Recombinant human erythropoietin (EPO) produced by Chinese hamster ovary cells and distributed by two different pharmaceutical companies were confirmed to contain about 1% N-glycolylneuraminic acid (Neu5Gc) in total sialic acid content. Since chickens, like humans, do not synthesize Neu5Gc, they were used to determine the immunogenicity of Neu5Gc epitope in EPO. Chickens immunized with EPO did not produce significant titer of antibody that was specific to GM3(Neu5Gc) as compared to antibody titers produced in chickens immunized with fetuin containing 7% Neu5Gc or GM3(Neu5Gc) containing 100% Neu5Gc. Results obtained by an ELISA inhibition test showed that EPO, compared to GM3(Neu5Gc), reacted almost one thousand times less strongly with a human Hanganutziu-Deicher (HD) antibody. This study implies that an increase of Neu5Gc content in a molecule enhances its HD antigenicity. The response to Neu5Gc in patients receiving therapeutic injections of EPO is currently under investigation.

Recombinant human erythropoietin (rHuEPO) was produced by Chinese hamster ovary cells and commercially distributed to hospitals by two pharmaceutical companies in Japan (‘ESPO’ by Kirin Brewery Co. Ltd., and Sankyo Co. Ltd., and ‘EPOGIN’ by Chugai Pharmaceutical Co. Ltd.) These products contained about 1% N-glycolylneuraminic acid (Neu5Gc) in total sialic acid content. Since humans do not synthesize Neu5Gc, successive injection of Neu5Gc-containing products was feared to lead to allergic-like symptoms. Therefore, serum levels of antibodies to Neu5Gc epitope in 90 patients who received repeated i.v. injections of ESPO or EPOGIN were determined by an enzyme immunoassay using Neu5Gcα2-3Galβ1 -4Glc-Cer, GM3(Neu5Gc), as an antigen and compared with those in 100 healthy persons. Either no or low antibody levels were detected in both groups with no significant difference. In 40 patients who received s.c. injections of ESPO or EPOGIN, serum HD antibody levels were determined before and after weekly therapeutic injections carried out for one to several months, but no significant elevations were detected in all patients. The above results indicated that therapeutic administration of rHuEPO to patients with chronic renal failure is safe from allergic-like side effects associated with the production of Neu5Gc-specific antibodies, and it was concluded that Neu5Gc epitope of rHuEPO is minimally antigenic in humans.

The specificities of five heterophile Hanganutziu and Deicher (HD) antibody-containing sera from four different cancer patients and one other diseased patients were compared. Three glycosphingolipids and one glycoprotein antigens and their chemically modified derivatives were used. The antibodies of all whole sera showed similar specificities. IgG and IgM antibody fractions of each serum were separated. Although antibodies of the same class showed similar specificities, differences were detected between the specificities of IgG and IgM. IgG antibody specificities were dependent on the hydrophobic (ceramide) group while IgM antibodies were directed more to the terminal sialic acid moiety of the glycosphingolipid antigens. The results suggested that a similar population of IgG-producing lymphocytes is stimulated in patients. Due to the similarities in specificities of HD antibodies, the results of this study will facilitate the future isolation of either IgG or IgM antibody-producing lymphocyte(s) from a patient with HD antibodies and the establishment of a monoclonal antibody through hybridization with a human myeloma cell line.

The activity of a free N-acetylneuraminic acid (Neu5Ac)-hydroxylating enzyme which converted Neu5Ac into N-glycolyl-neuraminic acid (Neu5Gc) was demonstrated in the soluble fraction of pig mandibular gland. The hydroxylation was possible only with NADPH as the electron donor. The apparent Km was 4.5 mM Neu5Ac. At 0.5 mM monovalent cations had no effect on the hydroxylation of Neu5Ac whereas bivalent cations gave varied inhibition capacities ranging from 14 to 75%. EDTA gave a time-dependent enhancement of activity. It was concluded that the enzyme does not require an exogenously added inorganic cofactor. Results from salt fractionation of the soluble fraction and the use of inhibitors such as mercurials suggested that the hydroxylation of Neu5Ac to Neu5Gc may involve other, as yet unknown, component(s) and the possibility of electrons donated by NADPH being transferred to activated molecular oxygen (second substrate). We propose to name this enzyme N-acetyl-neuraminic acid hydroxylase.

SUMMARY:A human Hanganutziu-Deicher (HD) antibody and a chicken anti-N-glycolylneuraminyllactosylceramide (HD3) antibody were compared in their reaction against HD antigen-active ganglioside (HD3) and a glycoprotein (GP) by radioimmunassay (RIA). The human antibody had a 50 times higher reactivity with the glycoprotein, while the chicken antibody reacted equally with both antigens. Both antibodies had a higher reactivities with HD antigen(s) in sera of five of eight lung cancer patients than 54 normal human sera. Since four of the above five sera had no abnormal titers to GP, it was concluded that their immunological status was antigen excess. The chicken antibody may be useful in follow-up studies of cancer patients to correlate the expression of HD antigen in tissues and sera with the elevation of HD antibodies, offering alternative methods of clinical prognosis of tumor growth and/or metastases.The human HD antibody may also be useful for the detection of HD antigens of glycoprotein nature.