Conjugates of superoxide dismutase with the Fc fragment of immunoglobulin G.

We constructed conjugates of superoxide dismutase (SOD) and the Fc fragment of human immunoglobulin G. The lysyl residues of bovine erythrocyte Cu,Zn-SOD were covalently linked with cysteine residues of the Fc fragment using N-succinimidyl 4-(N-maleimido)-butylate as a crosslinking agent. Analysis by gel filtration and SDS-PAGE revealed that the conjugates were composed of one molecule of SOD linked with one molecule of Fc [SOD-(Fc)1] and one SOD molecule linked with several Fc molecule [SOD-(Fc)n]. The resulting SOD-Fc conjugates retained more than 90% of the enzyme activity of SOD. When those conjugates were administered intravenously to mice, the half-lives of SOD activity in the circulation were 29 and 42 h for SOD-(Fc)1 and SOD-(Fc)n, respectively, while free SOD had a half-life of 5 min. Intravenous administration of the conjugates to mice markedly repressed the increase in serum glutamic-oxaloacetic transaminase (GOT) activity induced by paraquat. These results suggest that SOD-Fc conjugates, which have long half-lives, effectively perform dismutation of superoxide radicals and may be useful for preventing tissue injury caused by hazardous oxygen metabolites.     

Human B-cell differentiation by Fc fragment of IgG. I. Fc fragment from human IgG induces plasma cell generation but cannot induce lymphocyte proliferation

Circulating mononuclear cells (MNC) from normal donors were examined for lymphocyte proliferation and plasma cell differentiation following stimulation by Fc and Fab fragments or by intact IgG. Lymphocyte differentiation and DNA synthesis were examined as a function of culture duration and concentration of Fc, Fab fragments, and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin and with specific antisera (anti-mu, -gamma, -alpha, -delta, -kappa, and -lambda chains). DNA synthesis of mononuclear cells cultures was analyzed by measuring [3H]thymidine incorporation. The results indicated that only the Fc fragments are able to induce the differentiation of B cells. The polyclonal plasma cell response to Fc fragments was dose dependent, peaked on the sixth day of culture, and was isotypically diverse (IgM greater than IgA greater than IgG). This activity requires the presence of T helper cells and monocytes. In contrast, the Fc fragments were unable to induce a proliferative response.     

Enzymic degradation of the Fc fragment of rabbit immunoglobulin IgG

The digestion of the Fc fragment of rabbit immunoglobulin IgG by several proteolytic enzymes was investigated by using gel filtration and starch-gel electrophoresis in 8m-urea-formate as criteria of the extent of degradation. Though fragment Fc and mildly reduced fragment Fc proved resistant to tryptic hydrolysis, papain and pepsin cleaved the fragment at acidic pH values and appeared to give rise to a similar spectrum of products. A (limit) peptide comprising the C-terminal 113 residues of the heavy chain was isolated and identified from the pepsin-digest products of fragment Fc. The products of proteolytic digestion of fragment Fc were no longer able to inhibit passive cutaneous anaphylaxis by rabbit anti-(bovine serum albumin) or demonstrate reversed passive cutaneous anaphylaxis in the guinea pig. Nor were they able to inhibit the intestinal absorption of heterologous immunoglobulin IgG in the young mouse. These studies imply that the site or sites responsible for these biological properties of intact fragment Fc reside in the N-terminal 30-40% of the fragment.     

Subfragments from the Fc fragment of human immunoglobulin G. Isolation and physicochemical characterization

A fragment termed fragment Fc' and a related fragment termed fragment pFc' produced by the actions of papain and pepsin respectively on human immunoglobulin G have been isolated and characterized. Amino acid analyses and experiments utilizing cyanogen bromide to cleave the methionyl bonds of the Fc' and pFc' fragments make it possible to locate both fragments within the known chain structure of the immunoglobulin G molecule. The pFc' fragment is probably a non-covalently linked dimer situated at the C-terminal end of the molecule, containing about 232 amino acid residues and having a molecular weight of 26000. The Fc' fragment is a similar dimer of about 182 residues extending from near residue 14 to near residue 105 (numbered from the C-terminal end) of the gamma-chain and has a molecular weight of 21000.     

Intrachain disulphide bridges in immunoglobulin G heavy chains. The Fc fragment

The disulphide bridges of the Fc fragment (C-terminal half of the heavy chain) have been studied in several human immunoglobulins, containing heavy chains of different antigenic types (gamma1, gamma2, gamma3 and gamma4), and in heavy-chain-disease proteins. Two intrachain disulphide bridges were found to be present. The sequences appear to be identical in the Fc fragments of two types of chain studied (gamma1 and gamma3), and very similar to corresponding sequences of the Fc fragment in rabbit. These results suggest that the C-terminal half of the heavy chains is covalently folded (in a similar fashion to the light chains) with a C-terminal loop and an N-terminal loop. The similarity is emphasized by comparison of the sequence and location of the disulphide-bridged peptides of the C-terminal loop of heavy and light chains. The N-terminal loop, on the other hand, appears to be very different in Fc fragments and light chains. The C-terminal loop is the only one present in the F'c fragment.     

Dissection of the protein G B1 domain binding site for human IgG Fc fragment.

The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine-scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well-defined "knobs-into-holes" interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar "hot spot."     

A C-terminal interdomain disulfide bond significantly stabilizes the Fc fragment of IgG

We describe the stabilization of human IgG1 Fc by an engineered interdomain disulfide bond at the C-terminal end of the molecule. Covalently interconnecting the C-termini of the CH(3) domains led to an increase of the melting temperatures by 5.6 and 9.1°C respectively as compared to CH(3) domains in the context of the wild-type Fc. Combined with a recently described additional intradomain disulfide bond, both novel disulfide bonds led to an increase of the Tm by about 18.1°C to 100.7°C. The interdomain disulfide bond had no impact on the thermal stability of the CH(2) domain. Far- and near-UV CD spectroscopy showed very similar overall CD profiles, indicating that secondary and tertiary structure of the Fc was not negatively affected. When introduced into an Fc fragment that had been engineered to bind to Her2/neu via a novel antigen binding site located at the C-terminus of the CH(3) domain, the novel inter- and intra-domain bonds also brought about a significant increase in thermostability. Using them in combination, the Tm of the CH(3) domain was raised by 18°C and thus restored to the Tm of the wild-type CH(3) domain. Importantly, antigen binding of the modified Fc was not affected by the engineered disulfide bonds.     

Interference with tolerance induction of primed B cells by the Fc fragment of immunoglobulin

The capacity to interfere with tolerance induction in primed B cells was examined. Previous work had shown that TNP-specific splenic B cells from mice primed and boosted with TNP-KLH are highly susceptible to in vitro tolerization upon a brief exposure to TNP on a carrier unrelated to KLH. In the present work it was found that tolerance induction in these primed B cells could be partially disrupted by addition of the Fc fragment of immunoglobulin, a B-cell mitogen, and adjuvant, during exposure of the B cells to tolerogen. Addition of Fc fragments prepared by papain digestion of human IgG interfered with tolerization routinely in approximately 30-60% of the spleen cells susceptible to tolerogen. Addition of whole IgG or Fab fragments had no effect on tolerance induction. As little as 5 micrograms/ml of the Fc fragment preparation significantly interfered with tolerization and 32-64 micrograms/ml was optimal. Disruption of tolerization was most effective when the Fc fragment was added to the spleen cells either 4 hr prior to tolerogen or simultaneously with tolerogen; addition of the Fc fragment 4 hr after exposure to tolerogen was significantly less effective. Disruption of tolerization by the Fc fragment was not through polyclonal activation of B cells, as antigen was required for generation of significant numbers of PFC to TNP. Also, disruption was not through expansion of low avidity clones of B cells insusceptible to tolerogen, as the avidity of the antibody produced with and without Fc fragments present was approximately the same. These results show that the Fc fragment of IgG can partially interfere with tolerization of primed B cells. The manner in which Fc fragments may function to prevent tolerization through its lymphoid cell stimulatory capacities is discussed.     

Stabilisation of the Fc fragment of human IgG1 by engineered intradomain disulfide bonds

We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of T(m) of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the T(m) of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the T(m) of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule.     

A receptor for the Fc fragment of human IgG in the causative agent of tularemia

In 4 Francisella tularensis strains varying in virulence a receptor to Fc site of human IgG has been detected. This receptor consists of two active components with molecular weights of 67,000 and 40,000, competing for binding on Fc site of human IgG with Staphylococcus aureus protein A.     

Fc fragment from human IgG induces PGE2 secretion. II. Negative regulation in B cell differentiation

In humans, in vitro, Fc fragment of IgG at a low concentration induces plasma cell generation. However, Fc fragment at a high concentration induces PGE2 release of monocyte activation capable of inhibiting this differentiation. The levels of PGE2 in the supernatant culture from mononuclear cells from normal donors were examined as a function of culture duration and concentration of Fc, Fab fragments and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin(s). PGE2 levels, from mononuclear cell cultures, were analyzed by the RIA test. The results indicated that the Fc fragments are able to induce PGE2 secretion. The maximal release of PGE2 occurs after 24 hr of culture; this level is proportionate to the quantity of Fc fragments introduced. The addition of indomethacin in the cell culture stimulated by a high concentration of Fc fragments reestablishes the percentage of plasma cells. These results suggest the regulatory role of Fc fragment by PGE2 secretion in B cell differentiation.