Combination therapy strategies for improving PD-1 blockade efficacy: a new era in cancer immunotherapy.

Programmed death 1 (PD-1) is an immune checkpoint molecule that negatively regulates T-cell immune function through the interaction with its ligand PD-L1. Blockage of this interaction unleashes the immune system to fight cancer. Immunotherapy using PD-1 blockade has led to a paradigm shift in the field of cancer drug discovery, owing to its durable effect against a wide variety of cancers with limited adverse effects. A brief history and development of PD-1 blockade, from the initial discovery of PD-1 to the recent clinical output of this therapy, have been summarized here. Despite its tremendous clinical success rate over other cancer treatments, PD-1 blockade has its own pitfall; a significant fraction of patients remains unresponsive to this therapy. The key to improve the PD-1 blockade therapy is the development of combination therapies. As this approach has garnered worldwide interest, here, we have summarized the recent trends in the development of PD-1 blockade-based combination therapies and the ongoing clinical trials. These include combinations with checkpoint inhibitors, radiation therapy, chemotherapy and several other existing cancer treatments. Importantly, FDA has approved PD-1 blockade agent to be used in combination with either CTLA-4 blockade or chemotherapy. Responsiveness to the PD-1 blockade therapy is affected by tumour and immune system-related factors. The role of the immune system, especially T cells, in determining the responsiveness has been poorly studied compared with those factors related to the tumour side. Energy metabolism has emerged as one of the important regulatory mechanisms for the function and differentiation of T cells. We have documented here the recent results regarding the augmentation of PD-1 blockade efficacy by augmenting mitochondrial energy metabolism of T cell.

Isolation of new anti-CD30 scFvs from DNA-immunized mice by phage display and biologic activity of recombinant immunotoxins produced by fusion with truncated pseudomonas exotoxin.

To target CD30 on Hodgkin's disease and anaplastic large-cell lymphoma, anti-CD30 single-chain antibodies were obtained by DNA immunization of mice with the complete human CD30 cDNA. Spleens were isolated from mice with high anti-CD30 titer, and the RNA was used for the production of an scFv-displaying phage library. Specific phages were enriched by 3 rounds of panning on soluble CD30 or CD30+ K562 cells. Recombinant immunotoxins (rITs) were made from 3 ELISA-positive scFv phages by fusion to a 38 kDa truncated mutant of Pseudomonas exotoxin (PE38) with or without a KDEL mutant sequence at the C terminus. In vitro cytotoxicity of purified anti-CD30 rITs was measured on CD30-transfected A431 cells. IC50 values ranged from 3 to 7 ng/ml (50-110 pM) for PE38 rITs and 0.1 ng/ml (2 pM) for the PE38-KDEL IT on A431-CD30 cells. The parental A431 cells were resistant, indicating that the cytotoxicity was specific and CD30-mediated. rITs were tested for anti-tumor activity in a nude mouse model. A431-CD30 cells were injected s.c. on day 0; then, mice bearing measurable tumors were treated beginning on day 4 with 3 alternate daily doses i.v. Anti-tumor activity was dose-dependent and not found when irrelevant ITs were administered or when CD30- tumors were treated. Our data show that DNA immunization and antibody phage display may be useful in producing new rITs against hematologic malignancies. Published 2001 Wiley-Liss, Inc.

Generation of high titer antisera in rabbits by DNA immunization.

Mesothelin is a GPI-linked, membrane-associated differentiation antigen that is over-expressed in several forms of human cancers. Intradermal injection into rabbits of plasmid DNA encoding full length mesothelin resulted in antisera with titers as high as 1:100,000. Each immunization consisted of 320 microg of DNA delivered into 4 sites. After the initial three injections antisera titers were moderate (between 10 to 30,000) and fell over the course of about 7 weeks. When the titers had fallen, an injection of a booster dose of DNA resulted in very high titers of antisera. These antisera contained IgGs that could bind to both recombinant mesothelin made in Eschericha coli and to mesothelin present on human cells in Western blots and in immunofluorescence assays. These observations indicate that simple intradermal DNA immunization of rabbits can result in high titers of antibodies that can be used for a variety of purposes.

Circulating IgG response to stromelysin-3, collagenase-3, galectin-3 and mesothelin in patients with pharynx/larynx squamous cell carcinoma.

With the aim of identifying tumor-associated antigens that could be potential markers and/or targets of diagnostic and/or therapeutic approaches, we studied the occurrence of circulating IgG antibodies to human stromelysin-3, collagenase-3, galectin-3 and mesothelin, by Western blot against their purified recombinant forms, in the sera of 50 patients with pharynx/larynx squamous cell carcinoma (PLSCC), as well as in the sera of 50 healthy blood donors. Overall, antibodies to collagenase-3 were detected in 50% of all the cancer patients and 16% of the blood donors examined; this percentage difference was statistically significant (p = 0.00066). With respect to anti-galectin-3 antibodies, the percentages were 32% and 18%, respectively, but they were not statistically different (p = 0.16). Low levels of antibodies to stromelysin-3 and to mesothelin were detected in sera from only two cancer patients. No significant correlations were found in the present study between the presence of antibodies to these proteins and tumor site, clinical and T stages, lymph node involvement, DNA ploidy and histological grade of differentiation of the primary tumors. To our knowledge, this is the first report on the detection of circulating IgG to collagenase-3 in cancer patients. Some of the percentages found here in certain groups of patients are among the highest reported of circulating antibodies to any tumor component studied so far. The monitoring and the use of human antibodies to collagenase-3 could be of diagnostic and therapeutic interest.

Improving antibody affinity by mimicking somatic hypermutation in vitro.

In vivo affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve antibody affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in affinity compared with a single clone with a fourfold increased affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-affinity mutants of Fvs, Fabs, and other recombinant antibodies from antibody phage libraries that are small in size.

Analysis of cloned Fvs from a phage display library indicates that DNA immunization can mimic antibody response generated by cell immunizations.

BACKGROUND: Generation and cloning of antibodies against cell surface antigens can be simplified by combining DNA immunization which enables generation of antibodies against a protein in its natural configuration without the need for any protein purification step and antibody phage display which due to its immense screening power and physical coupling between the phenotype and genotype of antibodies simplifies the cloning of antibody genes. OBJECTIVES: Since DNA immunization is expected to elicit antibodies against a protein in its natural configuration, we wanted to see if it can mimic the antibody response generated by cell immunization. STUDY DESIGN: A phage display library made from splenic mRNA of a mouse immunized with mesothelin cDNA was panned on mesothelin-positive cells. The single-chain Fvs (scFvs) selected were then analyzed. RESULTS: We obtained several anti-mesothelin scFvs. One of these Fvs is almost identical to the Fv of a monoclonal antibody that was previously obtained from a hybridoma in which the mice were immunized with a mesothelin-positive ovarian cancer cell line. Another Fv was found to be specific for mesothelin present on human cells. CONCLUSION: Our results indicate that an antibody phage display library made from spleens of DNA-immunized mice is a rapid and efficient alternative to cell immunization for obtaining antibodies against different epitopes of a membrane antigen that is very difficult to purify in a native form.

Improved stability and yield of a Fv-toxin fusion protein by computer design and protein engineering of the Fv.

The conversion of the anti-mesothelin monoclonal antibody K1 to a single-chain Fv (scFv) that is fused to a truncated form of Pseudomonas exotoxin A (PE) results in a fusion protein (immunotoxin) that is unstable and refolds very inefficiently. We have devised a method that identifies candidate residues in the framework region of K1 Fv that, when mutated, improved the yield and stability of the protein. The method works by initially aligning the framework sequences of K1 VH and VL with those of other scFvs that are stable and give a good yield as immunotoxins. Then we assigned a character to each residue that indicates its state of exposure based on the known crystal structures of Fabs. This identifies residues that are not compatible with their environment in the folded state of the protein. Next we calculated the frequencies of different amino acids for each position of the Fvs based on the available sequence database. This identifies residues that are not commonly present in the conserved positions. If these residues are compatible with their exposure profile they are left unaltered. Otherwise, they are identified as candidate residues for mutation. We identified two such residues in the VH (T82 and A85) and two in the VL (H36 and V60) of K1 that did not seem appropriate for their respective positions. By mutating these residues in K1 into those that occur most commonly in the sequence database or in stable scFvs, we significantly improved the stability and yield of the K1 scFv immunotoxins. By making single and combined mutations we assessed the relative contribution of mutations at these four sites towards the stability and yield of K1 scFv immunotoxins. The method we devised is probably general and can be used to improve other scFvs.

Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity.

Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers. Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents. In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin. When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice. After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells. The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A. The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37 degrees C and is very cytotoxic to cells expressing mesothelin. It also produces regressions of tumors expressing mesothelin. This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent. This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.

Isolation of anti-mesothelin antibodies from a phage display library.

Phage display has been used to select single-chain Fvs (scFvs) against mesothelin, a surface antigen present on mesothelial cells as well as mesotheliomas and non-mucinous ovarian cancers. Several attempts to produce anti-mesothelin hybridomas from spleen cells of mice immunized with recombinant mesothelin were unsuccessful. This report describes the isolation of anti-mesothelin scFvs from a phage display library made from the mRNA of the same spleens. Panning on recombinant antigen produced in E. coli or on antigen positive cells was employed. Several scFvs which bind specifically to mesothelin were isolated. Panning on recombinant antigen yielded five different scFvs. Panning on cells selected two different scFvs which also differ from the scFvs selected by recombinant antigen. The heavy chains of the scFvs selected on recombinant antigen are derived from four different heavy chain gene families and the scFvs selected on cells are derived from two of these families. In contrast, the light chains of all of these scFvs are derived from family XI. Moreover, the light chains of the two scFvs selected on cells are very similar to the light chains of two of the scFvs selected by panning on recombinant mesothelin except for a few point mutations. One of these scFvs which have been studied in detail has been shown to bind specifically to mesothelin positive transfected cells.

Phage display of disulfide-stabilized Fv fragments.

Phage display of single-chain Fvs (scFvs) is a powerful tool to enrich and isolate specific antibody fragments from large pools (libraries) of Fv coding genes. However, many scFvs and scFv fusion proteins are unstable, not only as soluble proteins but also on the surface of phage. This limits and biases the recovery of specific Fv phage from display libraries to relatively stable scFvs. Also, the peptide linker in scFvs can diminish antigen binding of scFvs and scFv-fusion proteins. Disulfide-stabilized Fvs (dsFv) which have the VH-VL heterodimer stabilized by an interchain disulfide bond connecting framework regions in VH and VL rather than a peptide linker are more stable than scFvs and in some instances show better binding. To analyze whether these advantages can be utilized in a phage display system and to prove the feasibility of dsFv display, we constructed phage for dsFv display of the anti-Tac antibody and a dsFv-phage library. We find that dsFv phage can specifically bind antigen although the titer of dsFv phage in supernatants appears to be reduced compared to scFv phage. But this reduction in titer does not hamper the isolation of dsFv phages from large pools (libraries) as demonstrated by 'panning' of anti-Tac scFv and dsFv phages on living leukemia cells in suspension. In addition, dsFv phage are more stable than scFv phage. Therefore, display of dsFvs on phage is a useful alternative and addition to scFv-phage display.

Construction and characterization of M13 bacteriophages displaying functional IgG-binding domains of staphylococcal protein A.

Staphylococcal protein A (SPA) is ranked as a versatile probe in immunoassays because of its immunoglobulin G (IgG)-binding capability. However, poor binding of SPA to the IgG of some laboratory animals and its inability to bind human IgG3 restricts its universal utility. In the present study, DNA encoding the four IgG-binding domains of SPA (E, D, A and B) or the B domain alone has been fused, in separate phagemid vectors, to the 5' end of gene 111 of the phage M13. Upon infection by helper phage M13KO7, phagemid particles encapsulating single-stranded DNA were produced. Dot immunoblot and Western blot analyses showed the presence of fusion proteins on the M13 surface. Binding of rabbit IgG-horseradish peroxidase (IgG-HRP) complex to the phage particles confirmed that the fusion proteins possessed functional IgG-binding domains. The interaction of these phages with immobilised human IgG and its various subclasses was studied by the phage capture immunoassay where the captured phages were detected by a monoclonal antibody to the major coat protein encoded by gene VIII (gVIII). The phages showed maximal binding to IgG1 kappa, followed by IgG2 kappa, and showed negligible binding to the IgG3 kapa and IgG3 lambda subclasses. The specificity of IgG-binding phages was confirmed in a phage capture and elution assay where the binding of these phages to immobilised human IgG1 kapa weas abolished in the presence of excess of soluble protein A. Moreover, IgG-binding phages could be enriched approx. 1000-fold over non-specific phages in a single round of panning.(ABSTRACT TRUNCATED AT 250 WORDS)

Exocytosis from a single rat chromaffin cell by cholinergic and peptidergic neurotransmitters.

Secretion of catecholamines from chromaffin cells is mediated by cholinergic and peptidergic neurotransmitters. The cholinergic transmitter acetylcholine activates both nicotinic and muscarinic receptors to trigger catecholamine secretion in rat adrenal medulla. Vasoactive intestinal polypeptide (VIP) has been identified as the peptidergic transmitter in rat adrenal medulla and may also be the non-cholinergic transmitter in bovine adrenal. Pituitary adenylate cyclase activating polypeptide (PACAP), a VIP-like secretin peptide, is also found in the adrenal, and is a potent secretagogue. Thus, PACAP may be another peptidergic transmitter at the adrenal synapse. A most intriguing property of rat chromaffin cells is that stimulation of nicotinic, muscarinic, VIP or PACAP receptors are each able to produce robust catecholamine secretion on their own. This raises the question of whether a single chromaffin cell can respond to each of the above agonists or whether the secretion is due to subpopulations of chromaffin cells. This issue was addressed by using electrochemical techniques to monitor exocytosis from individual chromaffin cells in culture. We demonstrate that acetylcholine, nicotine, muscarine, VIP and PACAP are each able to evoke catecholamine secretion from a single chromaffin cell. Some cells only responded to acetylcholine. Furthermore, each agonist produced a distinct pattern of exocytosis. Muscarine-evoked secretion exhibited a latency of 0.5-2 s, but exocytosis persisted up to 30 s following 500 ms stimulation. Nicotine produced an immediate response which usually ended within 10 s. The secretory pattern following acetylcholine appeared to be the sum of the nicotinic and muscarinic patterns, showing both rapid onset and longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)

An expression system for secretion and purification of a genetically engineered thermostable chimera of protein A and alkaline phosphatase.

A chimera between gene segments of Protein A and a mutated alkaline phosphatase (lysine328 mutated to alanine) of Escherichia coli has been constructed. This chimeric gene was cloned in a T7 promoter-based IPTG-inducible expression vector. The chimeric protein was expressed in E. coli and was efficiently secreted into the periplasm, from which it could be easily purified by a combination of ion-exchange and gel permeation chromatography. The purified chimera was found to be thermostable and exhibited both IgG binding and high alkaline phosphatase activity. It was used as a probe in enzyme-linked immunosorbent assays and results indicate that it is a promising substitute for secondary antibodies in enzyme-linked immunoassays.

Massive exocytosis triggered by sodium-calcium exchange in sympathetic neurons is attenuated by co-culture with cardiac cells.

Entry of Ca2+ through voltage-dependent Ca2+ channels is known to be linked to the exocytotic release of transmitter from sympathetic neurons. In this paper we provide evidence that transmitter release can also be stimulated by Ca2+ influx via the Na-Ca exchanger. Furthermore, the release linked to Na-Ca exchange is regulated by cardiac target cells. Cultured sympathetic neurons of the chick embryo incubated in Ca2(+)-Mg(2+)-free Krebs solution for 20 min and then switched to Ca(2+)-containing solution exhibited 15-20-fold increases in [3H]noradrenaline release over the spontaneous release. Electrophysiologic studies showed that neurons were completely depolarized in Ca(2+)-Mg(2+)-free medium. Indo-1 fluorescence revealed a large and sustained increase in intracellular free Ca2+ concentration ([Ca2+]i) after addition of Ca2+ to Ca(2+)-Mg(2+)-free medium. The increased [3H]noradrenaline release and [Ca2+]i were dependent on external Na+ and Ca2+, but were not affected by the Ca2+ channel blockers lanthanum, cadmium, verapamil or omega-conotoxin. A conventional depolarizing stimulus (125 mM K+) produced a 13-fold increase in [3H]noradrenaline release over spontaneous release. However, K(+)-induced release and rise in [Ca2+]i declined rapidly and were sensitive to the Ca2+ channel blockers. When sympathetic neurons were co-cultured with embryonic cardiac cells the release induced by change from Ca(2+)-Mg(2+)-free to Ca(2+)-Krebs solution was dramatically reduced. The change from Ca(2+)-Mg(2+)-free to Ca(2+)-Krebs solution was ineffective in evoking [3H]noradrenaline release from sympathetic neurons in situ using perfused hearts of 15-day-old chick embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Sites of transmitter release and relation to intracellular Ca2+ in cultured sympathetic neurons.

Fluorescence imaging of indo-1 loaded cells was used to monitor influx and distribution of Ca2+ in cell bodies, neurites and growth cones of sympathetic neurons cultured from embryonic chick. Similar experiments on release of tritiated noradrenaline were performed to assess the relationship between intracellular Ca2+ concentration ([Ca2+]i) and transmitter release. Effects of Ca2+ channel antagonists on electrically stimulated rise in [Ca2+]i were dependent on the neuronal region examined. Cadmium and verapamil blocked Ca2+ entry in cell bodies but were less effective in neurites and growth cones. Nifedipine partially inhibited Ca2+ entry in cell bodies and was less effective in neurites and growth cones. Combination of cadmium and nifedipine blocked [Ca2+]i rise in all neuronal regions. Omega-conotoxin was an effective Ca2+ channel blocker in all regions. Ca2+ channel blockers had effects on [3H]noradrenaline release which paralleled effects on [Ca2+]i in neurites (and growth cones) but not cell bodies. Cadmium, verapamil and nifedipine each caused a partial, reversible block of the evoked release. Combination of cadmium and nifedipine completely blocked evoked [3H]noradrenaline release. Omega-conotoxin caused complete, irreversible block of electrically evoked release. During prolonged depolarization with 125 mM K+ Krebs solution, elevation of [Ca2+]i was maintained in cell bodies but was transient in neurites and growth cones. The amplitude and time course of [3H]noradrenaline release paralleled [Ca2+]i in neurites and growth cones, but not the cell body under the above conditions. A new method is described to study localized uptake and release of [3H]noradrenaline in cell bodies versus neurites of sympathetic neurons. Incubation of these modified cultures with [3H]noradrenaline showed that cell bodies had very low [3H]noradrenaline uptake (0.23 x 10(-6) c.p.m./mg protein), whereas neurites contained approximately 20 times more radioactivity. Depolarization of neurites by excess K+ and field stimulation caused a large increase in the net release of [3H]noradrenaline. The release was unaffected by removal of cell bodies. Neurites remained functionally viable for more than 2 h after separation from their cell bodies. [3H]Noradrenaline release could be evoked repeatedly over this time. [3H]Noradrenaline release from isolated neurites was partially blocked by nifedipine and fully blocked by combination of cadmium and nifedipine or by omega-conotoxin. The uptake and release of [3H]noradrenaline by neurites alone (expressed per mg protein) accounted for the total [3H]noradrenaline in intact cultures with neurites and cell bodies. Therefore, we conclude that neurites (and growth cones) are the prominent sites of uptake, storage and release of sympathetic transmitter.(ABSTRACT TRUNCATED AT 400 WORDS)

Barium-induced exocytosis is due to internal calcium release and block of calcium efflux.

The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([3H]NE) were monitored during Ba2+ stimulation of sympathetic neurons cultured from chick embryos. Ba2+ (2.5 mM in Ca(2+)-free medium) caused a rise in [Ca2+]i in all regions (cell bodies, neurites, and growth cones) of sympathetic neurons and evoked [3H]NE release in the absence of other stimuli. The increase in [Ca2+]i and release of [3H]NE were sustained for up to 30 min in the presence of Ba2+. When Ba(2+)-stimulated cells were immediately washed in Ca(2+)-free Ba(2+)-free EGTA solution, both the elevated [Ca2+]i and [3H]NE release returned to basal levels, with similar, fast, time courses. Ba2+ also blocked Ca2+ efflux from neurons loaded with 45Ca. We conclude from the parallel effects of Ba2+ on [Ca2+]i and [3H]NE release that Ba2+ stimulates exocytosis by a Ca(2+)-dependent mechanism. The Ba(2+)-induced rise in [Ca2+]i is a result of two separate actions: (i) the release of Ca2+ from intracellular sites and (ii) an effective block of Ca2+ extrusion. The ability of Ba2+ to release Ca2+ in growth cones that are insensitive to caffeine suggests that Ba2+ may displace Ca2+ from binding sites other than endoplasmic reticulum.

Activation of K+ channels by lanthanum contributes to the block of transmitter release in chick and rat sympathetic neurons.

We studied the effects of lanthanum (La3+) on the release of 3H-norepinephrine (3H-NE), intracellular Ca2+ concentration, and voltage clamped Ca2+ and K+ currents in cultured sympathetic neurons. La3+ (0.1 to 10 microM) produced concentration-dependent inhibition of depolarization induced Ca2+ influx and 3H-NE release. La3+ was more potent and more efficacious in blocking 3H-NE release than the Ca(2+)-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 microM, La3+ produced a complete block of the electrically stimulated rise in intracellular free Ca2+ ([Ca2+]i) in the cell body and the growth cone. The stimulation-evoked release of 3H-NE was also completely blocked by 3 microM La3+. However, 3 microM La3+ produced only a partial block of voltage clamped Ca2+ current (ICa). Following La3+ (10 microM) treatment 3H-NE release could be evoked by high K+ stimulation of neurons which were refractory to electrical stimulation. La3+ (1 microM) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K+ current (IA) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated 3H-NE release is a result of the unique ability of La3+ to activate a stabilizing, outward K+ current at the same concentration that it blocks inward Ca2+ current.

N-glycolylneuraminic acid specific lectin from Pila globosa snail.

A N-glycolylneuraminic acid-specific lectin (PAL) has been purified from an albumin gland extract of the apple snail, Pila globosa. Purification is conducted on a bovine submaxillary mucin-Sepharose 4B affinity matrix followed by gel filtration on a Sepharose 6B column. The lectin agglutinates rabbit erythrocytes. The hemagglutination activity is dependent on Ca2+ concentration in a significant manner but with a remarkable behaviour. The lectin is a trimeric glycoprotein of native Mr 440 kDa with 25% carbohydrate and is composed of three nonidentical subunits of molecular weights 190, 145, and 105 kDa. It has a pI of 7.0. The lectin exhibits a unique and strict specificity toward N-glycolylneuraminic acid and this phenomenon discriminates it from other known sialic acid binding lectins. The uniqueness indicates the absolute need for a glycolyl substitution on the amino residue and of a glyceryl side chain on the exocyclic part and an axial -COOH group in neuraminic acid. The presence of an acetyl substitution on the exocyclic part impedes lectin-ligand interaction.