Polyreactive Antibodies Research Articles

Structure and diversity of the heavy chain VDJ junctions in the developing Mexican axolotl.

The immune capacity of young and adult axolotls (Ambystoma mexicanum) was evaluated by examining the combinatorial and junctional diversity of the VH chain. A large number of VDJ rearrangements isolated from 2.5-, 3.5-, 10-, and 24-month-old animals were sequenced. Six JH segments were identified with the canonical structure of all known vertebrate JHs, including the conserved Trp103-Gly104-X-Gly106 motif. Four core DH-like sequences were used by most (80%) of the VDJ junctions. These G-rich sequences had structures reminiscent of the TCRB DB sequences, and were equally used in their three reading frames. About 25% of the Igh, VDJ junctions from 3.5-month-old axolotls were out of frame, but most rearrangements were in frame at 10 and 24 months, suggesting that there is active selection of the productively rearranged Igh chains in the developing animals. There was no significant difference between the size of CDR3 in young (3.5 months) and subadult (10 months) axolotls (mean: 8.5 amino acids). However, the CDR3 loop was 1 amino acid longer in 2-year-old adult animals (mean: 9.5 residues). Several pairs of identical VDJ/CDR3 sequences were shared between 3.5-month-old individually analyzed axolotls, or between groups of axolotl of different ages. These identical rearrangements might be provided by the selection of some B-cell clones important for species survival, although the probability that different 3.5-month-old axolotl larvae would produce identical junctions seems very low, considering their limited number of B cells (less than 10(5)). The high frequency of tyrosine residues and the paucity of charged residues in the axolotl CDR3 loops may explain the polyreactivity of natural antibodies, and also clarify why it is so difficult to raise specific antibodies against soluble antigens.

Characterization of thyroglobulin-directed and polyreactive catalytic antibodies in autoimmune disease.

Polyreactive and thyroglobulin (Tg)-directed proteolytic activities present in the serum IgG of healthy controls and patients with autoimmune disease were studied by electrophoretic separation of 125I-labeled Tg reaction products and spectrofluorometric measurement of Pro-Phe-Arg-methylcoumarinamide cleavage at the Arg-methylcoumarinamide bond. A decrease of the polyreactive proteolytic activity accompanying an increase of the Tg-cleaving activity in IgG from autoimmune thyroiditis (ATh) and systemic lupus erythematosus (SLE) patients was evident. The Tg, a known target of autoimmune reactions in ATh, was cleaved at lower levels by Abs from patients with this disease than from SLE patients. The Tg-cleaving and Tg-binding activities of the autoantibody preparations were not correlated. Enhanced rates of cleavage at saturating substrate concentrations (Vmax), not increased Tg-binding affinities, were evident in IgG preparations with the greatest Tg-cleaving activity. Similarly, diminution of the polyreactive proteolytic activity in IgG from the autoimmune disease patients was due to decreased Vmax values, not decreased substrate-binding affinities. No cleavage of Tg by IgG from subjects with HIV-1 infection, or from mice hyperimmunized with an albumin-hapten conjugate was evident, suggesting that generation of Tg-cleaving Abs does not accompany V region affinity maturation in response to unrelated Ags. These observations establish Tg as a target of catalytic autoantibodies in SLE and ATh, suggest a transition from polyreactive proteolytic activity to autoantigen-directed activity in autoimmune disease, and open the possibility that combining site chemical reactivity is a factor driving the expression of catalytic activity by autoantibodies.

The epitopes for natural polyreactive antibodies are rich in proline.

"Natural" polyreactive antibodies, which bind in a nonspecific manner to a range of biological molecules both of self- and nonself- origin, are normal constituents of serum and are a significant part of the immune repertoire in many species, including humans. Autoantibodies to sTNF-R (the 55-kDa extracellular domain of the human receptor to tumor necrosis factor alpha) were affinity purified from normal human sera using immobilized sTNF-R. The isolated anti-sTNF-R IgG bound both native and denatured forms of the receptor with low affinity. These antibodies also bound to different proteins and therefore are considered to be polyreactive. We used the anti-sTNF-R antibodies and purified polyreactive antibodies to mannose-specific lectin from garlic (Allium sativum) for screening a peptide library displayed on filamentous M13 phage. After the biopanning procedure, we failed to find epitopes with a consensus sequence; however, we found that proline is the most frequent amino acid in the selected phagotopes. Proline is commonly present at solvent-exposed sites in proteins, such as loops, turns, N-terminal first turn of helix, and random coils. Thus, structures containing proline can serve as conformation-dependent common "public" epitopes for polyreactive natural antibodies. Our findings may be important for understanding polyreactivity in general and for the significance of polyreactive natural antibodies in immunological homeostasis.

Human polyreactive IgM monoclonal antibodies with blocking activity against self-reactive IgG.

Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')2 fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.

Antibody epitopes probed by immunoselected phage-display library peptides in members of a family with various rheumatic manifestations

Objective. The random peptide combinatorial phage library approach overcomes the problem of lack of structural information about the aetiological agent or the antigen responsible for a given disease. Here, we used such a strategy to gain insight into the aetiology of rheumatoid arthritis (RA). Methods. We analyzed the reactivity of serum antibodies from a family with various rheumatic manifestations against RA-immunoselected nanopeptides displayed on phage particles. Results and conclusion. We found that within the same family, there was a difference in antibody reactivity against the peptides tested. The IgG isotype of the peptide reactive antibodies indicated that the observed reactivities were not related to the presence of polyreactive IgM antibodies. Furthermore, it is unlikely that the observed reactivity was due to rheumatoid factors (RF), since two patients who were positive for the immunoselected Pep3 peptide (LSSREPQAR) were RF negative. We also found that the serum of one patient with polyarthralgias also reacted with the same peptide bound by the RA serum, which may suggest the implication of a common aetiological agent in the apparition of this antibody reactivity. Finally, we noted that one patient with Sjogren's syndrome had antibodies to the RA peptide, which may indicate a potential relationship between these two autoimmune diseases.

Effect of temperature on the reactivities of polyreactive and monospecific monoclonal IgG antibodies

In this study, the reactivities of two groups of murine monoclonal antibodies (mAbs) of the IgG isotype were compared by ELISA at various temperatures (Ts) (range: 4-56 degrees C). The first group was constituted of 4 polyreactive mAbs that reacted with various antigens (Ags), such as actin, myosin and tubulin. The second contained 3 commercially available monoreactive mAbs specific to actin, myosin or tubulin. The binding of the monospecific mAbs to their Ags was modified only slightly at the other Ts compared with binding at 37 degrees C. In contrast, the activities of the polyreactive IgGs were considerably modified depending upon the T during incubation on Ag. In a second series of experiments, the effects of the T of the washes and conjugate incubation on the mAb/Ag interaction were evaluated. In these experiments, all steps and incubations were carried out at 37 or 4 degrees C. The values were then compared to those obtained when the washes and conjugate incubation were performed at room temperature. These two protocols generated very little difference in terms of monospecific mAbs. For polyreactive IgG, the values were generally lower when the incubations were carried out at 4 degrees C. However, on the whole, the effect of the T of the washes and conjugate incubation was negligible when the mAb/Ag complex had already formed on the polystyrene plate. Furthermore, at 4 degrees C, 2 of the polyreactive mAbs behaved like monospecific antibodies, while the other 2 remained polyspecific. It can be concluded from these experiments that the reactivities of polyreactive mAbs are more T-sensitive than those of monoreactive mAbs. This seems to indicate that they may possess a more plastic structure and thus may more easily undergo deformation when subjected to non-physiological conditions. In addition, the fact that the polyreactive mAbs showed the same variations for the 3 Ag tested suggests that the same paratope could be involved in all reactions.

Antibody epitopes probed by immunoselected phage-display library peptides in members of a family with various rheumatic manifestations

The random peptide combinatorial phage library approach overcomes the problem of lack of structural information about the aetiological agent or the antigen responsible for a given disease. Here, we used such a strategy to gain insight into the aetiology of rheumatoid arthritis (RA).We analyzed the reactivity of serum antibodies from a family with various rheumatic manifestations against RA-immunoselected nanopeptides displayed on phage particles.We found that within the same family, there was a difference in antibody reactivity against the peptides tested. The IgG isotype of the peptide reactive antibodies indicated that the observed reactivities were not related to the presence of polyreactive IgM antibodies. Furthermore, it is unlikely that the observed reactivity was due to rheumatoid factors (RF), since two patients who were positive for the immunoselected Pep3 peptide (LSSREPQAR) were RF negative. We also found that the serum of one patient with polyarthralgias also reacted with the same peptide bound by the RA serum, which may suggest the implication of a common aetiological agent in the apparition of this antibody reactivity. Finally, we noted that one patient with Sjögren's syndrome had antibodies to the RA peptide, which may indicate a potential relationship between these two autoimmune diseases.

Polyreactive binding of antibodies generated by polyclonal B cell activation. II. Crossreactive and monospecific antibodies can be generated from an identical Ig rearrangement by differential glycosylation.

In this work the authors tested the hypothesis that differential glycosylation may be one of the mechanisms by which B cells could be able to produce monospecific or polyreactive antibodies flexibly. The same genetic information will be used in both situations. To prove this hypothesis the authors used mice transgenic for the SP6 (mu + kappa) hybridoma cell line producing monoclonal antibodies (MoAbs) with specificity for the hapten 2,4,6-trinitrophenyl (TNP). In these animals most cells in the periphery express exclusively the transgene SP6. To obtain polyreactive antibodies (Abs) spleen cells from transgenic animals were stimulated in vitro with lipopolysaccharide (LPS). The authors used LPS derived B cell blasts to produce a hybridoma cell collection. A high proportion of the monoclonal antibodies were found to bind to TNP and to crossreact with unrelated antigens. Endogenous immunoglobulins (Igs) were not responsible for crossreactivity since the crossreactive clones only expressed the transgene products. This was demonstrated by polymerase chain reaction (PCR) amplification of cDNA and by sequencing analysis of the PCR products. The nucleotide sequences of the expressed mono- and crossreactive genes were identical to the sequences of the rearranged V(H) and V(K) SP6 which undoubtedly demonstrates that crossreactive Igs are derived from the same rearrangement and also that no mutations in the V(H) or V(K) or in the CDR3 could account for the observed crossreactivity. It is also shown here that the crossreactive antibodies bear the idiotype Id 20-5 characteristic of SP6 binding Abs. Crossreactive monoclonal antibodies were found to be slightly more glycosylated than the TNP-monospecific Abs. Furthermore, binding to TNP was not influenced by treatment with tunicamycin, an inhibitor of glycosylation, while, in the same molecule, other types of binding were considerably reduced. This supports the hypothesis of the importance of glycosylation in polyreactivity.

Polyreactive binding of antibodies generated by polyclonal B cell activation. I. Polyreactivity could be caused by differential glycosylation of immunoglobulins.

The aim of this study was to gain knowledge of the pre-immune repertoire with reactivity directed to the carbohydrate antigen dextran B512 (Dx). Polyclonal activation of spleen cells in mice has been estimated to be a method of revealing the available repertoire. Hybridoma cell lines derived from lipopolysaccharide (LPS)-stimulated C57BL/6 spleen cells were screened for reactivity with Dx and with the non-related protein bovine serum albumin (BSA). Despite the lack of structural similarity between Dx and BSA we observed that nearly all of the Dx positive monoclonal antibodies (MoAbs) cross-reacted with BSA. The Dx and BSA crossreactive MoAbs were also found to bind to several foreign-and auto-antigens, and therefore we concluded that these MoAbs fulfilled the criteria of polyreactivity. The Dx and BSA cross-reactive antibodies were produced in an apparently random fashion as judged by the use of kappa and lambda light chains and the use of V(H) J558 subfamily genes. There are two possible explanations for this type of polyreactivity: (1) LPS induces a randomly generated polyreactive and a randomly generated specific immunoglobulin (Ig) repertoire; (2) polyreactivity can be the result of post-translational modifications. Since immunoglobulins are glycoproteins we considered the possibility that post-translational modifications such as glycosylation could be responsible for the generation of the polyreactive pool. Dextran-specific and Dx/BSA cross-reactive MoAbs showed different degrees of sensitivity to inhibition of glycosylation performed by treatment with tunicamycin (Tm), an inhibitor of the formation of N-glycosidic linkages. Other polyreactive, connective and specific MoAbs were also tested. The authors found that Tm treatment had a more profound effect in reducing the binding capacity of the polyreactive antibodies (Abs), suggesting that the polyreactive Abs may be more dependent than the specific Abs on the carbohydrate content of the molecule for binding to the antigens. The authors propose that lymphocytes may use differential glycosylation as a means to generate polyreactive or monospecific Abs.

Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7-.

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or beta-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7- cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.

Characterisation and clinicopathological correlates of serum anti-p53 antibodies in breast and colon cancer

The humoral immune response to p53 was determined in 54 individuals with colon or breast cancer and 50 healthy subjects, in an attempt to better understand the origin and significance of anti-p53 serum antibodies. The presence of anti-p53 antibodies in serum was determined by enzyme-linked immunosorbent assay using purified recombinant human p53, and results were validated by immunoprecipitation of radiolabelled p53. Immunohistochemical analysis of 28 tumours was performed to detect the accumulation of p53 protein. Antibodies against p53 were significantly more common in patients with colorectal (10 of 42) and breast (2 of 12) cancer than in healthy individuals (2 of 50). They were of both the IgM (7 of 11) and IgG (4 of 11) isotypes. There was no significant difference in prevalence of serum antibodies against p53 with respect to the p53 immunohistochemical status of the tumour or to other pathological features, including the presence of lymph node and distant metastases. These findings provide indirect evidence that, rather than arising as a result of a specific immune response, anti-p53 antibodies in individuals with cancer may represent elevated levels of naturally occurring polyreactive antibodies.

Technical problems arising from the use of the immunoblot for determination of the reactivity of natural antibodies with different lipopolysaccharides (LPS)

Natural polyreactive antibodies (NPAB) appear to play an important role in the first-line defence against invading bacteria. The major constituent of the outer membrane of Gram-negative bacteria is the lipopolysaccharide (LPS). Therefore, reactivity against this structure could be of importance in protecting the organism from the harmful effects of LPS. Immunoblotting has become a common method to verify the specificity of antigen antibody interactions. Various immunoblot techniques for testing the reactivity of monoclonal antibodies with LPS have been published using nitrocellulose and detergent-free blocking buffer systems. These methods are not suitable for the investigation of NPABs due to the broad reactivity and a high background staining which gives rise to interpretational difficulties. In the present study we demonstrate an immunoblot technique using polyvinylidene difluoride (PVDF) membranes and a detergent-containing buffer system which permits to detect LPS reactivity of NPABs. The polyreactive monoclonal human antibody CB03 used was screened for lipid A LPS reactivity in ELISA experiments. The binding was confirmed in the described blot system and depends on the membranes and blocking agents used. The use of nitrocellulose versus PVDF was also tested for monospecific anti-LPS antibodies and the latter can be recommended due to the production of stronger reaction patterns without any background staining.

Polyreactivity of human monoclonal antibodies: human IgM anti-rhesus monoclonal antibodies are frequently polyreactive.

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.

Proteolytic Antibodies

AbstractCatalytic activities are known to arise naturally in antibodies. Several naturally‐occurring peptides, synthetic protease substrates, DNA, and esters are known to be cleaved by antibodies. There is increased production of antigen‐specific catalytic antibodies in autoimmune disease. Polyreactive catalytic antibodies are present in unimmunized donors. Antibody light chains isolated from multiple myeloma patients frequently express proteolytic activity. Immunization protocols using as immunogens the ground state of a naturally‐occurring polypeptide, anti‐enzyme antibodies, or the enzyme itself are known to provoke catalytic antibody synthesis. Active site residues in the light chain subunit serve as the catalytic residues in a model antibody with peptide bond cleaving activity. A split‐site model in which distinct amino acids serve as the essential catalytic residues and substrate ground‐state recognition residues has been developed from mutagenesis studies. Engineering of the available antibodies could potentially generate improved catalysts. The possible mechanisms underlying proteolysis by natural antibodies and evolution of the catalytic activity are reviewed.

Structure and function of natural antibodies.

Natural antibodies arise independently of known antigenic stimulation, are mostly IGM, polyreactive, and are generally encoded by V genes in germline configuration. Polyreactive IgM natural antibodies are produced by mainly B-1 cells which account for most of the B cell repertoire in the fetus and neonate, and possibly play a major role in the development and physiology of the human B cell repertoire. Although endowed with self-reactivity, natural antibodies also bind exogenous antigens [73, 74]. Exposure to environmental antigens is not necessary for the emergence of natural antibody-producing cell precursor clones to exogenous antigens, as suggested by the significant population of B cells capable of producing antibodies to a variety of bacterial antigens in germ-free animals. Because of their ability to bind a variety of exogenous antigens, including those on bacteria and viruses, natural antibodies play a major role in the primary line of defense against infections. A central issue related to the understanding of the physiopathologic roles of natural antibodies is whether precursors of cells producing natural antibodies, B-1a and B-1b lymphocytes, are capable of undergoing an antigen-driven clonal selection process, thereby producing autoantibodies with a high affinity for the selecting antigen. In this respect, we have clearly established that B-1 cells can express a hypermutation mechanism similar to that of conventional (B-2) cells. Furthermore, we have shown by gene shuffling, site-directed mutagenesis, and in vitro human Ig gene expression, that the main structural correlate for antibody polyreactivity is provided by the somatically generated H chain CDR3. We have also shown that this Ig V region provides the main structural correlate for antigen-binding in monoreactive antigen-induced autoantibodies. These findings in the human are at the basis of our proposed structure-function model in which the antigen binding features of the germline template antibody are dictated by the somatically generated H chain CDR3, and perhaps, but at a lower degree, L chain CDR3; the point-mutation changes underlying the antigen-driven affinity maturation process would impact mainly the Ig V gene encoded segments. This structure-function model is being tested in our laboratory by analyzing the antigen binding activity of somatically mutated polyreactive autoantibodies that have been structurally reverted to their original putative unmutated configuration. Precise identification of the Ig gene and/or somatic recombination products mediating recruitment of unmutated B cell clonotypes, as well as those that are preferentially modified by an antigen-dependent selection process, should further our understanding of the mechanisms that shape the B cell repertoire in development and disease.

Genetic control of the humoral immune response to xenografts. II. Monoclonal antibodies that cause rejection of heart xenografts are encoded by germline immunoglobulin genes.

The early phases of the rejection of xenografts exchanged between closely related species are dominated by a vigorous humoral immune response. We have recently used a linker-mediated polymerase chain reaction (LM-PCR) to generate Ig heavy and light chain-specific cDNA libraries to examine the Ig gene control of a prototypic IgM monoclonal antibody, HAR-1, that causes the hyperacute rejection of hamster xenografts. Recombinant clones from the library were screened directly from bacterial colonies by PCR and the nucleic acid sequences of the clones established. Our results demonstrate that the HAR-1 hybridoma is encoded by Ig VH and JH genes in a germline configuration. Comparison of the cDNA sequence for HAR-1 VH with the germline equivalent of this gene isolated from newborn LEW liver (provisionally designated VHHAR-1) showed that the two VH sequences share a nucleic acid identity of 99.3%. Similarly, the HAR-1 monoclonal uses a Ig JH gene that is 98.2% identical with the JH1 nucleic acid sequence available in the GeneBank. The use of Ig VH and JH genes in a germline configuration is similar to that seen with polyreactive natural antibodies to infectious agents and autoantibodies. These humoral responses are thought to be the result of the stimulation of a T cell-independent subset of B cells, the B-1a/B-1b subset, that is responsible for producing antibodies that serve as a primitive humoral (natural antibody) defense mechanism against infectious diseases. Our results suggest that the humoral component of the rejection of xenografts in the hamster-to-rat model may represent the stimulation of this type of B cell antibody response by xenogeneic target antigens that share antigenic epitopes with bacteria and other infectious agents.

Peritoneal cavity CD5 (Bla) B cells: cytokine induced IgA secretion and homing to intestinal lamina propria in SCID mice.

The mouse peritoneal cavity contains a unique population of B cells (Bla) with a high IgM/low IgD ratio, CD5+ (Ly1), MAC-1 + phenotype. These cells arise early in ontogeny, utilize a limited repertoire of immunoglobulin V genes, produce polyreactive IgM antibodies and have been implicated as the source of many auto-reactive immunoglobulins. Recent data from chimeric mice suggest that this B cell population also contains the precursors of many IgA plasma cells found in the lamina propria of the small intestine. In the present study we have investigated the potential of this cell population to secrete IgA (and IgG) in response to various cytokines. IL-5 alone, or in combination with IL-2, greatly enhanced secretion of both IgG and IgA. Cytokine-induced IgA secretion resulted from expansion of a subset of CD5 B cells co-expressing sIgA. Adoptive transfer of CD5 B cells while peripheral lymph nodes contained only IgM+ and some IgG+ B cells. Transfer of CD5+ B cells also reconstituted serum IgM, IgG and IgA and IgG, immunoglobulins characteristic of mucosal and anamnestic responses, when cultured in vitro with the appropriate cytokines. These cells also give rise to IgA plasma cells in the intestinal lamina propria following adoptive transfer to SCID mice, further supporting the hypothesis that cells of this lineage may be important in immune responses at mucosal surfaces.

Cell cluster formation during up-scaling of a human-mouse heterohybridoma producing a polyspecific human IgM antibody.

Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced. A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system. The process of fermentation has been carried out continuously over a period of 4 months. In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased. Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody. During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable. Furthermore, we detected formation of cell clusters in the fermentor system. These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies. Clusters were found to represent fusions of hybridoma cells using electron microscopy. Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas. We discuss these results in the content of the polyreactive binding properties of this particular antibody.

Unexpected presence of polyreactive catalytic antibodies in IgG from unimmunized donors and decreased levels in rheumatoid arthritis.

Polyclonal IgG from healthy humans and unimmunized mice was screened for peptide-methylcoumarinamide (peptide-MCA) hydrolyzing activity. The activity was detected in every IgG sample examined. Contaminant enzymes were precluded as an explanation, as the activity tracked exactly with the 150-kDa IgG peak separated by gel filtration in denaturing solvent (6 M guanidine hydrochloride) and with the 50-kDa Fab fragment peak produced by papain-digestion of the IgG. Patients with rheumatoid arthritis displayed a 3.2-fold reduced peptide-MCA hydrolyzing activity (mean) compared to healthy subjects. Control osteoarthritis patients showed no diminution in activity. A progressive decrease in the activity by 7.4-fold of the pre-immune levels was observed in mice over the course of hyperimmunization with SRBC, indicating that exogenous Ag challenge, like rheumatoid arthritis, is associated with decreased catalytic activity. Apparent Km values of the IgG for Pro-Phe-Arg-MCA were 0.39 to 0.53 mM, values approximately 3-orders of magnitude greater than observed previously for Ag-specific catalysis by Abs. The only common structural feature in peptide-MCA conjugates utilized by the Abs as substrates was the presence of Arg-MCA and Lys-MCA bonds. The IgG hydrolyzed Pro-Phe-Arg-Phe at the Arg-Phe peptide bond, showing that the activity is relevant to cleavage of peptide bonds in natural Ags. The universal occurrence of this polyreactive catalytic activity in unimmunized donors and its diminution in an autoimmune disease and nonspecific Ag challenge suggest that it may possess an important, but as yet unidentified, biologic role.

Structure of the VH‐D‐JH Segments of Human Natural Polyreactive IgM and IgG Antibodiesa

Structure of the V_H‐D‐J_H Segments of Human Natural Polyreactive IgM and IgG Antibodies^a