Shabnam Tangri

Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91–120 and Epo 126–155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.

Antigen-presenting function of mouse CD 1: one molecule with two different kinds of antigenic ligands

Summary: Mouse CDl (mCDl) is an antigen‐presenting molecule that is constitutively expressed by most bone marrow‐derived cells. Peptides with a hydrophobic binding motif can bind to mCDl, and the peptide‐CDl complex is recognized by CD8+ cytolytic T cells. In contrast, NK1.1+ T cells, which are CD8‐, are autoreactive for mCD 1 molecules. This autore‐activity, along with the ability of NK T cells to rapidly produce large amounts of cytokine, has led to the suggestion that these cells may be immunoregulatory We have shown that die mCD l ‐autoreactive T cells can distinguish between different cell types that express similar levels of mCDl, suggesting that mCDl ‐bound autologous ligands may be critical for T‐cell stimulation. Consistent with this, some of these mCDl‐restricted T cells can recognize the glycolipid a‐galactosyiceramide presented by mCDl, while others do not respond. The mCDl crystal structure reveals a deep and narrow hydrophobic antigen‐binding site which can more easily bind lipid antigens than the long hydrophobic peptides that we have defined as mCDl antigens. The ability of mCDl to bind and present two different types of ligands raises the question as to how mCDl can accommodate both types of antigens.

Antigen‐presenting Function of the TL Antigen and Mouse CD1 Molecules

The hallmark of all the nonclassical antigen-presenting molecules, including nonclassical class I and nonclassical class II (Karlsson et al. 1992) molecules, is their lack of polymorphism. It is presumed, therefore, that these nonclassical molecules must have a distinct antigen-presenting function in which polymorphism is not advantageous. In some cases this may involve presentation of a nonpeptide antigen, as has been demonstrated for human CD1b. It is possible that a molecule adapted to present bacterial lipids would remain relatively nonpolymorphic, because a lipid, which is the end product of a complex biosynthetic pathway, is likely to evolve less rapidly than a short stretch of amino acid sequence containing a T-cell epitope. Alternatively, the lack of polymorphism could reflect the presentation by these molecules of relatively invariant peptides, such as those derived from heat shock proteins. It also is possible that a nonpolymorphic molecule could be selected for the presentation of modified peptides. An example of this is the M3 molecule, which can bind even short peptides as long as they have a formylated N-terminus (Fischer Lindahl et al. 1991). Based upon their structural differences, we believe it is likely that the TL antigen and mCD1 are likely to present different types of ligands. The presence in the TL antigen of the conserved amino acids, which in class I normally from hydrogen bonds with peptides, suggests that the TL antigen also can present nanomeric peptides. A peptide antigen-presenting function also is suggested by the expression of the TL antigen by at least one antigen-presenting cell type, the epithelial cell of the intestine, and by the ability of alloreactive T cells to recognize the TL molecule. While we favor the hypothesis that the TL antigen presents peptides, the data cited above do not constitute formal proof of any kind of antigen-presenting function, and it remains possible that the TL antigen does something else. As noted above, no attempts to elucidate the structure of the ligands bound to the TL antigen have so far succeeded, including the screening of bacteriophage display libraries (Castaño, A.R., Miller, J.E., Holcombe, H.R., unpublished data). In contrast, our recent work has demonstrated that mCD1 presents relatively long peptides with a structured motif distinct from classical class I molecules. This mCD1-binding motif, which is present in a wide range of proteins, does not by itself provide a simple explanation for the lack of mCD1 polymorphism and, as noted above, it remains possible that the natural ligand for mCD1 is a nonpeptide structure. Besides their lack of polymorphism, the TL antigen and mCD1 molecules share two additional features in common which might give insight into their their biological role. First, their surface expression does not depend upon the presence of a functional TAP transporter, and they probably can reach the cell surface as empty molecules. Second, both molecules are expressed by epithelial cells in the intestine. This leads to the speculation that these two nonclassical class I molecules could be involved in sampling or uptake of lumenal peptides for their ultimate presentation to cells of the systematic immune system. For example, longer lumenal peptides could be taken up by mCD1, and perhaps by the TL antigen, and then further processed to nonamers for presentation by classical class I molecules. They also could be transported across the epithelial cell by the TL antigen or mCD1 and subsequently presented by either class I or class II molecules expressed by cells in the lamina propria. This sampling or uptake mediated by either the TL antigen or mCD1 could play a role in the induction of immune responses, or more likely perhaps, in the induction of systemic oral tolerance to peptide antigens.(ABSTRACT TRUNCATED)

Immunodystrophism, T Cells, Cytokines, and Pregnancy Failure

It has been abundantly clear for some time now that pregnancy can be compromised by a variety of factors, endocrinologic, genetic, anatomic and infectious. What has become apparent recently is that pregnancy can be affected both in positive and negative ways by immunologic factors. The maternal immune system can promote or inhibit the growth and survival of the feto-placental unit, but the precise mechanisms involved, the key mediators, their modes of action and the extent of the influence of the immune system on gestation are far from clear. The availability of murine models of pregnancy failure has made it possible to elucidate many of the mechanisms involved in interactions between the immune system and the reproductive system, and such studies should pave the way for similar studies in humans where we understand even less about these interactions. This article discusses our observations on immunologically mediated fetal resorptions in mice and speculates on the implications these studies may have for pregnancy failure in humans. The most common complication of pregnancy is spontaneous abortion, with about 30% of implanted embryos in humans aborting before 14 weeks of gestation.’,’ While a proportion of spontaneous abortions is clearly attributable to anatomical, chromosomal and infectious etiologies, it is argued that recurrent spontaneous abortions are not likely to be caused by anatomic or chromosomal Therefore, as much as 60% of recurrent spontaneous abortions (RSA) are due to unknown or unexplained e t i~ logy;~ it has long been suspected, with supportive evidence coming in only recently, that immunological factors may contribute to the pathogenesis of many of the unexplained cases of RSA.

Antigen-presenting function of the mouse CD1 molecule.

CD1 molecules are distantly related to major histocompatibility complex (MHC)-encoded class I molecules, and they are coexpressed with beta2 microglobulin (beta2m). In the mouse, CD1 is expressed by intestinal epithelial cells and also by some cells in spleen and lymph node. We have shown that surface expression of mouse CD1 (mCD1) is not dependent upon a functional transporter associated with antigen processing (TAP). This, and other data, suggest that mCD1 may acquire peptides in an intracellular compartment other than the endoplasmic reticulum, where classical class I molecules bind peptide. mCD1 molecules also are distinct from classical class I molecules with regard to the types of peptides that they bind. We have demonstrated that mCD1 molecules preferentially bind peptides much longer than the 8-9 amino acids typical of the peptides that bind to classical class I molecules. The sequence motif for mCD1 peptide binding is characterized by the presence of bulky and hydrophobic amino acid side chains. We have generated mCD1-restricted and peptide-specific T-cell lines, thereby demonstrating the immunologic relevance of peptide binding to mCD1. The reactive T cells are TCR alphabeta+ and CD8+, a phenotype typical of many lymphocytes in both lymph node and intestinal mucosae. We speculate that mCD1 molecules may be capable of sampling peptides from the gut lumen and presenting them to mucosal T lymphocytes. In this way, they may function in the maintenance of normal mucosal homeostasis, and perhaps also in the induction of systemic tolerance to antigens delivered by the oral route. In summary, CD1 molecules are a novel category of antigen-presenting molecules that have features in common with class I molecules, features in common with class II, and properties distinct from either subset of antigen-presenting molecules. Further studies of the antigen-presenting function of these molecules are certain to yield new insight into immune regulation and perhaps also into the mechanism of oral tolerance.

Lymphocyte—Epithelial Cross-Talk in the Intestine: Do Nonclassical Class I Molecules Have a Big Part in the Dialogue?

Publisher Summary This chapter focuses on the behavior and properties of nonclassical class I molecules expressed by mouse intestinal epithelial cells, and the possible role these molecules may have in the development and function of mouse intestinal intraepithelial lymphocytes (IEL). IEL have a phenotype that is distinct from other T-cell populations, and these lymphocytes may be the product of a separate, thymus-independent lineage. If such a lineage exists, it would be likely that the intestinal epithelium plays a major role in its development. It is also possible, however, that some IEL are derived from thymus-derived T cells. This is evidenced by the ability of conventional CD4+ cells to home to the gut epithelium in scid mice, and to acquire characteristics of the T cells that normally reside in the intestine. This chapter shows that nonpolymorphic class I molecules expressed in the intestine have unique properties, most notably, their lack of a requirement for TAP in order to be expressed on the cell surface. This lack of a TAP requirement may provide some insight into the specialized function of the TL antigen and mCDl in the mucosal immune system. This chapter further demonstrates that mCDl can function as an antigen-presenting molecule, and that it presents peptide antigens distinct from those presented by classical class I molecules.