The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc region thereof.

Polypeptide variants with altered effector function

Fibronectins are large glycoproteins that have been implicated in a wide variety of cellular properties, particularly those involving the interactions of cells with extracellular materials. These properties include cell adhesion, morphology, cytoskeletal organization, migration, differentiation, oncogenic transformation, phagocytosis, and hemostasis. During the past several years, investigations in many laboratories have analyzed the expression, functions, and structure of fibronectins. These studies have revealed that fibronectins have a complex molecular structure consisting of multiple specific binding sites, and the complex biological phenomena in which fibronectins participate can now be considered in terms of this structure. In this brief article we will review the current understanding of the structure and properties of fibronectins. Because a number of comprehensive reviews on various aspects of fibronectins have been published (1-13), we shall focus on overall concepts, recent developments and promising future research directions in this rapidly expanding field.

/pdf/fibronectins-multifunctional-modular-glycoproteins-4x0ehu2vrh.pdf

Fibronectins: multifunctional modular glycoproteins.

The DNA encoding an antibody with an altered function, e.g. altered affinity for an effector ligand such as Fc receptor (FcR) on a cell or the Cl component of complement is produced by replacing the nucleic acid encoding at least one amino acid residue in the constant portion of the antibody with nucleic acid encoding a different residue.

DNA encoding antibodies with altered effector functions

Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes

It is currently thought that immune responses are initiated by pathogen-associated molecular patterns or by tissue-derived danger/alarm signals. Here, we propose that these two groups of molecules might not be mutually exclusive. Many of them might be part of an evolutionarily ancient alert system in which the hydrophobic portions of biological molecules act, when exposed, as universal damage-associated molecular patterns to initiate repair, remodelling and immunity.

Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses

Conditions are described whereby human Clq may be enzymatically radioiodinated with full retention of hemolytic activity. A comparison is made between the chloramine T (CT) and lactoperoxidase (LP) methods of iodination which indicates the following: 1) Clq may be iodinated to a level of 0.2 atoms iodine/molecule by using LP with no detectable loss in hemolytic activity, whereas, at a level of 0.02 atoms incorporated/molecule with CT, Clq is only 50% active. Thus the hemolytic activity of Clq is quite labile to either low levels of iodide incorporation or the oxidizing conditions of the labeling procedure. 2) Its ability to bind immune complexes, conversely, is virtually resistant to the levels and conditions of iodination reported here. 3) With either labeling method, iodide is incorporated exclusively into only the smallest of the three covalently bonded subunits.

Effect of Chemical and Enzymatic Radioiodination on in Vitro Human Clq Activities

Fibronectin immobilized to plastic tubes binds soluble C1q with a Kd of 82 +/- 2.6 nM. The binding of fibronectin to C1q is relatively insensitive to pH but is sensitive to ionic conditions. C1q covalently bound to Sepharose selectively binds cellular fibronectin produced by a hamster fibroblast cell line. The globular head regions of C1q have no effect on the binding of C1q to fibronectin but the collagenous tails of C1q interfere competitively with a Ki of 59 nM. We conclude that fibronectin binds C1q via its collagen-like tail region and thus the process resembles the binding of fibronectin to gelatin. This is further emphasized by our observation that gelatin binds to fibronectin immobilized on plastic tubes with a Kd of 131 nM. Because fibronectin stimulates endocytosis in several systems and promotes the clearance of particulate material from the circulation, these results suggest the possibility that fibronectin could function in the clearance of C1q-coated material such as immune complexes or cellular debris.

http://www.pnas.org/content/79/13/4198.full.pdf

Fibronectin binds to the C1q component of complement.

Lipid vesicles containing phospholipids known to be present in substantial amounts in mitochondrial membranes were tested for their capacity to activate C1. Among them, only cardiolipin (CL) was highly efficient in C1 activation; no such effect was observed with phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. CL was shown to bind specifically C1q, because only unlabeled C1q competed with 125I-C1q for binding to CL. The requirement for C1q was confirmed by the finding that only fully reconstituted macromolecular C1, containing C1q, was activated by CL. The specificity of CL-induced activation of C1 was also demonstrated by introducing adriamycin, an agent known to interact with CL. Whereas adriamycin did not decrease C1 activation induced by immune complexes, it abrogated C1 activation by CL. The latter was shown to be a strong nonimmune activator of C1, because C1-INH did not inhibit CL-induced activation. When the concentration of CL in vesicles was decreased in the presence of phosphatidylcholine, C1 activation was detected only above a critical level of 35 mol% CL, compatible with a minimal density or clustering of CL molecules in the plane of the membrane. Moreover, C1 activation by CL was modulated by the addition of cholesterol. The threshold of CL required for C1 activation was lowered by the incorporation of more than 35 mol% cholesterol into the vesicles. These results show that CL incorporated into liposomes can be a potent nonimmune activator of C1. The negatively charged phosphate groups in CL are likely candidates for Clq-binding.

Antibody-independent activation of C1, the first component of complement, by cardiolipin.

Human C1q was shown to be sensitive to digestion by trypsin, chymotrypsin and collagenase, but not by elastase. Treatment with trypsin and chymotrypsin caused a rapid loss of the hemolytic activity of C1q and its ability to fix to immune complexes. Collagenase digestion of C1q resulted in a rapid loss of the hemolytic activity also, whereas the ability to bind to immune complexes decreased slowly and remained at a constant value of 15%. In contrast to trypsin and chymotrypsin, collagenase treatment of 125I-labeled C1q led to well defined fragments, only some of which were labeled. Adsorption of collagenase-digested C1q on ovalbumin-antiovalbumin complexes indicated the larger non-covalently bound peripheral subunits of C1q to be involved in fixation to immune complexes. None of the fragments was able to initiate lysis in the presence of RC1q. Immunologic analysis of the digest revealed at least two different fragments with respect to antigenicity.

Henri Isliker

Papers

Effect of Chemical and Enzymatic Radioiodination on in Vitro Human Clq Activities

Fibronectin binds to the C1q component of complement.

Antibody-independent activation of C1, the first component of complement, by cardiolipin.

Enzymatic digestion of the first component of human complement (C1q).

Studies on the complement-binding site of rabbit immunoglobulin G—II. The reaction of rabbit IgG and its fragments with Clq