Primary Response Antibodies Research Articles

The synergistic effects of the constant region and variable heavy chain families of multimeric immunoglobulin M on its interaction with Fc-mu receptor and antigen

Aim: As the primary response antibody with increasing use as a therapeutic immunoglobulin (Ig) format, IgM is also the largest antibody structure among the five major human isotypes. Spontaneously formed pentamers and hexamers of IgM have avidity effects that could compensate for weaker interactions in monomeric Igs. However, this advantage is counterbalanced by potential steric clashes when binding to multiple large antigens. Recent findings have challenged the expected canonical independence of Fc receptor (FcR) binding at the heavy chain constant (C)-region where the heavy chain C-region isotypes affected antigen binding at the variable (V)-regions, and the variable heavy (VH) families of the V-region affected FcR engagement at the antibody C-regions. With such effects found on other Ig isotypes, IgM candidates need to be investigated with regards to such effects, especially when considering its natural oligomerisation at the C-region that can amplify or modulate such allosteric effects. Methods: Through a panel of 14 recombinant complementarity determining regions (CDRs)-grafted trastuzumab and pertuzumab VH1-7 IgMs subjected to bio-layer interferometry measurements, the interactions with the antigen human epidermal growth factor receptor 2 (Her2), Fc-mu receptor (FcµR), and superantigen Protein L (PpL) were investigated. Results: Significant effects from the V-regions to mitigate FcµR binding and the IgM C-region bidirectional effect modulating Her2 antigen engagements at the V-regions were found. Additional modulatory effects from superantigen PpL binding on the V-region of the kappa chain (Vκ) mitigating antigen binding were also found, revealing possible novel mechanisms of antibody superantigens that can be moderated by the antibody VH frameworks.

Expression, characterization and crystallization of the Fv fragment of mouse antibody 3B62 from the secondary immune response.

Affinity of antibodies increases in the course of the immune response. Mouse anti-nitrophenol antibody 3B62 from the secondary immune response shows higher affinity than the primary-response antibodies. An expression system for the 3B62 Fv fragment was constructed by introducing coding regions for the V(L) and V(H) into the genome of the methylotrophic yeast Pichia pastoris. Each of the coding regions was placed downstream of the coding region for the secretion signal of the yeast alpha-factor. The alpha-factor signals were cleaved off from the expressed proteins and the Fv was secreted as a heterodimer consisting of the V(L) and V(H) domains. The binding constant of the expressed Fv against the (4-hydroxy-5-iodo-3-nitrophenyl)acetate ligand was comparable to that of the Fab fragment. Crystals of the Fv were obtained in the presence of the ligand and diffracted X-rays to 1.8 A resolution. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 46.48 (9), b = 34.99 (4), c = 77.76 (17) A, beta = 101.47 (14) degrees, and contain one Fv molecule per asymmetric unit.

Production of Antibodies to Dextran Using Liposome as Adjuvant

The kinetics and nature of the immune response to dextran-BSA entrapped into liposomes was studied in rabbit. The antibody titer in the secondary immunization was found to be higher as compared to that in the primary response. In the primary response antibodies were of both IgG and IgM type but following secondary immunization the IgG level was increased. The conjugate entrapped into liposomes could establish an immunological memory for dextran. In order to eliminate the carrier effect of BSA, dextran was either entrapped or coupled to liposome and the immune response was studied in mice. The results revealed that dextran entrapped into liposomes or coupled to liposome surface induced IgM type immune response and antibody titer did not increase on secondary immunization.

The amino acid residues at the VH-D-JH junctions affect the affinity of anti-p-azophenylarsonate antibodies.

Murine A/J anti-p-azophenylarsonate (Ars) antibodies sharing a predominant idiotype are encoded by a single combination of germ-line V region gene segments. The dominance of this idiotype among secondary immune response anti-Ars antibodies has been explained by the Ag-driven selection of favorable somatic mutants of this gene segment combination, associated with an intrinsic Ars-affinity of the germ-line V region higher than that of other possible combinations. To determine the effect of junctional diversity upon affinity for Ag, independently of somatic mutation, we determined the V region sequences and affinity for Ars of five primary response antibodies. These antibodies share identical unmutated V regions but differ only at the D gene junctions. Among the five antibodies, Ars-affinity differed up to 10-fold depending upon the identity of the amino acid residues at the VH-D and the D-JH junctions. The combination of junctional residues observed in two primary response antibodies with relatively low Ars-affinity has not been observed among secondary response antibodies. Thus the identity of junctional residues resulting from gene rearrangement prior to antigen stimulation must be taken into account in hypotheses which account for idiotype dominance by selection on the basis of affinity.

Antibody Responses to Human Erythrocytes and Ovalbumin as Marker Traits of Disease Resistance in Dairy Calves

Microhemagglutination procedures were used to quantify antibody titers against human erythrocytes and ovalbumin in calf sera. Calves were also monitored for the prevalence of pneumonia and diarrhea. Calves, 72 males and 82 females, were the progeny of 15 AI bulls. Blood was sampled weekly for 2 wk after primary and secondary immunizations. Antibody response peaks to both antigens occurred by 14 and 7 d postimmunization, respectively. There were significant effects of season and birth and location of rearing on antibody titers against both test antigens. Diarrhea prevalence was negatively associated with high primary response antibody titers against human erythrocytes, but no trends were observed for pneumonia prevalence and for antibody titers to ovalbumin. Paternal half-sib heritability estimates ranged from 0 to .40±.32 for primary antibody responses and from 0 to .87±.50 for secondary antibody responses, depending on antibody specificity, and those for average titer were higher for antiovalbumin antibody (h2 = .48±.39) than for antihuman erythrocyte antibody (h2 = .31±.21). Although the environmental component of the humoral immune response is substantial, heritabilities of the magnitude in this study suggest the feasibility for successful genetic manipulation of antibody response profiles of young calves, and these may contribute to enhanced disease resistance.

Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP.

The nucleotide sequences of the variable regions of lambda 1 chain bearing anti-NP antibodies from the secondary response of C57BL/6 mice were determined. The data indicate that the V186.2 VH gene which dominates the primary anti-NP response is expressed in nine out of 10 secondary response antibodies and is extensively mutated. In the V lambda 1 regions somatic mutations are less frequent. While point mutations predominate, there is suggestive evidence for two conversion events, one involving a one-codon deletion. Most, but not all, secondary response antibodies have a higher affinity (up to 10-fold) for the hapten than is seen in the primary response. The increase in affinity correlates with 'parallel' mutations in CDRs of H and L chains, likely to play a role in hapten binding. The analysis of VDJH rearrangements demonstrates that the secondary response lambda 1 chain-bearing antibodies are produced by a diverse set of B cell clones, which are only rarely expressed in primary responses. These clones are characterized by N-sequence-mediated heterogeneity in the 3' half of CDR3, where the germ line sequence of the D element DFl16.1 predominates in primary response antibodies. The antibodies analyzed in this and in previous work were isolated from idiotypically suppressed mice in order to evaluate whether, intraclonally, idiotype suppression selects antibody mutants into the memory pool, through suppression of the wild-type. A selection of this type was not detectable. However, idiotype suppression may control the pattern of clonotypes expressed in the primary versus the secondary response.

IgG and IgM antibodies in normal and leukaemic cattle

Normal cattle were immunized with a native protein, human serum albumin (HSA), and a multichain synthetic polypeptide, poly(Tyr, Glu)-poly( dlAla)-poly(Lys), (T,G)-A—L. The synthetic antigen provoked precipitable antibody formation when administered in complete Freund's adjuvant but not when injected in incomplete adjuvant. Intradermal and subcutaneous injections resulted in a similar pattern of antibody production. HSA was tested only in complete Freund's adjuvant. The temporal pattern of the immune response was similar in all cattle to both antigens. In the primary response antibodies of IgG and IgM classes could be detected. In anti-(T,G)-A—L sera there was a relatively high percentage of IgM antibodies after primary immunization. After secondary immunization the antibodies were elicited for a longer period, and were exclusively of the IgG class. Leukaemic cattle were injected with the same antigens. In each responding cow antibody appearance occurred late, was low, and the antibodies produced were mainly of the IgG class. Even though all the leukaemic cattle possessed IgM in their sera, they produced very little IgM class antibody upon antigenic stimulation, compared with normal cattle. A synthetic antigen, (T,G)-A—L, which is capable of eliciting antibodies of the IgM class in almost equal amounts to those of the IgG class in normal cattle, was not capable of causing the production of more than traces of IgM antibodies. It seems, therefore, that the mechanism of IgM synthesis must be significantly impaired in leukaemic cattle in addition to a general depression of the immune response.

γ-Globulin and Antibody Formation in Vitro

Summary The spleens of rabbits, injected either with 10 mg of BGG intravenously or with a similar dose of BGG simultaneously with 10 µg of endotoxin, were examined histologically at various days after injection. “Red” and “white” pulp of these spleens were cultured separately to study their ability to form antibody in vitro. It was found that an increased number of hemocytoblasts occurred in the white pulp starting 1 day after the antigen injection. Antibody formation in vitro was demonstrated at the time of immature plasma cell proliferation (day 4 to 7), and was much more pronounced in the “red” than in the “white” pulp. Histologically, most of the immature and mature plasma cells were found on the border of red and white pulp. Endotoxin enhanced the plasmacellular reaction, the serum antibody titers, and the formation of secondary nodules arising in the white pulp between days 5 and 13 after antigen. Since only slight production of antibodies was observed in the white pulp of some rabbits in the later phase of the response, the effect of endotoxin on the primary antibody response seemed mediated rather through its effect on plasma cell proliferation than through stimulation of secondary nodule formation. Most of the serum antibody formed by the rabbits in this study was destroyed by treatment with mercaptoethanol, indicating that the early primary response antibody belongs to the 19 S fraction of γ-globulin.