Inhibition of in vitro tumor cell growth by RP215 monoclonal antibody and antibodies raised against its anti-idiotype antibodies

RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and the corresponding rat anti-id Mabs were generated and characterized. Following immunizations in mice, antisera raised against anti-id antibodies were analyzed by typical immunoassays. It was observed that mouse anti-anti-id sera (Ab3) revealed binding affinity and specificity to CA215 that are comparable to those of RP215. Both RP215 and Ab3 were shown to induce apoptosis of cultured cancer cells in vitro by TUNEL and MTT assays. These experimental observations were consistent with that of in vivo tumor growth inhibition by RP215 in previous nude mouse experiments. Therefore, heterologous or homologous anti-id antibodies of RP215 that contain the internal image of its specific epitope in CA215 may serve as effective anti-cancer vaccines for therapeutic treatments of various cancers in humans. The relative stability of RP215-specific carbohydrate-associated epitope was compared to that of human IgG at extreme pH’s (≤2 or ≥12) or following NaIO₄ treatments. The major molecular forms of CA215 were further documented with various enzyme immunoassays and found to have similar secondary structures to those of normal human immunoglobulin G.     

Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein

The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI x RNase 1 complex was determined at a resolution of 1.95 A, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI.RNase 1 complex was found to be extremely stable (t(1/2)=81 days; K(d)=2.9 x 10(-16) M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10(3)-fold lower association rate for complex formation and 5.9 x 10(9)-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.     

Stabilization of human pancreatic ribonuclease through mutation at its N-terminal edge

Enzyme stability can be an important parameter in the design of recombinant toxins because unstable proteins are often degraded before they can reach their cellular target. There is great interest in the design of human pancreatic ribonuclease variants that could be cytotoxic against tumoral cells. To this end, some residues in the protein need to be substituted, but this may result in a loss of stability. Previous papers have reported the production of N- and C-terminal human pancreatic ribonuclease variants with increased thermal stability. Here, we investigated the contribution of the different amino acid changes at the N-terminus of the protein to its thermostability increase. We show that this increase correlates with the helical propensity of the first alpha-helix of the protein. On the other hand, deletion of the four last residues of the protein does not affect its thermal stability. These results set the basis for the design of a human pancreatic ribonuclease template on which amino acid substitutions can be made that could render the enzyme cytotoxic, without an important loss in its stability.     

Stabilization of pancreatic ribonuclease A by immobilization on Sepharose-linked antibodies that recognize the labile region of the enzyme.

The stabilizing potential of the antibodies recognizing the labile region of pancreatic ribonuclease A (RNase) has been investigated. The dodecapeptide SRNLTKDRAKPV corresponding to the labile region 32--43 on RNase was synthesized by the solid-phase method. Antiserum raised against the dodecapeptide-bovine serum albumin conjugate showed good cross-reactivity with the peptide and native RNase. RNase immobilized on Sepharose support precoupled either with the antipeptide immunoglobulin (IgG) or anti-RNase IgG proved to be more resistant to thermal inactivation than the soluble enzyme. Besides, stability against inactivation by trypsin at 55 degrees C was markedly high when enzyme was immobilized on the antipeptide IgG support, as compared to the soluble and other immobilized preparations. These results suggest that matrices bearing antibodies recognizing specific labile regions of enzyme may be useful in selectively improving their stability against specific forms of inactivation.     

Inhibition of Fc-receptor dependent platelet aggregation by monoclonal antibodies against the glycoprotein IIb-IIIa complex]

A murine monoclonal antibody (MoAb) VM16a specifically binding to human platelets has been produced. Approximately 56,000 molecules of VM16a bound per platelet at saturation (Kd = 7.9 nM) but no binding to platelets from Glanzmann's thrombasthenia patients was detected. VM16a precipitated two proteins with molecular masses corresponding to those of glycoproteins (GP) IIb and IIIa from solubilized surface-labelled platelets. However, after dissociation of the GPIIb--IIIa complex with EDTA VM16a did not bind to platelets and precipitated nothing from their lysate, thus evidencing that its determinant is complex-dependent. VM16a had no effect on ADP-, thrombin- and ristocetin-induced platelet aggregation but inhibited the aggregation induced by collagen. This inhibitory effect was more pronounced in the presence of plasma. VM16a completely blocked the Fc-receptor-mediated aggregation induced by aggregated human IgG, aggregated murine IgG1 and the previously described MoAb VM58. F(ab')2 fragments of VM16a were also able to inhibit this aggregation by decreasing the rate of aggregation induced by aggregated IgG and by extending the lag phase of VM58-induced aggregation. These results suggest that the platelet Fc-receptor may be topographically associated with the GPIIb-IIIa complex.     

Oligomerization and aggregation of bovine pancreatic ribonuclease A: characteristic events observed by FTIR spectroscopy

Nonnative protein aggregation, which is a common feature in biotechnology, is also a clinical feature in more than 20 serious degenerative diseases. We studied the specific events of bovine pancreatic ribonuclease A thermal aggregation by a combination of second derivative infrared analysis and two-dimensional infrared correlation spectroscopy. By comparing the events that occur in reversible and irreversible thermal unfolding processes, certain events that were related to protein aggregation were characterized. Particularly, a band that appeared at high temperatures was assigned to the cross beta-structures in oligomers. The effect of pH, NaCl, and ethanol on ribonuclease A oligomerization as well as further aggregation induced by heat were studied and dissimilar effects of these additives were found. Basic pH and NaCl could accelerate the thermal aggregation but did not affect the formation of oligomers, whereas ethanol could increase both the aggregation rate and the population of oligomers. Our results suggested that the aggregation of RNase A might be initiated by hydrophobic interactions, controlled by oligomerization and mediated by electrostatic interactions. Moreover, the strategy of using second derivative and two-dimensional infrared analysis might provide a potential powerful tool to study the events that are directly related to the initiation of protein aggregation.     

Oligomerization and aggregation of bovine pancreatic ribonuclease A: backbone hydration probed by infrared band-shift

Protein hydration plays a crucial role in almost all aspects of biomolecular processes. In this research, we studied the hydration/dehydration-induced infrared amide I band-shift by using poly-L-lysine and bovine pancreas ribonuclease A as model polypeptides. It was found that a 1-4 cm(-1) shift could be clearly distinguished for all regular secondary structures during protein thermal unfolding. This shift was proven to be due to backbone hydration but not from experimental error, temperature effect or possible incomplete hydrogen/deuterium exchange of the samples. Moreover, we also found that protein aggregation was closely associated with the backbone hydration/dehydration status of proteins. In conditions favoring aggregation, a significant shift to a higher wavenumber of the band from the intermolecular beta-sheet structures in aggregates was observed. The present study suggested that the changes of the amounts of regular secondary structures could be monitored by the intensity changes, while the changes of the hydration status could be monitored by the shift of the infrared bands.     

Probes of beta-galactosidase structure with antibodies. Reaction of anti-peptide antibodies against native enzyme.

Antibodies were prepared against 18 tryptic and cyanogen bromide peptides from beta-galactosidase ranging in size from 15 to 96 amino acid residues representing more than 80% of the polypeptide chain. They were tested for binding capacity and affinity toward their homologous antigens and toward the whole native protein. Nine antisera bound to beta-galactosidase; these had been raised against certain peptides from the central and carboxyl-terminal regions of the poly-peptide chain. Based on these results a preliminary model of the three-dimensional structure of the folded protein is suggested.     

Selective stabilization of a labile mutant form of bovine pancreatic ribonuclease A by antibodies

The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a dramatic destabilizing effect (T_m=9 °C). Antibodies raised against a peptide corresponding to the sequence of the labile region, S32-V43, of RNase were effective in stabilizing L35S-RNase against thermal inactivation (65 °C for 2 h) and surpassed the stabilization effect of antiRNase antibodies. An 11% contribution to the stabilizing effect of antiRNase antibodies resulted from antibodies recognizing the unfolding region of the enzyme.     

The processing of interleukin-1 beta studied with antibodies raised against synthetic peptides from the precursor N-terminal region

The exact sequence of events during processing of human interleukin-1 beta (IL-1 beta) and the fate of the N-terminal region are unknown. We have used anti-peptide sera specific for the precursor and mature regions of IL-1 beta to study biosynthesis. These were raised against peptides corresponding to amino acids 1-15, 17-32, and 43-54 of the precursor and a peptide corresponding to the C-terminal 33 amino acids of mature human IL-1 beta. Antiserum to the mature region peptide immunoprecipitated the 35-kD precursor from cell lysates and 17-kD mature IL-1 beta and a 31-kD protein from the culture supernatants from radiolabeled human peripheral blood monocytes stimulated with lipopolysaccharide (LPS). Antisera to peptides from the precursor region also immunoprecipitated the 35-kD IL-1 beta precursor but not the 31-kD or 17-kD forms. Of the precursor-specific sera, only antiserum to amino acids 1-15 specifically recognized any other proteins; a peptide of 18 kD and a low molecular weight peptide, both of which accumulated in the medium. The 18-kD protein was not recognized by any of the other antisera and is unlikely to be the N-terminal region of the precursor removed during processing. Pulse-chase experiments indicated that the 31-kD protein could be a processing intermediate and also that it was itself an end product along with full-length precursor. Only 17-kD mature IL-1 beta had biological activity.     

Inhibition of a plasminogen activator from oncogenic virus-transformed mouse cells by rabbit antibodies against the enzyme

Antisera were raised in rabbits against an electrophoretically pure 48 000 dalton plasminogen activator from mouse cells transformed by an oncogenic virus. The IgG fraction of the antisera inhibited 48 000 dalton mouse plasminogen activators from a variety of sources (neoplastic and nonneoplastic), a 29 00) dalton plasminogen activator from mouse urine and a 48 000 dalton plasminogen activator from rat urine. No inhibition was observed of a 75 000 dalton plasminogen activator extracted from mouse lung, of mouse plasmin or of plasminogen activators from human urine and from oncogenic-virus transformed chicken cells. The IgG antibodies were stronger and more specific inhibitors of the 48 000 dalton mouse plasminogen activator than any previously tested compounds.     

Inhibition of biological activities of the aerobactin receptor protein in rough strains of Escherichia coli by polyclonal antiserum raised against native protein

The aerobactin iron-uptake system of plasmid ColV-K30, genetically isolated from other plasmid determinants by molecular cloning, was sufficient to restore full virulence in a mouse peritonitis model to a clinical Escherichia coli isolate, D551 (O78:H-), whose resident aerobactin-encoding ColV plasmid had been lost by curing. Antiserum was raised in rabbits against live E. coli K12 cells expressing the outer-membrane aerobactin receptor protein and absorbed with an isogenic strain lacking the receptor. This antiserum inhibited binding of aerobactin, cloacin DF13 and bacteriophage B74K to the native protein in whole E. coli K12 bacteria expressing the receptor, or in membranes prepared from such organisms. However, it did not react with the native receptor protein in several wild strains unless lipopolysaccharide was first removed by treatment with trichloroacetic acid, nor did it protect mice in experimental infections with strain D551. Antisera raised in rabbits against partially or fully denatured forms of the aerobactin receptor reacted only in assays involving denatured protein; they showed no inhibition of the biological activities of the native receptor.     

Isolation and characterization of monoclonal antibodies against ribonuclease inhibitor

Mouse monoclonal antibodies were generated against human ribonuclease inhibitor, an intracellular regulatory protein. A total of four antibodies were isolated, all of which were of the immunoglobulin G1 subtype. Western blot analysis of the antibodies suggested monospecificity. Based on immunoradiometric competition assays two of the antibodies were determined to be directed against the same antigenic epitope, while the other two were against a second and possibly third epitope. None of the antibodies appeared to be directed against the ribonuclease binding site of the antigen. Data is presented suggesting that ribonuclease inhibitor is present in normal human serum. The potential significance of these findings is discussed.     

Formation of native structure by intermolecular thiol-disulfide exchange reactions without oxidants in the folding of bovine pancreatic ribonuclease A

It has been shown previously that the oxidative folding of bovine pancreatic ribonuclease A proceeds through parallel pathways with two major native-like three-disulfide (3S) intermediates. We show here that, under some conditions, the native disulfide bonds can also be regenerated through disproportionation reactions; in other words, the protein can serve as its own redox reagent. The results also show that disulfide species of the unstructured 3S ensemble have a strong propensity to participate in intermolecular interactions. These interactions are favored at high protein concentration, temperature and pH, and lead to formation of the native structure during disulfide reshuffling in the rate-determining step.