Naoya Tsurushita

An Engineered Human IgG1 Antibody with Longer Serum Half-Life

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was ∼2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.

E-Selectin Appears in Nonischemic Tissue During Experimental Focal Cerebral Ischemia

BACKGROUND AND PURPOSE E-selectin participates in leukocyte-endothelial adhesion and the inflammatory processes that follow focal cerebral ischemia and reperfusion. The temporal and topographical patterns of microvascular E-selectin presentation after experimental focal cerebral ischemia are relevant to microvascular reactivity to ischemia. METHODS The upregulation and fate of E-selectin antigen during 2 hours of middle cerebral artery occlusion (n = 4) and 3 hours of occlusion with reperfusion (1 hour, n = 4; 4 hours, n = 6; 24 hours, n = 6) were evaluated in the nonhuman primate. E-selectin and E:P-selectin immunoreactivities were semiquantitated with the use of computerized light microscopy video imaging and laser confocal microscopy. RESULTS Three patterns of microvascular E-selectin expression, defined by the antibody E-1E4, were confirmed by complete elimination of E-1E4 binding after incubation with soluble recombinant human E-selectin: (1) Low immunoperoxidase intensity was observed in ischemic microvessels at 2 hours of occlusion extending to 4 hours of reperfusion (E-selectin/laminin = 0.32 +/- 0.10). (2) A significant fraction of ischemic microvessels displayed high-intensity E-selectin signal by 24 hours of reperfusion (0.61 +/- 0.17) compared with control and nonischemic tissues (2P < .003). (3) In the contralateral nonischemic basal ganglia and other nonischemic tissues, low but significant E-selectin levels appeared by 24 hours of reperfusion (2P = .0005). The latter were further confirmed by an E:P-selectin immunoprobe. CONCLUSIONS E-selectin antigen is distinctively and significantly upregulated in nonhuman primate brain after focal ischemia and reperfusion. The late appearance of E-selectin in nonischemic cerebral tissues suggests stimulation by transferable factors generated during brain injury.

Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries

New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.

The Integrity of the Ball-and-Socket Joint Between V and C Domains Is Essential for Complete Activity of a Humanized Antibody

AF2 is a high affinity murine Ab possessing potent neutralizing activity against human IFN-γ. In carrying out the modifications to humanize this Ab, we discovered that an initial version displayed affinity for IFN-γ that was slightly less than that of AF2, but exhibited IFN-γ-neutralizing activity that was severely diminished. Characterization via site-directed mutagenesis revealed that the majority of this loss in IFN-γ-neutralizing activity was due to altering the VH framework residue at position 11. VH position 11 is distal to the binding surface of the Ab; however, it, along with residues 110 and 112, have been identified as forming the socket of a molecular ball-and-socket joint between the V and C domains of the Ig Fab, which influences the elbow angle between these domains. To determine whether disrupting the structure of this joint was the basis for reduced IFN-γ-neutralizing capacity, we altered residue 148 of CH1, which with residue 149 comprises the corresponding ball portion of the joint. Changing this single CH1 domain residue diminished the ability of the Ab to neutralize IFN-γ to a level similar to that observed with the VH alteration. Thus, an intact ball-and-socket joint between the V and C domains in AF2 is required for potent neutralization of IFN-γ. These results suggest the importance of the elbow angle between Ig V and C domains in Ab activity, and support the hypothesis that this joint can be an important functional element of Ab structure.

Amphiregulin and Epidermal Hyperplasia: Amphiregulin Is Required to Maintain the Psoriatic Phenotype of Human Skin Grafts on Severe Combined Immunodeficient Mice

Overexpression of amphiregulin has been shown to induce psoriasiform changes in the skin of transgenic mice shortly after birth. Therefore, amphiregulin has been suggested as a target for anti-psoriatic therapy. To test this theory, a humanized monoclonal antibody capable of neutralizing human amphiregulin was examined for anti-proliferative effects in the human skin-severe combined immunodeficient (SCID) mouse transplant model. The anti-amphiregulin antibody reduced epidermal thickness of transplanted psoriatic skin and also inhibited the hyperplastic response that developed in nonpsoriatic skin after transplantation. The same antibody also suppressed keratinocyte proliferation in monolayer culture in a dose-dependent manner. Under the same conditions in which keratinocyte proliferation was inhibited, the antibody had little effect on proliferation of human dermal fibroblasts and no effect on type I procollagen production by these cells. Taken together, these data indicate an important role for amphiregulin in psoriatic hyperplasia and suggest that inhibition of amphiregulin activity could be an efficacious therapeutic strategy for psoriasis. These data also suggest that the hyperplastic response occurring in nonpsoriatic human skin on transplantation to the SCID mouse is mediated, in large part, by amphiregulin.

Fine mapping of the epitopes of humanized anti-l-selectin monoclonal antibodies HuDREG-55 and HuDREG-200

Blocking the function of L-selectin with a monoclonal antibody (mAb) is a promising way to prevent neutrophils from causing tissue damage during inflammation. HuDREG-55 and HuDREG-200 are humanized mAb which bind to human L-selectin and block its function as an adhesion molecule. To understand the mechanism of the action of HuDREG-55 and HuDREG-200, we determined their epitopes on L-selectin at the amino acid level. The analysis of human E- and L-selectin chimeric proteins demonstrated that the lectin domain of L-selectin is necessary for the binding of HuDREG-55 and HuDREG-200. Mutational analysis of Escherichia coli-expressed L-selectin showed that HuDREG-55 binding is sensitive to amino acid changes at positions 11, 56, 87, 89, 105, 107 and 111 (counting from the amino-terminus of mature L-selectin) while HuDREG-200 binding is sensitive to amino acid changes at 45, 46 and 47. Both epitopes are located close to the predicted carbohydrate binding site, indicating that HuDREG-55 and HuDREG-200 block the function of L-selectin by directly inhibiting the binding to carbohydrate ligands.

PCR cloning of the cDNA encoding baboon l-selectin

The cDNA encoding baboon L-selectin was isolated from baboon peripheral blood lymphocytes-derived cDNA and its nucleotide (nt) sequence was determined. The deduced 372-amino acid (aa) sequence of baboon L-selectin is 95% identical to that of human L-selectin.