G. E. Francis

PEGylation of Adenovirus with Retention of Infectivity and Protection from Neutralizing Antibody in Vitro and in Vivo

Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

Synergistic interaction between differentiation inducers and DNA synthesis inhibitors: A new approach to differentiation induction in myelodysplasia and acute myeloid leukaemia

Numerous agents induce differentiation and maturation of neoplastic and dysplastic myeloid cells in vitro and some of these agents have been used with limited success in the treatment of patients with myelodysplastic syndromes (MDS) and myeloid leukaemias. We recently proposed that physiological and pharmacological agents which enhance differentiation and maturation in vitro act by two fundamentally different routes: (1) by hastening the progression through various differentiation/maturation steps; (2) by slowing proliferation (usually by inhibition of DNA synthesis). In order to test this thesis we looked for synergistic effects on differentiation using pairs of agents from the two groups in cultures of cells from myelodysplastic and acute myeloid leukaemia (AML) patients and from normal marrow donors. The results with three MDS, two AML and three normal samples show that combinations of differentiation inducing agents (retinoic acid, N-methylformamide) with DNA synthesis inhibitors (6-mercaptopurine, cytosine arabinoside and aphidicolin) produce a differentiation inducing effect equivalent to that of 10-100, or even 1000 fold higher concentrations of single agents. Myelotoxic effects in vitro were not synergistic. The use of these synergistic combinations should greatly enhance the usefulness of differentiation inducers in the therapy of MDS and myeloid leukaemia.

ELIMINATION OF T‐LYMPHOCYTES FROM HUMAN BONE MARROW WITH MONOCLONAL T‐ANTIBODIES AND CYTOLYTIC COMPLEMENT

Complement‐mediated cytolysis has been used to remove T‐lymphocytes from suspensions of human peripheral blood and bone marrow. Selective T‐cell removal was investigated by three monoclonal antibodies, OKT3, MBG6 and OKT11A. All three removed > 90% of T‐cells but combinations were necessary to kill > 99% of T‐cells in vitro. The macrophage‐granulocyte and erythroid colony forming cells of the bone marrow were spared. The method can be applied on bulk BM samples during clinical BM transplantation and will be useful to establish whether the virtually complete removal of T‐lymphocytes totally prevents transplant associated graft‐versus‐host disease in man.

USE OF BONE-MARROW CULTURE IN PREDICTION OF ACUTE LEUKAEMIC TRANSFORMATION IN PRELEUKAEMIA

Bone-marrow granulocyte-macrophage progenitor cell proliferation and regulatory factor (colony-stimulating activity; CSA) production were assessed at presentation and, if possible, subsequently in twenty-one patients with dysmyelopoiesis and less than 5% bone-marrow blasts. Seven patients underwent acute leukaemic transformation 1-35 months after the first marrow culture. Assay of bone-marrow endogenous CSA proved the most useful prognostic test. The rate of transformation in the seven patients with raised CSA at presentation was significantly greater (five transformed and one died at or before 4 months) than that in the fourteen patients with normal, low, or undetectable CSA (two transformed at 27 and 35 months). In one of the latter an increase in bone-marrow CSA occurred 6 weeks before transformation (serial marrow samples were not available in the other case). No other marrow culture feature measured, including the presence or absence of granulocyte-macrophage colony-forming cells and total clone numbers (colonies and smaller clusters) was as useful.

Quantitative analysis of polyethylene glycol (PEG) in PEG-modified proteins/cytokines by aqueous two-phase systems

Covalent attachment of poly(ethylene glycol) (PEG) to proteins produces conjugates with altered/improved physicochemical and biological properties which depend upon the number of PEG chains linked. Quantification of the attached PEG is however not a trivial issue. The partition coefficient, K, of the PEG-protein conjugate in PEG/dextran two-phase systems provides a quantitative measure for the degree of modification. A linear relationship between log K and the number of PEG chains was observed in fractionated PEG-modified-granulocyte-macrophage colony stimulating factor conjugates having 1 to 3 substitutions. Furthermore, in mixtures of PEG-bovine-serum-albumin conjugates with increasing degrees of modification, a linear relationship was found between log K and n, the average substitution. The increment in log K per PEG chain added is protein specific and this suggests that the interactions between the PEG-protein conjugate and the polymers in the phase system are more complex than just a simple affinity of the PEG for the PEG-rich top phase. Increasing the polymer concentration in the phase system produces larger increments in log K per PEG molecule attached and the proportionality between log K and number of PEG molecules is only compromised for conjugates with high degree of substitution when partitioned in biphasic systems of high concentration of polymers.

Stimulation of Human Haemopoietic Cells by Colony Stimulating Factors: Sensitivity of Leukaemic Cells

Summary. The sensitivity of populations of human granulocyte precursors to factors with colony stimulating activity (CSA) was assessed in agar culture in vitro. The mean threshold of stimulation was estimated by analysis of dose‐response curves of clone growth against concentration of CSA. This method of assessment has the advantage that CSA production by cells contaminating the population under test does not affect results. Marrow cells from patients with acute myeloid leukaemia were less sensitive to CSA than marrow cells from normal individuals. Sensitivities of cells from chronic granulocytic leukaemia and from chronic myelomonocytic leukaemia were within the normal range, but this group also tended to require more CSA than controls. In addition, sensitivities of granulocyte precursors from patients with acute myeloid leukaemia were closely related to the culture pattern, and thus to the remission probability. The significance of this relationship is discussed.

Reactivity of Monoclonal Antibodies with Human Myeloid Precursor Cells

Summary. Five monoclonal antibodies have been tested for their ability to bind to myeloid precursor cells in normal human bone marrow. Indirect immunofluores‐cence and the fluorescence activated cell sorter was used to separate cells according to their reactivity for trial culture in vitro in order to grow granulocyte‐macrophage colony forming cells (CFUc). Two antibodies (OKT3 and OKT11) which react strongly with bone marrow T lymphocytes were found to be unreactive with CFUc. YD 1/23 reacts very strongly with both T and B lymphocytes but is only weakly reactive with CFUc. In contrast, OKT10 and YE2/36 did react with CFUc. The consequences of these findings and the potential clinical use of these antibodies in bone marrow transplantation are discussed.

Stimulation of Human Haemopoietic Cells by Colony Stimulating Factors: Adherent Cell Dependent Colony Stimulating Activity in Human Serum

The number of granulocyte‐macrophage clones formed in agar culture of bone marrow is dependent on levels of colony stimulating activity (CSA) a proposed in vivo haemopoietic regulator. A dose‐response relationship for stimulation of human haemopoietic cells by CSA is demonstrated, which could be explained by thresholds of stimulation to cell division following a normal distribution. A simple method for the comparison of activities of test and control sources of CSA is presented. The apparent potentiating effect of the addition of two sources of CSA is explained by this dose‐response relationship. Haemopoietic cells from patients with chronic granulocytic or acute myeloid leukaemia showed the same dose‐response relationship. CSA levels in normal human sera were greatly reduced by assay in the absence of adequate numbers of bone marrow ‘adherent cells’(cells adherent to nylon or plastic) or peripheral blood leucocytes, suggesting the presence of two kinds of CSA in human serum, one dependent on the presence of bone marrow adherent cells and one effective in their absence. Reduction of numbers of nonspecific esterase positive cells in the bone marrow sample correlated with reduction in the stimulating effect of serum. In all sera tested, adherent cell dependent CSA was the major component.

Chronic myeloid leukaemia and the philadelphia translocation: Do the C-SIS oncogene and platelet-derived growth factor provide the link?

The study of cellular oncogenes and of chromosomal abnormalities in human tumours has, in several instances, suggested a link between a specific oncogene translocation and oncogenesis. It was recently suggested that the translocation of the c-abl gene (the human cellular homologue of the transforming sequence of Abelson murine leukaemia virus) from chromosome 9 to 22 in Philadelphia translocation, might have a role in the generation of chronic myeloid leukaemia (CML). We propose an alternative hypothesis and suggest that the translocation of another gene, c-sis, may be more important.

Proliferative capacity, sensitivity to colony stimulating activity and buoyant density: linked properties of granulocyte-macrophage progenitors from normal human bone marrow.

Abstract Phenotypic differences between acute myeloid leukaemia (AML) cells and normal cells can only be attributed to abnormal AML cell properties if comparisons have been made with the precise normal counterpart cell. When AML clonogenic cells are compared with the normal bone marrow cells which form clones of granulocytes and macrophages in agar culture, differences are detected. These include lower average buoyant density and varying degrees of reduced sensitivity to the specific regulator granulocyte-macrophage colony stimulating activity (CSA). The degree of reduction in sensitivity to CSA is inversely related to the proliferative capacity of the individuals AML cells. This association suggests that the phenotypes of different AML cell populations might be a parody of the phenotypic heterogeneity of the normal granulocyte-monocyte progenitor population; heterogeneity which possibly reflects the maturation hierarchy of the normal population. The aim of this study was to test this thesis. The results show that buoyant density, proliferative capacity and sensitivity to CSA are heterogenous properties of normal granulocyte-monocyte progenitor cells which are linked in an orderly manner. Buoyant density and CSA sensitivity differences between AML cells and normal progenitor cells may thus indicate shifts in the location of the majority of progenitor cells on the granulocyte-monocyte pathway in AML rather than abnormal cell properties.