Victor Ghetie

FcRn: the MHC class I-related receptor that is more than an IgG transporter

Recent data have indicated that the major histocompatibility complex (MHC) class I-related receptor FcRn plays a role in regulating serum IgG levels, in addition to transferring maternal IgGs across the rodent neonatal gut. The isolation of a human homolog of FcRn from placenta suggests that the studies in rodents have relevance to understanding similar processes in humans. This has implications for the engineering of improved antibodies for therapy.

Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn

The analysis of the pharmacokinetics of wild‐type and mutated Fc fragments derived from human IgG1 indicates that Ile253, His310 and His435 play a central role in regulating serum half‐life in mice. Reduced serum half‐life of the recombinant, mutated fragments correlates with decreased binding to the MHC class I‐related neonatal Fc receptor, FcRn. In addition, the analysis of an Fc fragment in which His435 is mutated to Arg435 demonstrates that the sequence difference at this position between human IgG1 (His435) and IgG3 (Arg435) most likely accounts for the shorter serum half‐life of IgG3 relative to IgG1. In contrast to His310 and His435, the data indicate that His433 does not play a role in regulating the serum half‐life of human IgG1. Thus, the interaction site of mouse FcRn on human and mouse IgG1 involves the same conserved amino acids located at the CH2‐CH3 domain interface of the IgG molecule. The sequence similarities between mouse and human FcRn suggest that these studies have direct relevance to understanding the factors that govern the pharmacokinetics of therapeutic IgG.

Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice

Vascular leak syndrome is a major and often dose-limiting side effect of immunotoxins and cytokines. We postulated that this syndrome is initiated by damage to vascular endothelial cells. Our earlier studies identified a three–amino acid motif that is shared by toxins, ribosome-inactivating proteins, and interleukin-2, all of which cause this problem. We have now generated a panel of recombinant ricin A chains with mutations in this sequence or in amino acids flanking it in the three-dimensional structure. These have been evaluated alone and as immunotoxins for activity, ability to induce pulmonary vascular leak in mice, pharmacokinetics, and activity in tumor-xenografted mice. One mutant was comparable to the ricin A chain used before in all respects except that it did not cause vascular leak at the same dose and, when used as an immunotoxin, was more effective in xenografted SCID mice.

Transcytosis and catabolism of antibody

This review describes the evolution of our knowledge of the transmission of immunoglobulin G (IgG) from mother to infant and the factors which regulate the persistence of IgG in the circulation. These apparently unrelated processes involve the same Fc receptor, FcRn (n=neonatal). FcRn appears to carry out these diverse roles by binding to IgG and then either transporting the bound IgG across cells (transcytosis) or recycling its cargo back to the cell surface (control of catabolism). IgG that is taken up by cells in the absence of binding to FcRn undergoes degradation. Thus, FcRn is the “protective” receptor that servesto maintain IgG homeostasis and deliver IgGs across cellular barriers.

The stoichiometry and affinity of the interaction of murine Fc fragments with the MHC class I-related receptor, FcRn

The binding of recombinant wild type and mutant Fc-hinge fragments to soluble, FcRn expressed in insect cells has been analysed. The mutant Fc-hinge fragments are derived from murine IgG1 with mutation of residues located at the CH2-CH3 domain interface (Ile253, His31O, Gln311, His433 and Asn434; EU numbering). These mutant Fc-hinge fragments have previously been shown to be deficient in neonatal transcytosis in suckling mice and also have abnormally short serum half lives. The mutated residues are highly conserved in human and rodent gammaglobulins (IgGs) and are also involved in binding to staphylococcal protein A. This study demonstrates that the Fc mutants have lower binding affinities for recombinant FcRn and mutations in the CH2 domain have a greater effect than those in the CH3 domain. There is an excellent correlation between affinity and transcytosis or the control of catabolism, and this provides further evidence in support of the close overlap of the sites of IgG/Fc involved in these processes. The stoichiometry of the FcRn:Fc interaction has also been investigated and has been found to be 1:1, indicating that binding of FcRn to one CH2-CH3 domain interface site precludes an FcRn:Fc interaction at the second site.

IDENTIFICATION AND FUNCTION OF NEONATAL FC RECEPTOR IN MAMMARY GLAND OF LACTATING MICE

In addition to its proposed function in regulating serum IgG levels, the MHC class I‐related neonatal Fc receptor (FcRn) is known to play a role in IgG transfer across rodent yolk sac and neonatal intestine. In contrast to humans, for which transplacental transfer of IgG appears to be the only mechanism of maternal IgG delivery, the transmission of IgG in mice occurs both antenatally (yolk sac) and neonatally (transport from mothers milk across intestinal epithelial cells). In the current study, a possible role for FcRn in regulating IgG transfer into milk has been investigated. FcRn has been shown to be present in functional form in the mammary gland of lactating mice, and is localized to the epithelial cells of the acini. Analysis of the transfer of Fc fragments and IgG which have different affinities for FcRn indicate that, unexpectedly, these proteins are transferred in inverse correlation with their binding affinity for FcRn. Thus, in the lactating mammary gland FcRn appears to play a role in recycling IgG in a mode that may have relevance to FcRn trafficking during the maintenance of constant serum IgG levels.

A novel recombinant vaccine which protects mice against ricin intoxication.

Ricin toxin (RT) is a plant-derived toxin of extraordinary toxicity; a single molecule successfully internalized into the cytoplasm of a cell is lethal for that cell. An estimated dose of 1-10 microg/kg is lethal to humans, making aerosolized ricin a potential agent for bioterrorism. Vaccination against ricin using either denatured toxin or its modified A chain subunit (RTA) has been successful in experimental animals but both vaccines have potential toxicities. Recombinant (r) RTA has not been evaluated as a vaccine. However, the advantage of such a vaccine is that these potential toxicities can be deleted by appropriate mutations. In this study we have generated three mutants and shown that two lack toxicity as compared to the wild type rRTA. These mutants induce protective humoral immune responses in mice. One or both should be considered for use in humans.

Catabolism of the Murine IgGl Molecule: Evidence that Both CH2-CH3 Domain Interfaces are Required for Persistence of IgGl in the Circulation of Mice

Site‐directed mutagenesis of a recombinant Fc‐hinge fragment has previously been used to identify a region of the murine IgG 1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this ‘catabolic site’ has been further analysed using recombinant techniques. Fc‐hinge, CH2‐hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice. The CH2‐hinge has been analysed as both a monomer and dimer, and the dimer has a longer β phase half‐life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc‐hinge fragment comprising a heterodimer of one Fc‐hinge with the wild type (WT) IgGl sequence and a mutant Fc‐hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a β phase half‐life of 37.9 h and this is significantly shorter than that of the WT Fc‐hinge fragment (82.9 h). In contrast to the CH2‐hinge dimer, the CH3 domain is cleared rapidly (β phase half‐life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgGl molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.

A Phase 1 Study of Combotox in Pediatric Patients With Refractory B-lineage Acute Lymphoblastic Leukemia

Background Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Combotox is a 1:1 mixture of RFB4-dgA and HD37-dgA which are immunotoxins that target the CD22 and CD19 antigens, respectively. Combotox has different toxicities and targets than chemotherapy and is, thus, a new candidate for the treatment of patients with relapsed ALL. Preclinical data have demonstrated which Combotox is effective in killing pre-B-ALL cell lines and cells from patients with pre-B ALL. Methods We designed and conducted a Phase 1 dose-escalation study using Combotox in children with refractory or relapsed B-lineage-ALL. Seventeen patients aged 1 to 16 years were enrolled in this multi-institution study. They were treated at 4-dose levels: 2 mg/m2, 4 mg/m2, 5 mg/m2, and 6 mg/m2. Results The maximum tolerated dose was 5 mg/m2 and graft versus host disease defined the maximum tolerated dose. Three patients experienced complete remission. Six additional patients experienced a decrease of >95% in their peripheral blood blast counts, and 1 patient experienced a decrease of 75%. Conclusions Combotox can be safely administered to children with refractory leukemia. It has clinically important anticancer activity as a single agent. The recommended dose for future studies is 5 mg/m2/dose.

Generation of mutated variants of the human form of the MHC class I-related receptor, FcRn, with increased affinity for mouse immunoglobulin G.

Much data support the concept that the MHC class I-related receptor FcRn serves to regulate immunoglobulin G (IgG) concentrations in serum and other diverse body sites in both rodents and humans. Previous studies have indicated that the human ortholog of FcRn is endowed with unexpectedly high stringency in binding specificity for IgGs. In contrast to mouse FcRn, which binds promiscuously to IgGs across species, human FcRn does not bind to mouse IgG1 or IgG2a, and interacts weakly with mouse IgG2b. Here, we investigate the molecular basis for this high-level specificity. We have systematically mutated human FcRn residues to the corresponding mouse FcRn residues in the regions that encompass the FcRn-IgG interaction site. Notably, mutation of the poorly conserved residue Leu137 of human FcRn to glutamic acid (L137E) generates a human FcRn mutant that binds to mouse IgG1 and mouse IgG2a with equilibrium dissociation constants of 13.2 microM and 14.4 microM, respectively. From earlier high-resolution structural analyses of the rat FcRn-rat Fc complex, residue 137 of human FcRn is predicted to contact residue 436 of IgG, which can be either His436 (mouse IgG1, mouse IgG2a) or Tyr436 (human IgG1, mouse IgG2b). The simplest interpretation of our data for the L137E mutant is therefore that replacement of the Leu137-Tyr436 (human) by the Glu137-His436 (mouse) pair generates a receptor that can bind to mouse IgG1 and mouse IgG2a. The L137E mutation reduces the affinity of human FcRn for human IgG1 by about twofold, consistent with the introduction of a less favorable Glu137-Tyr436 interaction. However, the analysis of the effects of other mutations on the binding to different IgGs indicates that the contribution to binding of the interaction of FcRn residue 137 with IgG residue 436 can vary. This suggests the existence of distinct docking topologies that are accompanied by variations in contacts between these two residues for different FcRn-IgG pairs. Our observations are of direct relevance to understanding the molecular nature of the human FcRn-IgG interaction. In turn, understanding human FcRn function has significance for the optimization of the serum half-lives of therapeutic and prophylactic antibodies.