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Background: This study was conducted to evaluate the significance of serum level of immunoglobulins (Igs) and particularly IgG for leukemic cell persistence in peripheral blood (PB) and prognosis for childhood acute lymphoblastic leukemia (ALL)., Procedure: Human sera were obtained from 68 children with primary B-lineage ALL at diagnosis and 46 healthy children (control). Serum level of IgM, IgG, IgA, IgG1, IgG2, IgG3, IgG4, antitumor antibody, homogeneous IgG were quantified by turbidimetric or enzyme-linked immunosorbent assays., Results: The mean values of serum IgM, IgG, IgA at diagnosis were not differed significantly in ALL patients and control children. The level of IgM and IgG1 inversely correlated with white blood cell (WBC) count in PB of patients. Normal range of serum IgG, separated by 25th and 75th percentiles of IgG variables, was associated in patients with decreased WBC count in PB but not in bone marrow (BM) versus patients with low concentration of IgG. Normal range of IgG also favors low frequency of homogeneous IgG and antitumor antibodies. Patients with high level of IgG, besides increased frequency of homogeneous IgG and antitumor antibodies, had worse 3-year overall survival (OS) rate as compared to patients with normal level of IgG (58.8 vs. 91.2%, P = 0.014)., Conclusions: The normal level of serum IgG at diagnosis is a beneficial prognostic factor associated with lower rate of leukemic cell persistence in PB and better outcome of childhood B-lineage ALL., (Copyright 2004 Wiley-Liss, Inc.)

Two IgG2a monoclonal antibodies (G10 and F11) are described which have similar affinity for human spleen ferritin and identical protein A-binding affinity. The two mAbs display changes in protein A-binding affinity following binding of the antigen to its specific recognition site in the variable domains. However, while antigen-induced conformational changes in G10 enhance its affinity to protein A, interaction of F11 with ferritin results in a significant decrease in protein A-binding affinity. In contrast to the IgG2a antibodies, using a mouse IgG1 antiferritin antibody (C5) high-affinity binding of the antigen does not change an inherently low ability to bind protein A. Differential scanning calorimetry revealed that the enthalpy and Gibb's free energy of thermal unfolding for G10 was 19% and 23% higher, respectively, than the corresponding parameters for F11. The lower structural energetics of F11 are associated with the absence of a calorimetrically revealed folding unit, which may be responsible for interactions between the antigen-binding site and the protein A-binding site. This study provides the first demonstration that functionally significant interactions between two recognition sites in antibodies of the same subclass can be modulated by subclass-independent structural variations associated with different thermodynamic stability.

The two anti-ferritin monoclonal antibodies of mouse IgG2a subclass, G10 and F11, are described that have similar affinity to human spleen ferritin and identical protein A-binding affinity. Antigen binding was shown to change significantly the protein A-binding parameters of the IgG2a antibodies. Antigen-induced conformational changes result in enhanced protein A-binding affinity of the G10 antibody while reduced affinity of the F11 antibody. Antigen binding does not change inherently low affinity of the anti-ferritin IgG1 antibody C5 to protein A. Differential scanning calorimetry revealed that the enthalpy and Gibbs free energy of denaturation for G10 was respectively by 19 and 29% higher than the corresponding parameters for F11. The lower structural energetics of F11 is associated with the lack of a calorimetrically revealed folding unit that may be responsible for distinct interaction between the antigen-binding and protein A-binding sites. This work provides experimental demonstration of the fact that functionally significant interactions between the two spatially remote recognition sites in antibodies of the same heavy chain isotype can be modulated by relatively small structural variations that also result in different thermodynamic stability.

Metalloporphyrin-antibody conjugates provide a significant advantage over other types of conjugates in biomedical use for phosphorescence immunoassay, targeted immunotherapy, and internal imaging of malignant tumors. Monoclonal HSF102 antibody of mouse IgG2a subclass and monospecific rabbit IgG, both antibodies directed to human spleen ferritin, were modified with the new reagent Pd(II) coproporphyrin I tetra-N-hydroxysuccinimide ester. Functional study of the conjugates obtained revealed a 7- and 1.4-fold increase in the antigen binding activity for monoclonal HSF102 and monospecific IgG antibodies, respectively. For rabbit monospecific IgG, a concomitant increase in binding to anti-rabbit IgG antibodies directed to the epitopes of the CH2 domain in the Fc fragment was observed. In all cases, the maximum functional activation was found after conjugating one mole porphyrin per mole antibody. These results suggest that functional activation of the conjugates might be due to an increase in conformational flexibility of an antibody molecule after the modification. This increase in flexibility involves the Fab fragments and a pair of the CH2 domains in the Fc fragment and might be due to a significant charge shift (minus 5 charge units per modified amino group) that occurs after conjugation of an antibody with tetracarboxylic porphyrin.

By changing pH within the pH range 2-7, the processes of intramolecular rearrangement in the multidomain structure of rabbit IgG were induced which resulted in the formation of four IgG conformers - N, NA, I and NI, differing in the amount of structure in the CH2 domain and as well as in its interaction with the neighboring domains. The stability of the IgG conformers was studied by differential scanning calorimetry and expressed in terms of thermodynamic parameters, delta H, Tm, and delta G. The pH-dependent changes in thermal stability of IgG and the range of stability for the conformers were described by conformational (phase) diagrams. The native N-conformer exists at pH 7.0-5.5. At pH < 5.5 the formation of the NA conformer showing significant decooperation (lower extent of interaction) of the domains occurs. Decooperation permits the CH2 domain in the NA conformer to undergo conformational transitions independently of the rest of the molecule-the property which cannot be observed for the more cooperative N-conformer of IgG. The formation of the intermediate I-state occurs at pH < 3 as a consequence of unfolding of the tertiary structure of the CH2 domain, while its secondary structure and compact tertiary structure of the remainder of the IgG domains remain unchanged. Such partially structured and stable states of immunoglobulins have not yet been described. Reversibility of pH-induced transitions in IgG was studied using calorimetry and ligand-binding assays involving the C1q component of the complement, protein A, antigen and monospecific anti-CH2 domain antibodies as conformational probes reporting local conformational changes in the CH2 domain and its interdomain interfaces. Refolding of IgG from the I-state by increasing pH results in the formation of a functionally active N1 conformer differing from native IgG by a changed balance of interdomain interactions. In addition to previously reported data suggesting a greater extent of the CH2 and CH1 domain interactions in the NI conformer, this conformer was shown to possess a lower extent of interaction of the CH2 and CH3 domains. The changed stability and a balance of domain interactions in the NI conformer results in changes in some of the effector functions including enhanced two-site binding to protein A and antigen-dependent binding to C1q with a concomitant decrease in the affinity of protein A binding site and reduced antigen binding capacity of the high affinity IgG. Low affinity IgG does not change the antigen-binding parameters after refolding to the N1 conformer.

Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra-N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the CH2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1-40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.

At pH2, rabbit IgG adopts a partially structured state that exhibits loss of thermal unfolding transition, tentatively assigned to the CH2 domain, whilst retaining a well-defined tertiary structure for the rest of the molecule and extensive secondary structure. Renaturation of IgG from this state yields a stable conformer that differs from native IgG by a lower degree of interaction between the CH2 and CH3 domains, and stronger interaction between the CH1 and CH2 domains, as judged by differential scanning calorimetry and probing the IgG conformation with specific ligands (C1q component of complement, protein A and monospecific antibodies to the CH2 domain and hinge region.

Rabbit IgG after exposure to 0.05 M glycine-HCl, pH 2.0, and native IgG were compared by differential scanning calorimetry (DSC) at pH 3.5 and C1q binding studies at pH 7.8. For acid-treated IgG, a large increase (by approx. 12-15 degrees C) in thermal stability of the CH2 domain occurs and this domain no longer demonstrates a separate and thermodynamically independent unfolding at 56 degrees C seen for native IgG. The results suggest that stabilization of the CH2 domain in acid-treated IgG arises from stronger, relative to the native protein, interaction of the CH2 domain with adjacent and more stable IgG domain(s). Conformational differences of the two forms of IgG were confirmed at neutral pH by a 4-fold increase of C1q-binding affinity of acid-treated IgG.

Thermal stability and functional activity of rabbit IgG in its native conformation and after incubation at pH 2.0 were studied using differential scanning calorimetry and binding of conformational probes, i.e., the C1q component of the complement and two monospecific anti-IgG antibodies. The latter reacted selectively with the "hinge" region joining the Fab and Fc fragments of IgG or with the CH2 domain in the Fc fragment. At pH 2.0 complete unfolding of rabbit IgG did not occur: the protein demonstrated the presence of secondary and compact tertiary structures but differed from the native conformation by decreased overall enthalpy and TM of thermal denaturation as well as by changed secondary structure parameters as could be evidenced from CD spectroscopy and scanning calorimetry data. Incubation at pH 2.0 followed by renaturation at neutral pH led to irreversible conformational changes in IgG. The most significant differences between the two IgG conformers were demonstrated by calorimetry at pH 3.5 which revealed that the acid-treated conformer differs from the native one by enhanced thermal stability of the CH2 domain. Using the combination of thermodynamic and functional studies, it was shown that the origin of stabilization was the increase in the extent of interaction between the CH2 domain in the Fc fragment and the CHI domain in the Fab fragment. This resulted in the increase of the functional link between the antigen-binding domain and the C1q binding site in the CH2 domain of the acid-induced IgG conformer. In parallel with the increase in stability of the CH2 domain, conformational changes in the "hinge" region were found, together with the absence of intrinsic conformational changes in the CH2 domain proper as could be judged from C1q and monospecific anti-IgG binding assays. The results obtained demonstrate one of possible mechanisms whereby functionally significant rearrangements in the IgG molecule can be induced by changes in the interactions between invariably folded domains rather than by intrinsic changes in the domain conformation.

To localize essential epitopes of rabbit IgG, a series of proteolytic IgG fragments obtained by papain (Fab, Fc) or pepsin (pFc', F(ab')2) proteolysis have been prepared and their interaction with sheep antibodies against rabbit IgG has been studied. The data obtained suggest that essential immunoreactive epitopes of rabbit IgG are located in the CH2 domain and hinge region. This finding is in line with the results obtained by computing the antigenic sites of immunoglobulins. However, the deviation from the computed antigenic structure was deduced from the complete lack of immunoreactivity of the pFc fragment, it being a dimer of the terminal CH3 domain of the Fc fragment. The hinge region comparable in size with the dimensions of the epitope reveals high affinity binding to anti-IgG, thus testifying to the localization of the expressed epitope or its essential part in the hinge region. Proteolytic cleavage of this region leads to a significant decrease in the binding of the IgG fragment to anti-IgG. In addition to the CH2 domain and hinge region, a relatively low interaction of the antigen-binding antibody fragments with anti-IgG was found.