Geoffrey T. Yarranton

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

An engineered human antibody fab fragment specific for Pseudomonas aeruginosa PcrV antigen has potent antibacterial activity.

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.

Tumour targeting of humanised cross-linked divalent-Fab' antibody fragments: a clinical phase I/II study.

Antibody engineering has made it possible to design antibodies with optimal characteristics for delivery of radionuclides for tumour imaging and therapy. A humanised divalent-Fab′ cross-linked with a bis-maleimide linker referred to as humanised divalent-Fab′ maleimide was produced as a result of this design process. It is a humanised divalent antibody with no Fc, which can be produced in bacteria and has enhanced stability compared with F(ab′)2. Here we describe a clinical study in patients with colorectal cancer using humanised divalent-Fab′ maleimide generated from the anti-carcinoembryonic antigen antibody A5B7 radiolabelled with iodine-131. Ten patients received an i.v. injection of iodine-131-labelled A5B7 humanised divalent-Fab′ maleimide, and positive tumour images were obtained by gamma camera imaging in eight patients with known lesions, and one previously undetected lesion was identified. True negative results were obtained in two patients without tumour. Area under the curve analysis of serial blood gamma counting and gamma camera images showed a higher tumour to blood ratio compared to A5B7 mF(ab′)2 used previously in the clinic, implying this new molecule may be superior for radioimmunotherapy. MIRD dose calculations showed a relatively high radiation dose to the kidney, which may limit the amount of activity that could be administered in radioimmunotherapy. However the reduction in immunogenicity was also a major advantage for A5B7 humanised divalent-Fab′ maleimide over murine versions of this antibody suggesting that humanised divalent-Fab′ maleimide should be a useful vehicle for repeated therapies.

Targeting EphA3 Inhibits Cancer Growth by Disrupting the Tumor Stromal Microenvironment

Eph receptor tyrosine kinases are critical for cell-cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow-derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3(+)/CD90(+)/Sca1(+) mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell-cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy.

PcrV antibody–antibiotic combination improves survival in Pseudomonas aeruginosa -infected mice

The type III secretion system (TTSS) of Pseudomonas aeruginosa, associated with acute infection, facilitates the direct injection of cytotoxins into the host cell cytoplasm. Mab166, a murine monoclonal antibody against PcrV, a protein located at the tip of the injectisome, has demonstrated efficacy against P. aeruginosa infection, resulting in reduced lung injury and increased survival in murine models of infection. We hypothesised that the administration of Mab166 in combination with an antibiotic would further improve the survival of P. aeruginosa-infected mice. A murine model of P. aeruginosa acute infection, three clinically relevant antibiotics (ciprofloxacin, tobramycin and ceftazidime) and the Mab166 antibody were used for this study. Consistently, compared to other treatment groups (antibiotic or antibody administered in isolation), the combination of Mab166 and antibiotic significantly improved the survival of mice infected with three times the lethal dose (LD90) of the highly cytotoxic ExoU-secreting strain, PA103. This synergistic effect was primarily due to enhanced bactericidal effect and protection against lung injury, which prevented bacterial dissemination to other organs. Hence, the combination of Mab166 with antibiotic administration provides a new, more effective strategy against P. aeruginosa airway infection, especially when large numbers of highly virulent strains are present.

Granulocyte-Macrophage Colony-Stimulating Factor Antibody] Suppresses Microglial Activity: Implications for Anti-inflammatory Effects in Alzheimer's Disease and Multiple Sclerosis

The objective of our study was to determine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) activity in the brain following GM‐CSF induction. We injected recombinant mouse GM‐CSF into the brains of 8‐month‐old C57BL6 mice via intracerebroventricular injections and studied the activities of microglia, astrocytes, and neurons. We also sought to determine whether an anti‐GM‐CSF antibody could suppress endogenous microglial activity in the C57BL6 mice and could also suppress microglial activity induced by the recombinant mouse GM‐CSF in another group of C57BL6 mice. Using quantitative real‐time RT‐PCR, we assessed microglial, astrocytic, and neuronal activity by measuring mRNA expression of pro‐inflammatory cytokines, GFAP, and the neuronal marker NeuN in the cerebral cortex tissues from C57BL6 mice. We performed immunoblotting and immunohistochemistry of activated microglia in different regions of the brains from control (phosphate‐buffered saline‐injected C57BL6 mice) and experimental mice (recombinant GM‐CSF‐injected C57BL6 mice, GM‐CSF antibody‐injected C57BL6 mice, and recombinant mouse GM‐CSF plus anti‐GM‐CSF antibody‐injected C57BL6 mice). We found increased mRNA expression of CD40 (9.75‐fold), tumor necrosis factor‐alpha (2.1‐fold), CD45 (1.73‐fold), and CD11c (1.70‐fold) in the cerebral cortex of C57BL6 mice that were induced with recombinant GM‐CSF, compared with control mice. Further, the anti‐GM‐CSF antibody suppressed microglia in mice that were induced with recombinant GM‐CSF. Our immunoblotting and immunohistochemistry findings of GM‐CSF‐associated cytokines in C57BL6 mice induced with recombinant GM‐CSF, in C57BL6 mice injected with the anti‐GM‐CSF antibody, and in C57BL6 mice injected with recombinant mouse GM‐CSF plus anti‐GM‐CSF antibody concurred with our real‐time RT‐PCR findings. These findings suggest that GM‐CSF is critical for microglial activation and that anti‐GM‐CSF antibody suppresses microglial activity in the CNS. The findings from this study may have implications for anti‐inflammatory effects of Alzheimer’s disease and experimental autoimmune encephalomyelitis mice (a multiple sclerosis mouse model).

Dosimetric evaluation and radioimmunotherapy of anti-tumour multivalent Fab« fragments

SummaryWe have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab′)2) which have been used previously in clinical radioimmunotherapy (RIT) studies in colorectal carcinoma. Comparative biodistribution studies of similar sized DFM and F(ab′)2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model (Casey et al (1996) Br J Cancer 74: 1397–1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab′)2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM.

Humanization of an Anti-Mucin Antibody for Breast and Ovarian Cancer Therapy

Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.

Antibody to granulocyte macrophage colony-stimulating factor reduces the number of activated tissue macrophages and improves left ventricular function after myocardial infarction in a rat coronary artery ligation model.

Granulocyte macrophage colony-stimulating factor (GM-CSF) promotes infarct expansion and inappropriate collagen synthesis in a myocardial infarction (MI). This study was designed to determine if treatment with anti-GM-CSF will inhibit macrophage migration, preserve function, and limit left ventricular (LV) remodeling in the rat coronary artery ligation model. Treatment with a monoclonal antibody to GM-CSF (5 mg/kg) was initiated 24 hours before coronary artery ligation and continued every 3 days for 3 weeks. Left coronary arteries of rats were ligated, animals were recovered, and cardiac function was evaluated 3 weeks postligation. Tissue samples were processed for histochemistry. Anti-GM-CSF treatment increased LV ejection fraction (37 ± 3% vs 47 ± 5%) and decreased LV end systolic diameter (0.75 ± 0.12 vs 0.59 ± 0.05 cm) with no changes in LV systolic pressure (109 ± 4 vs 104 ± 5 mm Hg), LV end diastolic pressure (22 ± 4 vs 21 ± 2 mm Hg), LV end diastolic diameter (0.96 ± 0.04 vs 0.92 ± 0.05 cm), or the time constant of LV relaxation tau (25.4 ± +2.4 vs 22.7 ± 1.4 milliseconds) (P < 0.05). Significantly lower numbers of tissue macrophages and significant reductions in infarct size were found in the myocardium of antibody-treated animals (81 ± 21.24 vs 195 ± 31.7 positive cells per 0.105 mm2, compared with controls. These findings suggest that inhibition of macrophage migration may be beneficial in the treatment of heart failure after MI.

EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia.

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.