Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described

CD33/Siglec-3 Binding Specificity, Expression Pattern, and Consequences of Gene Deletion in Mice

Mouse CD33/Siglec-3 (mCD33) is the apparent ortholog of human CD33/Siglec-3 (hCD33), a member of the Siglec (sialic acid-binding Ig superfamily lectin) family of sialic acid-recognizing cell-surface lectins. We examined the binding specificity and expression pattern of mCD33 and explored its functions by generating mice deficient in this molecule. Like hCD33, mCD33 is expressed on myeloid precursors in the bone marrow, albeit mostly in the more mature stages of the granulocytic lineage. Moreover, unlike hCD33, mCD33 in peripheral blood is primarily expressed on granulocytes. Also, unlike hCD33, mCD33 did not bind to α2-3- or α2-6-linked sialic acids on lactosamine units. Instead, it showed distinctive sialic acid-dependent binding only to the short O-linked glycans of certain mucins and weak binding to the sialyl-Tn epitope. Binding was enhanced by removal of 9-O-acetyl groups and attenuated by truncation of the glycerol-like side chain of sialic acids. Mice deficient in CD33 were viable and fertile in a controlled-access specific-pathogen-free vivarium, showed no major morphological or histological abnormalities, had no changes in bone marrow or peripheral leukocyte subpopulations, and had very minor differences in biochemical and erythrocyte parameters. Cellular responses to intraperitoneally injected proinflammatory stimulants, as well as subsequent interleukin-6 secretion, were also apparently unaffected. These results indicate substantial species differences in CD33 expression patterns and ligand recognition and suggest functional degeneracy between mCD33 and the other CD33-related Siglec proteins expressed on cells of the myeloid lineage

Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products. PLoS One 4: e4241

Abstract Background: Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac)

Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis.

<p>Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.</p

Sensitive and specific detection of Neu5Gc in mouse and human tissues by immunohistochemistry.

<p>A. Direct scan of glass slides with frozen sections of mouse embryos d16.5 or paraffin sections of adult mouse tissues, and detected using biotinylated anti-chicken antibody followed by HRP-Streptavidin. B. Frozen sections of Human Placenta immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin (top) or CY3-Streptavidin (bottom). (400× magnification) C. Frozen sections of normal human tissues immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin. (400× magnification) D. Frozen sections of examples of human tumors immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin (400× magnification). E. Frozen sections of human ovarian carcinoma immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin (top) or CY3-Streptavidin (bottom) (200× magnification).</p

Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies.

<p>Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.</p

Sensitive and specific detection of Neu5Gc on cells by flow cytometry.

<p>CHO-K1 cells were detached from the tissue culture dish using 10 mM EDTA in PBS, pH 7.3. The cells were immediately washed in blocking buffer (0.5% gelatin from cold water fish skin in PBS, pH 7.5) containing 5 mM EDTA and counted by hemocytometer. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll Paque Plus (GE Healthcare), washed in blocking buffer and counted. 1×10⁶ cells were used for each staining which was done at 4°C. The cells were washed with 1 ml cold blocking buffer then pelleted at 500×g for 5 min. The supernatant was carefully removed and discarded. The cell pellet was gently suspended in 100 µl of either affinity purified chicken anti-Neu5Gc antibody or control non-specific IgY antibody diluted 1∶4000 in blocking solution and incubated on ice for 1 hr. The cells were washed by adding 1 ml of blocking buffer, mixed gently, and pelleted as above. The cell pellet was gently suspended in 100 µl Donkey-anti-chicken IgY Cy5-conjugated diluted 1∶4000 in blocking buffer and incubated on ice for 1 hr. The cells were washed as above, resuspended in 400 µl PBS, analyzed on a FACSCalibur (BD Biosciences Immunocytometry Systems, San Jose, CA) and the data analyzed using Flowjo software (Tree Star, Ashlan, OR). Human PBMCs were negative for Gc (bottom), while mouse PBMCs (data not shown) and CHO cells were positive for Gc (top). The gray peak represents cells stained with total IgY of un-immunized chickens, and the black trace represents cells stained with anti-Neu5Gc.</p

Affinity purified Chicken IgY antibody recognizes Neu5Gc with various linkages to underlying glycans.

<p>A. Detection of Neu5Gc on synthetic and semi-synthetic molecules. Reactivity of the affinity-purified anti-Neu5Gc antibody was tested in triplicate, by ELISA against synthetic and semi-synthetic Neu5Gc- and Neu5Ac-target pairs, using Neu5Ac-glycans for background subtraction (the Neu5Ac A490 value was subtracted from the corresponding Neu5Gc A490 value). B. Detection of Neu5Gc on natural glycoproteins. The affinity-purified anti-Neu5Gc antibody was tested in triplicates by ELISA, against natural glycoproteins containing Neu5Gc and Neu5Ac, using buffer only for background subtraction. Error bars indicate the standard deviation of triplicates. Gc, alpha-linked Neu5Gc; GM3, GM3 ganglioside; HSA, Human serum albumin; PAA, polyacrylamide; and, SPG, sialylparagloboside.</p

Lack of Neu5Gc in a biotherapeutic agent prepared in the human PER.C6® cell line under Neu5Gc-free/serum-free conditions.

<p>PER.C6® cells were stably transfected with cDNA encoding human EPO and co-transfected with ST3GalIV for full sialylation of the glycans of EPO. EPO-expressing PER.C6® cells were cultured under serum-free conditions (VPRO medium) and EPO was purified from the medium. The sialic acid content of PER.C6®-rEPO and of CHO-rEPO (Eprex) was similar as determined by IEF (data not shown). A. The presence of Neu5Gc on CHO-rEPO (Eprex) or PER.C6®-rEPO was examined by DMB-HPLC according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004241#pone.0004241-Hara1" target="_blank">[38]</a>. B. The presence of Neu5Gc on both CHO-rEPO (Eprex) and PER.C6®-rEPO was examined by the highly sensitive Western blot as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004241#s2" target="_blank">Methods</a>. 1 and 2 µg of EPO and 5 µg of positive control (bovine fetuin) were run on a 4–12% SDS-PAGE gel and blotted onto nitrocellulose membrane. Immunostaining for Neu5Gc was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004241#s2" target="_blank">Methods</a> and as above under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004241#pone-0004241-g002" target="_blank">figure 2</a>.</p

A common solution to group 2 influenza virus neutralization

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccine