Nicholas F. Landolfi

Activation of T lymphocytes results in an increase inH-2-encoded neuraminidase

The endogenous neuraminidase activity of various mouse lymphoid subpopulations and tissue compartments was examined by a sensitive fluorometric assay. These analyses indicated that activated T lymphocytes possessed a significantly higher level of intracellular neuraminidase than activated B or resting T or B lymphocytes. Examination of the level of neuraminidase in bone marrow, thymus, lymph node, and unfractionated spleen indicated that these lymphoid tissues contained significantly less neuraminidase than was detected in stimulated T cells. Kinetic studies revealed that the majority of the increase in neuraminidase activity occurred between 24 and 48 h following stimulation. Analysis of activated T lymphocytes prepared from a panel of inbred mouse strains indicated that cells from mice of theH-2v haplotype, which possess theNeu-1a allele and are deficient in liver neuraminidase, exhibited a level of activity which was significantly lower than that detected in stimulated T cells from other mouse strains. These results indicate that the endogenous neuraminidase activity of T lymphocytes increases upon stimulation, and that the level of this enzyme activity in lymphoid cells is also controlled by theNeu-1 locus, which is located in theH-2 region of the major histocompatibility complex.

Activated T-lymphocytes express class I molecules which are hyposialylated compared to other lymphocyte populations

Various H-2 and Qa/Tla region encoded class I glycoproteins expressed on the surface of resting and activated T- and B-lymphocytes were compared by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The isoelectrophoretic patterns of the H-2K, H-2D, Qa-2 and Qa-1 molecules isolated from activated T-lymphocytes were more isoelectrically heterogeneous and/or possessed species with a more basic pI than the same molecules isolated from resting T- and B-cells or activated B-lymphocytes. The differences in charge heterogeneity of class I molecules between activated T-cells and the other cell subpopulations were abolished by treatment with: (1) endoglycosidase F which removes N-linked oligosaccharides from glycoproteins, and (2) neuraminidase which removes sialic acids from carbohydrate side chains. Thus, the increased charged heterogeneity of class I molecules expressed by activated T-cells is due to altered sialylation of their N-linked oligosaccharides. These results indicate that a mechanism exists, upon activation of T-lymphocytes, for alteration (desialylation) of the carbohydrate moieties of class I molecules.

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.

Comparison of the three-dimensional structures of a humanized and a chimeric Fab of an anti-gamma-interferon antibody.

The objective of this work is to compare the three‐dimensional structures of “humanized” and mouse–human chimeric forms of a murine monoclonal antibody elicited against human γ‐interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen‐binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8000 by nearly identical microseeding procedures. Their structures were solved by X‐ray analyses at 2.9 Å resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity‐determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N‐terminal segment (designated strand 4‐1) to be closely juxtaposed to an adjacent strand (4‐2) and form hydrogen bonds typical of an antiparallel β‐pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an α‐helix involving residues 25–32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 Å to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space‐saving α‐helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use. Copyright

Three-dimensional structures of a humanized anti-IFN-γ Fab (HuZAF) in two crystal forms

Three-dimensional structures were determined for two crystal forms (orthorhombic P2(1)2(1)2(1) and monoclinic C2) of the Fab from the humanized version of a murine monoclonal antibody (AF2) that possesses binding and potent neutralizing activity against human interferon gamma (IFN-gamma). This humanized antibody (HuZAF; USAN name fontolizumab) is currently in phase II clinical trials for the treatment of Crohns disease. HuZAF exhibits binding and IFN-gamma neutralizing capacities that closely approximate those of the original antibody. It is shown that HuZAF, whose VH domain was designed using a best-sequence-fit approach, is closer structurally to its mouse precursor than is a version whose VH was constructed using a human sequence with lower homology to the original mouse sequence. This work thus offers direct structural evidence in support of the best-sequence-fit approach and adds to previous results of biological and biochemical evaluations of distinctly engineered antibodies that also favored the use of a best-sequence-fit strategy. A second crystal type appeared during attempts to crystallize the Fab-IFN-gamma complex. The antibody-antigen complex that existed in solution dissociated in the crystallization mixture. A conformationally altered but unliganded HuZAF protein crystallized in a different space group (C2), with two Fab molecules in the asymmetric unit. In this crystal lattice, no space was available for accommodating the IFN-gamma antigen. Thus, there are currently three slightly different structures of the HuZAF Fab.

A potent neutralizing monoclonal antibody can discriminate amongst IFNγ from various primates with greater specificity than can the human IFNγ receptor complex

A monoclonal antibody (AF2) generated against recombinant human interferongamma (IFNgamma) exhibited potent IFNgamma neutralizing activity and prevented human IFNgamma from binding to the cell surface IFNgamma receptor complex. The AF2 antibody also neutralized IFNgamma from higher primates (superfamily Hominoidea) but did not react with IFNgamma from rhesus or other primates in the suborder Anthropoidea IFNgamma from all primates tested, however, could signal via the human IFNgamma receptor complex, as indicated by the ability to upregulate the level of MHC class II molecule expression on the surface of a responsive human cell line. We cloned and sequenced the IFNgamma gene from chimpanzee, gorilla, orangutan, and gibbon, and compared those with the previously reported IFNgamma sequences of human, rhesus, baboon and marmoset. This comparison revealed that, of the primate IFNgammas that were not reactive with AF2, rhesus IFNgamma was most homologous to human IFNgamma, differing at only nine amino acids and containing a one amino acid deletion. Comparing the sequence of human IFNgamma with that of rhesus IFNgamma suggested residues of the human IFNgamma molecule that were involved in the formation of the epitope recognized by the AF2 antibody. Constructing human/rhesus chimeric IFNgamma molecules, combined with site-directed mutagenesis of both human and rhesus IFNgamma revealed that this epitope was dependent upon two non-contiguous amino acids that are juxtaposed in the tertiary structure of IFNgamma. The determinant recognized by AF2 antibody resides in a portion of IFNgamma that is proximal to, but distinct from the surface that interacts with the IFNgamma receptor. Therefore, this neutralizing monoclonal antibody reacts with a conformational determinant that distinguishes primate IFNgammas serologically, but not functionally.

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1a. Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1b- and Qa-1c-encoded determinants. Qa-1d target cells are unique in that they express determinants recognized by anti-Qa-1a CTL and by anti-Qa-1b CTL. Killing of Qa-1d targets by anti-Qa-1a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1d cells by one anti-Qa-1b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1a CTL and Qa-1.1-specific antibodies did not have identical specificities.

Molecular weight diversity among murine class I antigens: both the mature cell surface forms and the unglycosylated polypeptides vary significantly in molecular weight

The molecular weights of the fully glycosylated cell surface form and the unglycosylated polypeptide of five murine class I antigens (H-2Kb, Db, TL, Qa-1.1, and Qa-2) were compared by SDS-PAGE. Significant molecular weight diversity was observed for both forms among these molecules. The size of the fully glycosylated forms ranged from approximately 52,000 daltons (H-2Db) to 41,000 daltons (Qa-2), whereas the unglycosylated polypeptides ranged from 43,000 daltons (H-2Kb and TL) to 33,000 daltons (Qa-2). The magnitude of the size variation observed in the unglycosylated polypeptides implies that there are differences in the gene organization, RNA processing or post-translational modifications of various class I glycoproteins.