Loss of secretion in mouse-human hybrids need not be due to the loss of a structural gene.

Three cloned mouse-human lines (B1-29, E2-42, and A2-31) secreting human immunoglobulin (Ig) were obtained from a fusion between the mouse myeloma line NS-1 and human tonsillar lymphocytes stimulated in vitro with pokeweed mitogen. One line, B1-29, has continued to secrete human IgG for a period of 2 yr in culture. This line was recloned three times to give a panel of secreting and nonsecreting subclones. Most of the nonsecreting subclones had also lost surface Ig. The structural genes for human Ig heavy chains have been provisionally assigned to chromosome 14, which also encodes the enzyme nucleoside phosphorylase. Human nucleoside phosphorylase was detected in all secreting and nonsecreting B1-29 subclones, indicating the presence of human chromosome 14. The retention of chromosome 14 in nonsecreting clones implied that the structural genes for human Ig were A2-31 and E2-42, which had stopped secreting, an attempt was made to restimulate the secreting of human Ig with mitogens A2-31 was unique among the cell lines examined, in that chromosome 14 could not be detected by an isoenzyme marker. Lipopolysaccharide, at an optimum dose of 10 micrograms/ml, restimulated these nonsecreting hybrid lines to secrete human IgG in levels up to 0.7 micrograms/ml. Loss of Ig secretion may not therefore be caused by loss of Ig structural genes.

A tumor-associated antigen specific for human kappa myeloma cells.

A monoclonal antibody (K-1-21) raised against a kappa Bence Jones protein exhibits unique binding properties to malignant plasma cells. K-1-21 is an IgG1 kappa antibody that reacts with human kappa light chains in free form, but shows no reactivity with heavy chain-associated kappa light chains. By immunofluorescence, K-1-21 binds to the surface of LICR LON/HMy2 (HMy2) kappa myeloma cells and to plasma cells from a majority (8/11) of patients with various types of kappa myeloma; it did not bind to the surface of normal cells, nor to malignant cells of non-kappa myeloma origin. Flow cytometry analysis of K-1-21 binding to HMy2 cells indicated that the surface reactivity of K-1-21 could be completely inhibited by preincubation of the antibody with purified kappa light chains, whereas no inhibition occurred after preincubation with lambda chains or intact human IgG. Thus, the epitope recognized by K-1-21 on the cell surface may be similar, if not identical, to the determinant recognized on soluble free kappa light chains, and constitutes a tumor-associated antigen with selectivity for kappa myeloma cells. K-1-21 may therefore have clinical potential in patients with kappa myeloma.

Three-dimensional structure of an Fv from a human IgM immunoglobulin.

An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an example of one dominant complementarity-determining region (CDR) essentially negating the diversity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.

Conformation dependence of a monoclonal antibody defined epitope on free human kappa chains.

Chemically and enzymatically modified kappa chains were tested by inhibition radioimmunoassay for their ability to block the binding of antibody K-1-21 with native kappa chains. Complete reduction and carboxymethylation of intrachain disulphide bonds destroyed the free kappa-chain epitope, a result confirmed by Western blotting of unreduced and reduced kappa monomers and dimers. Purified V kappa fragments failed to block the homologous interaction while inhibition was obtained with a pepsin digest yielding predominantly the C kappa region. Dimeric kappa chains were less effective than monomers in the inhibition assay, although HPLC analysis of immune complexes demonstrated the binding of two antibody molecules per molecule of dimer. Thus, the epitope on free kappa chains recognized by K-1-21 is dependent upon conformational integrity of the C kappa domain, the decreased binding activity of dimeric chains possibly being due to minor conformational changes induced by C-domain interactions.

Localization of an idiotope on the L chain dimer and intact IgG1 immunoglobulin from the patient Mcg.

A monoclonal anti-idiotype (M3.9) raised against the covalently linked Mcg lambda chain dimer binds with a similar affinity to the Mcg IgG immunoglobulin and covalent heterodimers of Mcg with other human L chains. Despite having identical amino acid sequences, the two light chains in the Mcg dimer adopt different conformations with monomer 1 acting as a heavy chain analog and monomer 2 behaving like a light chain component of an Fab. As the lambda chain in the Mcg IgG and at least one hybrid L chain dimer (Mcg X Weir) assumes a conformation similar to that of monomer 2 and the binding of anti-idiotype requires only the presence of a single Mcg lambda chain, we conclude that the idiotope is restricted to the monomer 2 type of the Mcg lambda chain conformational isomer. Cooperative binding of two molecules of rhodamine 123 in the main cavity of the Mcg dimer block the binding of the anti-idiotype whereas the binding of one molecule of bis(DNP)lysine has no significant effect on the idiotype-anti-idiotype system. Previous crystallographic analyses indicated that bound rhodamine 123 protrudes outside the rim while bis(DNP)lysine is completely immersed in the cavity. At high concns bis(DNP)lysine penetrates through the floor of the main cavity and forms a virtually irreversible complex with the dimer. Production of this complex is accompanied by conformational changes, which are presumed to be correlated with observed inhibition of binding with the anti-idiotype M3.9. Expression of the idiotope probably involves more than one linear sequence since reduction and alkylation of the intra- and inter-chain disulphide bonds in 8 M urea leads to a complete loss of binding of the anti-idiotype. The inhibition data suggest involvement of residues on or near the rim of the main cavity. Distribution of potential contact residues for rhodamine 123 is asymmetric only in the case of aspartic acid 97, which is located on the cavity rim in only one conformational isomer (monomer 2). The homologous residue in monomer 1 is directed away from the cavity and is unlikely to participate in the epitope recognized by M3.9. Attempts to define the epitope in more detail by simulation with multiple peptides have been initiated in collaboration with the laboratory of H. M. Geysen.

Purification of two allergens from Dermatophagoides pteronyssinus using monoclonal antibodies.

Fractionation of soluble extracts from the house dust mite Dermatophagoides pteronyssinus (DP) by SDS-PAGE and crossed immunoelectrophoresis (CIE) revealed at least fifty distinct protein components. Western blotting and crossed radioimmunoelectrophoresis (CRIE) indicated that fewer than one quarter of these components were allergens as determined by their ability to bind IgE from allergic individuals. Following immunization with a crude extract, two monoclonal antibodies were raised against distinct components which exhibited IgE binding capacities in Western blot and CRIE. Affinity chromatography using these monoclonal antibodies yielded components which elicited positive skin test reactions in patients allergic to DP.

Effect of monoclonal anti-neuraminidase antibodies on the kinetic behavior of influenza virus neuraminidase.

Neuraminidase from the recombinant influenza virus A/NWSHA-Tokyo/3/67NA HON2 has been shown to exhibit non-Michaelis-Menten kinetics. The multiphasic behaviour was demonstrated for both the isolated neuraminidase heads and for the intact virus. Interaction of the enzyme with two monoclonal anti-neuraminidase antibodies (WANA 1 and RANA 1), which recognize separate antigenic determinants on the molecule, resulted in hyperbolic kinetic behaviour. While both antibodies abolished the multiphasic kinetics of the enzymic reaction, only WANA 1 altered the Vmax and Km values, indicating that it may in some way inhibit the interaction of enzyme and substrate.

Monoclonal antibody against human somatomedin-C/insulin-like growth factor-I.

A monoclonal IgG3 antibody produced by mouse hybridoma shows high specificity for human somatomedin-C (SM-C) and insulin-like growth factor-I (IGF-I). The apparent Ka for SM-C is 1.7 x 10(10) M-1. At 1:2000 final dilution, culture medium from antibody-producing cells binds 35-40% of radioiodinated SM and IGF-I tracers, while similar binding is seen with ascites fluid from tumor-bearing mice at 1:200,000 dilution. Rat SM, multiplication stimulating activity, IGF-I C-peptide and human insulin show little or no crossreactivity, while IGF-II has 7%, and IGF-I 70%, of the potency of SM-C. Of a variety of species tested, acid-ethanol extracted guinea pig serum has the greatest immunoreactivity, and extracts of rabbit, rat and mouse serum have the lowest activity. Human plasma extracts give displacement curves parallel to purified SM, and vary in potency depending upon the GH status of the donor. These results suggest that this antibody will be of value for SM-C/IGF-I radioimmunoassay.

A new microsphere-based immunofluorescence assay for antibodies to membrane-associated antigens.

The screening of panels of hybridoma supernatants for specific secreted monoclonal antibodies is often achieved by cellular immunofluorescent staining and flow cytometric analysis. In some circumstances such assays are difficult because the required antigen-bearing cell population is not suitable for use in flow cytometry, has limited cell cycle expression or poor in vitro growth. A method is presented here that provides a solution to these difficulties. A system was developed, using polyacrylamide microspheres coupled with cell membranes, which permitted the production of an easily stored, standardised antigen source which could be used in subsequent flow cytometric assays. Studies comparing the binding of antibodies to whole cells and cell membrane-coupled microspheres indicate a strong qualitative and quantitative correlation in the expression of surface antigens. It is shown here that membrane antigens can be stored coupled to microspheres for months and still retain good reactivity with the appropriate antibodies. This same technique could be used in studies of antigens other than those of the mammalian cell membrane - for example, membrane antigens of sub-cellular organelles such as the mitochondrion and endoplasmic reticulum, plant or bacterial membrane antigens, or antigens not associated with membranes such as viral proteins.

Single-step purification of immunoglobulin M on C1q-Sepharose.

A rapid and simple affinity chromatography method for purifying IgM from myeloma serum and ascites fluid is described. Complement protein C1q is coupled to Sepharose with an efficiency of 35%, giving 1.7 mg of C1q bound/ml of gel. This C1q-Sepharose selectively binds IgM from crude samples at 5 degrees C, with a capacity of 0.4 mg of IgM/ml of gel. The bound IgM may be eluted simply and isocratically by bringing the gel to room temperature for 2 h, or by washing with buffer containing 0.5 M KI. The eluted IgM is highly pure by SDS-PAGE and double immunodiffusion analysis, although IgG may be a potential contaminant. The C1q-Sepharose is stable for at least 18 months.

Quantitation of free kappa light chains in serum and urine using a monoclonal antibody based inhibition enzyme-linked immunoassay.

The murine monoclonal antibody K-1-21 with specificity for free but not heavy chain-associated kappa chains was used to develop an inhibition enzyme immunoassay for quantitation of free kappa chains in serum and urine. The assay utilizes a biotin conjugate of the monoclonal antibody and has an effective working concentration range of 0.5-50 micrograms/ml free kappa chain. Normal serum and urine yielded mean values of 1.2 and 4.9 micrograms/ml respectively using this assay system. Serum levels in patients with kappa chain myelomatosis ranged from 2 to 1700 micrograms/ml while serum from patients with lambda chain malignancies exhibited normal free kappa chain levels. The importance of the ability to readily quantitate light chains in serum was demonstrated by the finding in one patient of an absence of free kappa chains in the urine despite elevated levels of serum kappa chains. A significant drop in serum-free kappa chain levels was observed in this patient following a course of chemotherapy. The assay should prove valuable in the diagnosis, prognosis and monitoring of B cell neoplasias characterized by free kappa light chain secretion.

Immunoglobulin-bearing blood leucocytes in the Pacific hagfish.

The occurrence of immunoglobulin (Ig) bearing leucocytes in the blood of the Pacific hagfish, Eptatretus stoutii , was examined using a murine monoclonal antibody (45.3) and a rabbit antiserum specific for hagfish serum Ig. Binding of antibody 45.3 to hagfish leucocytes assessed by radioimmunoassay was inhibited by preincubation of antibody with purified serum Ig thus verifying the presence of cell surface Ig cross reactive with serum Ig. The monoclonal antibody identified approximately 65% of blood leucocytes as Ig+ve while the rabbit antiserum indicated 81% Ig+ve cells. Both antibody preparations failed to react specifically with cells from mouse, horned shark, tunicate or sea star; this indicates the distinctive nature of hagfish Ig. The high percentage of blood cells bearing surface Ig in the hagfish raises the possibility that lymphocyte divergence to separate B and T pathways may not have occurred in this most primitive vertebrate. Alternatively, an Ig-like specificity characteristic of both "T" and "B" lymphocytes may have been detected. In any event, a subset of Ig negative leucocytes is evident in hagfish.

Evolutionary development of lymphocyte heterogeneity: leucocyte subpopulations in the Pacific hagfish.

Flow cytometric analysis of forward angle versus 90 degree scatter patterns of hagfish peripheral blood revealed two distinct leucocyte populations with size characteristics analogous to mammalian monocytes/granulocytes (hagfish large leucocytes) and small lymphocytes (hagfish small leucocytes). A cell population enhanced for the small leucocytes was obtained by density gradient centrifugation. Over 70% of the small leucocyte population consistently stained with a rabbit antiserum directed against polypeptide determinants on hagfish immunoglobulin, while staining of the large cell population was greatly reduced (less than 10%). A panel of monoclonal antibodies raised against a crude hagfish leucocyte preparation distinguished the two cell populations and revealed the existence of subpopulations of both small and large leucocytes.

Identification of a homologue of CD59 in a cyclostome: implications for the evolutionary development of the complement system.

We have employed a COS cell expression cloning procedure to isolate a full length cDNA clone encoding a hagfish leukocyte-associated membrane protein (HLMP1). The protein, which is identified by a monoclonal antibody (JB3) generated in our laboratory, is present on the majority of hagfish leukocytes and is also expressed on erythrocytes. The cDNA clone contained an open reading frame encoding a 120 residue polypeptide which exhibits 33% amino acid sequence identity with the precursor protein of human CD59, a leukocyte-associated membrane protein which regulates the action of the complement membrane attack complex on homologous cells. CD59 belongs to a family of structurally related glycoproteins which includes the Ly-6 proteins expressed on mouse lymphocytes. In addition to significant overall sequence homology HLMP1 shows conservation of 8 key cysteine residues with members of the CD59/Ly-6 family. Comparison of the hagfish sequence with that of the mature human CD59 protein suggested a processed protein consisting of 74 amino acids associated with the cell membrane via a GPI anchor. The latter was confirmed by immuno-flow cytometry following treatment of transfected COS cells with phospholipase. Phylogenetic analysis and tissue distribution of this protein in the hagfish are consistent with HLMP1 being a homologue of CD59. A three-dimensional model of HLMP1, constructed using the NMR-determined structure for human CD59 as a template, indicated conservation of a core structure of five strands of beta-sheet and a short helix stabilised by four disulfide bonds. These findings, when taken together with our previous identification of C5a-like chemotactic activity in LPS-activated serum, provide indirect evidence for the existence of the terminal lytic complement pathway (C5 to C9) in these primitive vertebrates.

Chemotactic responses of hagfish (Vertebrata, Agnatha) leucocytes.

The chemotactic responses of hagfish leucocytes were tested using a variety of chemoattractants. Leucocyte migration was significantly enhanced by purified mammalian complement anaphylotoxin (C5a) and LPS-activated hagfish plasma. Checkerboard analyses confirmed that the responses of leucocytes to both of these chemoattractants were directed along concentration gradients (chemotaxis) and did not result from accelerated random movement (chemokinesis). Chemotaxis was undertaken by leucocyte fractions that were enriched in granulocytes, the predominant phagocytic cells of hagfish. The data suggest that chemotactic mechanisms may have been conserved during evolution to such a degree that mammalian chemoattractants can bind and activate chemotactic receptors on hagfish leucocytes. Moreover, hagfish appear to express plasma proteins that are structurally and functionally homologous to mammalian complement anaphylotoxins.

In vivo binding of mouse IgG via polyreactive surface IgM abrogates progressive lymphocytosis in prolymphocytic leukemia.

Surface IgM expressed by malignant CD5+ B-cells from patients with B-chronic lymphocytic leukemia (B-CLL) has previously been shown to bind mouse Ig in what appears to be an example of polyreactive antigen-binding activity. This report demonstrates the in vitro and in vivo binding of mouse Ig to the surface of malignant B-cells from a patient with B-cell prolymphocytic leukemia (B-PLL). In vitro studies showed that K121, a mouse monoclonal antibody, bound to the B-PLL cells via the same low-affinity binding interaction demonstrated to occur between mouse Ig and surface IgM expressed by B-CLL cells rather than in the conventional sense against a specific antigen via its antigen-binding site. With the view to using this phenomenon to target malignant B-cells, it was important to determine whether the low-affinity interaction also occurred in vivo. Infusions of K121 totalling 286 mg were administered to a B-PLL patient over 7 days. Binding of K121 to circulating B-PLL cells was demonstrable after the administration of 36 mg of antibody and was preceded by the appearance of free antibody in the serum. Throughout the period of the infusion, the rapid rise in the peripheral blood white cell count normally observed after leukopheresis was abrogated. However, the count rose markedly after cessation of the antibody infusion in parallel with a decrease in both free and cell-bound K121. There were no observable side effects and no host immune response to either species specific or idiotypic determinants on the mouse Ig was detected. The in vivo binding of mouse Ig together with the previous in vitro data suggest the potential for a novel targeting mechanism using a region of the mouse Ig molecule to target polyreactive Ig expressed by malignant cells in B-CLL and B-PLL.

Interleukin-10 inhibits the in vitro proliferation of human activated leukemic CD5+ B-cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterised by the proliferation and accumulation of sIgM+/CD5+ B-cells that fail to progress to the final stages of B-cell development. Despite their developmental arrest, leukemic CD5+ B-cells can undergo proliferation in vitro in the presence of different activators including phorbol esters, antibodies to cell surface antigens and human cytokines. Interleukin-10 (IL-10) has recently been found to inhibit CLL B-cell function in vitro by inducing apoptosis and down-regulating expression of bcl-2. Here, we examined the effect of IL-10 on proliferation, RNA synthesis, immunoglobulin (IgM) secretion and viability of leukemic CD5+ B-cells induced by activation with the phorbol ester PMA, alone or in combination with anti-Ig. IL-10 reduced PMA and PMA/anti-Ig induced proliferation and RNA synthesis by 50-80% and 15-40% respectively. Although proliferation and RNA synthesis induced by PMA/anti-Ig could be enhanced by the addition of IL-2, IL-4, IL-13, IFN-gamma or TNF-alpha, the presence of these cytokines failed to abrogate the IL-10-mediated inhibition of leukemic CD5+ B-cell activation. In contrast to the effects on proliferation and RNA synthesis, IL-10 did not inhibit IgM secretion, and had only a minimal effect on the viability of activated cells. Our results indicate that IL-10 inhibits proliferation of leukemic CD5+ B-cells by a mechanism distinct from induction of apoptosis and support the proposal for the utilisation of IL-10 in the therapy of B-CLL.

Radioimmunolocalization and selective delivery of radiation in a rat model system: comparison of intact and fragmented antibody.

Monoclonal antibody (MoAb) fragments are known to have advantages over intact immunoglobulins for radioimmunoscintigraphy. It is less clear whether they are as effective in the delivery of radioimmunotherapy. The imaging and dosimetric properties of an intact MoAb, K-1-21, reactive against human kappa light chains (LC) were compared with that of its F(ab')2 and Fab fragments using a normal rat model system. Two days after injection of 131I-K-1-21 into rats bearing antigen-sepharose implants, gamma camera images showed specific localization of the MoAb to the target (kappa LC) but not to the control (lambda LC) implant. Better images were obtained with K-1-21 F(ab')2 than with Fab or intact antibody. Mean kappa implant: blood ratios were 8.6 +/- 3.9 for Fab, 7.9 +/- 1.8 for F(ab')2 and 2.0 +/- 0.3 for intact K-1-21. The improvement associated with the use of 131I-K-1-21 fragments was, however, achieved at the expense of lower absolute values of activity at the target site. Thus the absorbed dose delivered to the implant by the intact K-1-21 was double that delivered with F(ab')2 and six times that delivered with Fab. As intact K-1-21 also delivered a greater radiation dose to normal tissues, F(ab')2 fragments may have the greatest overall advantages for therapy with radionuclide MoAb conjugates.

A monoclonal antibody with selectivity for human kappa myeloma and lymphoma cells which has potential as a therapeutic agent.

K-1-21 is a monoclonal antibody which binds to human free kappa light chains and recognises a determinant, KMA selectively expressed on kappa myeloma and lymphoma cells. KMA is absent on plasma cells and resting B cells from normal adults but can be detected on some foetal B cells and a small proportion of activated B cells. Expression of KMA is greatest on cycling cells. K-1-21 is an IgG1 antibody that elicits ADCC but will not cap the KMA determinant unless a second ligand is present. K-1-21 has potential for practical application.

Monoclonal antibody against sheep kappa light chain.

The production of a monoclonal antibody specific for sheep kappa light chain protein is described. The monoclonal antibody was designated McM11 and its specificity was verified using western blots of sheep IgG and slides of efferent lymph cells. The specificity of McM11 was confirmed by specific recognition of fusion proteins expressed by recombinant phage containing sheep kappa cDNAs. N terminal sequence of the light chain recognized by McM11 showed homology to kappa type light chains. McM11, together with McM6, a lambda specific monoclonal antibody, was shown by two color FACS analysis of sheep blood lymphocytes to recognize all sheep light chains.