation doses which ranged from 2 to 5cGy per injected μCi of ¹³¹iodine-labelled A6H.     

Molecular cloning and chimerisation of an inhibitory anti-cathepsin B antibody and its expression in Chinese hamster ovary cells

The lysosomal cysteine protease cathepsin B is one of several proteases that have been linked to tumour progression. Its increased expression and secretion in tumour cells may facilitate the degradation of extracellular matrix proteins, leading to tumour cell invasion and metastasis. Specific inhibitory monoclonal antibodies are a possible alternative to synthetic inhibitors as a therapeutic tool for cancer treatment. An inhibitory monoclonal antibody, which binds to an epitope near the active site of cathepsin B and inhibits its proteolytic activity, was prepared and its effect on invasion of ras-transformed MCF-10A neoT cells was tested in vitro. Here we present the nucleotide sequences of the heavy and light chains of the inhibitory antibody and compare them to the murine immunoglobulin germline sequences for possible somatic hypermutations. Since no harmful mutations were found, a mouse/human chimeric antibody was constructed by fusing murine V(H) and V(L) variable regions of the inhibitory antibody with human gamma 1 and K constant regions, respectively. Chinese hamster ovary K1 cells were co-transfected with expression vectors pcD-NA3L and pcDNA3H and the reactivity of the isolated chimeric antibody was tested by ELISA and Western blotting. We could demonstrate an inhibitory effect of the chimeric antibody.     

Localization of an anti-CEA monoclonal antibody in colo-rectal carcinoma xenografts

Summary A mouse monoclonal anti-CEA antibody (11.285.14) has been examined for tumour localization potential by assessing its distribution in immunodeprived mice with xenografts of human colon carcinoma cell lines HCT-8, HRT-18, HT-29, and LS174T and a xenograft (HRVB) established from a primary rectal carcinoma. With four carcinomas (HCT-8, HT-29, LS174T, and HRVB) preferential tumour localization of ¹²⁵I anti-CEA was seen. Compared with ¹³¹I normal IgG₁ localization indices of up to 4.4:1 were achieved. Up to 10% of the injected dose of ¹²⁵I anti-CEA was present/g of tumour tissue and with the largest xenografts examined (3–4 g) up to 40% of the total body reactivity was localized in tumour tissue. The tumour localization of ¹³¹I labelled antibody was visualized by external gamma camera imaging. Overall antibody localization correlated with the CEA content of the xenografts and the fourth colon carcinoma xenograft (HRT-18) and an osteogenic sarcoma xenograft (791T), both with very low CEA levels, showed no localization of the monoclonal antibody.     

Rat serum antibodies binding to high-molecular-weight glycoprotein(s) on syngeneic colon carcinomas,demonstrated by competition with rat monoclonal antibody

Summary Sera from rats immunized to syngeneic 1,2-dimethylhydrazine colon carcinomas were analyzed for their ability to inhibit the binding of a syngeneic rat IgM monoclonal antibody (10B12) specific for high-molecular-weight glycoprotein(s) from rat colon carcinoma. Immunization with irradiated tumour cells or with tumour tissue extracts resulted in the appearance of a strong inhibiting activity. Sera of animals with established growing tumours and of females shortly after partus also inhibited binding of the monoclonal antibody, while unimmunized animals or animals immunized with irrelevant antigens had no inhibiting antibodies in their sera. Dimethylhydrazinetreated animals showed an increased titer of antibodies binding to the high-molecular-weight glycoprotein, but showed no inhibition of binding of the 10B12 monoclonal antibody. The syngeneic 10B12 rat antibody obviously does not reflect a rare event captured from a hyperimmune animal by the hybridoma technique but rather represents an antibody specificity frequently appearing in the immune response to tumours expressing the high-molecular-weight glycoprotein.     

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Treatment of low-grade non-Hodgkin's lymphoma with continuous infusion of low-dose recombinant interleukin-2 in combination with the B-cell-specific monoclonal antibody CLB-CD19

Seven patients with low-grade non-Hodgkin's lymphoma were treated with a combination of a murine monoclonal antibody directed against the B-cell-specific antigen CD19 (CLB-CD10), given twice weekly, and continuous infusion of low-dose recombinant interleukin-2 (rIL-2). We demonstrated stable serum CLB-CD19 levels throughout the 12 weeks of treatment, and homing of the antibody into the tumour sites. A variable degree of antigenic modulation was noted. Prolonged treatment resulted in a sustained increase in the number of natural killer cells in the circulation with enhanced cytotoxic capacity, including antibody-dependent cellular cytotoxicity. During the first weeks of treatment, T cell activation occurred in the majority of patients. Toxicity was related to the rIL-2 treatment and consisted of transient constitutional symptoms and a flu-like syndrome without organ dysfunction. A partial remission occurred in one patient, and in another patient who was primarily leukaemic a greater than 50% reduction of circulating B cells was noted. An antitumour effect occurred early during treatment and could not be related to rIL-2-induced modulation of natural killer cell or T lymphocyte activation.     

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.     

Immunocytochemistry and image cytometry of progesterone receptors in breast carcinoma imprints. Expression of tissular and cellular heterogeneity: relations to biochemical assay, clinical and histological parameters and DNA analysis

Breast cancer imprints from 93 patients were assayed for the presence of progesterone receptors (PgR) using a monoclonal antibody (Transbio) and an immunocytochemical assay (ICA) method which stains only the epithelial cell nuclei. Results were compared with conventional biochemical PgR determinations (DCCA) and were in qualitative agreement in 86% of the cases. Quantitative analyses were done on PgR-ICA- and Feulgen-stained imprints from 32 tumours using a SAMBA 2005 cell image processor. Results obtained showed a high correlation between DCCA values and the P product derived from the mean PgR concentration of marked tumour cells and the percentage of marked cells. Intra-tumoral and intra-cell heterogeneity were featured and showed relation to tumour differentiation and size.     

抗人肝素酶单克隆抗体的制备与鉴定

目的:肝素酶在白细胞游走和恶性肿瘤转移的过程中发挥重要作用,肝素酶抗体的制备对于自身免疫病和肿瘤的良恶性鉴别诊断具有重要意义。制备抗人肝素酶单克隆抗体,用于肝素酶的研究及临床恶性肿瘤的鉴别诊断。方法:通过杂交瘤技术将分泌抗人肝素酶单抗的小鼠B细胞与小鼠骨髓瘤细胞Sp2/0融合,获得稳定分泌抗人肝素酶单抗的杂交瘤细胞;用有限稀释法获得单克隆,以重组人肝素酶及含肝素酶的血小板裂解液对抗体进行Western印迹检测。结果:Western印迹结果显示制备的单抗与人肝素酶具有特异性免疫识别特性。结论:获得了能够特异性免疫识别人肝素酶的分泌性抗人肝素酶单克隆抗体。     

免疫印迹法检测牛海绵状脑病和羊瘙痒病

用大肠杆菌表达的牛朊病毒正常成熟蛋白 (BoPrPC)免疫新西兰白兔 ,获得了与朊病毒蛋白 (PrP)反应的抗体T1。根据致病型朊病毒 (PrP^SC)能抵抗蛋白酶消化的特性 ,用蛋白酶K消化脑组织提取物 ,以抗体T1进行免疫印迹反应 ,结果表明从接种羊瘙痒病朊病毒 2 6 3K的金黄地鼠脑组织提取物内检测到抗蛋白酶K消化的致病型PrPSC ,而正常金黄地鼠脑组织中没有抗蛋白酶消化的蛋白。以我国正常牛羊为材料 ,制备其脑组织提取物 ,用上述方法和抗体T1进行检测 ,结果没有发现抗蛋白酶K的任何蛋白存在 ,说明没有牛海绵状脑病和羊瘙痒病存在。用 1A8抗体也获得了同样的结果。这些结果表明可以用自制的抗血清检疫牛海绵状脑病和羊瘙痒病 ,防止其传入我国     

Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2'' of methicillin-resistant Staphylococcus aureus

The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.     

Radiolocalisation of an anti-CEA monoclonal antibody (FO23C5) and its fragments in a colon carcinoma xenograft model

A new monoclonal antibody designated FO23C5 against a protein component of carcinoembryonic antigen (CEA) has been developed. A xenograft system of human colon cancer was used to compare the intact monoclonal IgG with its fragments (Fab')2 and Fab) and with an established anti-CEA antibody (MAb35) and the antibody AUA1 raised against the colon carcinoma cell line. We demonstrate that FO23C5 compares well with the existing anti-CEA antibody and with AUA1, and that F(ab')2 fragments perform best in achieving optimal tumour to normal tissue ratios compared with intact IgG and Fab fragment.     

Early transmembrane events in tumour cell responses observed by stopped-flow fluorometry

Early transmembrane events of tumour cells (mouse myeloma X5563 and lymphoma RDM4) after binding of a monoclonal antibody against mouse MHC antigen and a mitogenic lectin, Con A, were examined by stopped-flow fluorometry with 3 different fluorescent probes. The results showed that membrane fluidities of the cells increased first after binding of anti H-2Kk monoclonal antibody (11-4.1), then calcium was released from intracellular stores into the cytoplasma, and lastly calcium influx occurred from the external medium into the cytoplasma. While Con A only induced calcium influx from the external medium into the cytoplasma.     

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.     

A Monoclonal Antibody to Rabbit Brain GABA Transaminase

A monoclonal antibody of class IgG (subclass IgG1) has been prepared to rabbit brain GABA transaminase (GABA-T). This antibody reveals a single band of molecular weight 52,000 on a nitrocellulose filter blotted with purified GABA-T. On a filter blotted with unfractionated rabbit brain supernatant a major band of molecular weight 58,000 is revealed. An immunoaffinity column was prepared by coupling proteins from ascites fluid containing anti-rabbit GABA-T antibody to Bio-Rad Affi-Gel 15. This column bound purified GABA-T and extracted from unfractionated rabbit brain supernatant a protein of molecular weight 58,000, which was almost homogeneous and which had GABA-T enzyme activity. Using immunoaffinity chromatography, therefore, a high degree of purification of GABA-T may be achieved in a single step. Further, this technique may preserve an authentic form of the enzyme that is lost during the conventional purification procedure. The antibody inhibits GABA-T enzyme activity, up to a maximum of 35%.     

Tumour eradication and induction of memory against murine mesothelioma by combined immunotherapy

Numerous immunotherapy treatments for cancer are undergoing clinical trials or are already approved for use. One particular area of interest is targeting mechanisms of immune tolerance. Using a murine model of mesothelioma, we investigated the roles of regulatory T-cells, intratumoural transforming growth factor (TGF)-β and the negative regulator molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in immune tolerance to tumours. It was found that treatments targeting a single negative regulator molecule mechanism were not as effective against tumours as targeting multiple mechanisms simultaneously. Most importantly, it was found that a combined triple treatment of anti-CD25 monoclonal antibody (mAb), anti-CTLA-4 mAb and TGF-β soluble receptor resulted in long-term clearance of tumours and memory against tumour rechallenge. These data suggest that clinical application of immunotherapies against tumours may be improved by simultaneously targeting multiple mechanisms of immune suppression.     

Iodine sensitivity of bacteria isolated from iodinated water systems

Fourteen bacterial isolates, predominantly Pseudomonas sp., from two water systems disinfected by iodinated anion-exchange resins were studied and compared with an isolate of Pseudomonas aeruginosa from a povidone-iodine solution and four other isolates. Pseudomonas cepacia and P. aeruginosa grown in brain heart infusion were 3 to 5 logs less sensitive to 1 mg/L I2 (pH 7.2, 1 min) when compared with cultures grown in phosphate buffer. Another P. cepacia isolate was the least sensitive culture when grown in brain heart infusion (1 log decrease) but was more sensitive after cultivation in phosphate buffer (5 logs). Isolates from an iodinated potable water system, including P. cepacia, Staphyloccus warneri, and a Bacillus sp., were all less sensitive to iodine than a "resistant" P. aeruginosa and three other isolates when grown in brain heart infusion. A clinical isolate of P. aeruginosa exhibited intermediate sensitivity. The sensitivity of bacteria to iodine is thus highly variable, depending on the organism as well as the growth conditions.     

Resurrection of a clinical antibody: template proteogenomic de novo proteomic sequencing and reverse engineering of an anti-lymphotoxin-α antibody

A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-α), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning; however, biochemical assays with the ascites showed this antibody against LT-α (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing MS analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high-throughput informatic proteomic de novo sequencing with reverse engineering to reestablish monoclonal antibody-expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai virus) M protein

Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.