Soluble divalent and multivalent heterodimeric analogs of proteins

Abstract

Specificity in immune responses is in part controlled by the selective interaction of T cell receptors with their cognate ligands, peptide / MHC molecules. The discriminating nature of this interaction makes these molecules, in soluble form, good candidates for selectively regulating immune responses. Attempts to exploit soluble analogs of these proteins has been hampered by the intrinsic low avidity of these molecules for their ligands. To increase the avidity of soluble analogs for their cognates to biologically relevant levels, divalent peptide / MHC complexes or T cell receptors (superdimers) were constructed. Using a recombinant DNA strategy, DNA encoding either the MHC class II / peptide or TCR heterodimers was ligated to DNA coding for murine Ig heavy and light chains. These constructs were subsequently expressed in a baculovirus expression system. Enzyme-linked immunosorbant assays (ELISA) specific for the Ig and polymorphic determinants of either the TCR or MHC fraction of the molecule indicated that infected insect cells secreted approximately 1 .mu.g / ml of soluble, conformnationally intact chimeric superdimers. SDS PAGE gel analysis of purified protein showed that expected molecular weight species. The results of flow cytometry demonstrated that the TCR and class II chimeras bound specifically with high avidity to cells bearing their cognate receptors. These superdimers will be useful for studying TCR / MHC interactions, lymphocyte tracking, identifying new antigens, and have possible uses as specific regulators of immune responses.

Description

[0001] This application is a continuation-in-part of Provisional Application Ser. No. 60 / 014,367 which was filed Mar. 28, 1996.TECHNICAL FIELD[0002] This invention is directed to compositions comprising soluble divalent and multivalent heterodimeric analogs of proteins that are involved in immune regulation and methods of making and using the same. The high affinity that these complexes have for their cognate ligands enables them to be effective competitors to T cell receptors and MHC molecules normally involved in transplant rejection and autoimmune disease. Molecules such as divalent T cell receptors may also have an impact on the diagnosis and treatment of cancer in that they may be used to augment antitumor responses, or may be conjugated to toxins which may then be used to help eliminate tumors. Use of such compositions will allow one to accomplish selective immune modulation without compromising the general performance of the immune system.BACKGROUND OF THE INVENTION[0003] The...

AI Technical Summary

Benefits of technology

[0031] Accordingly, also included in the invention are methods of identifying unknown antigens or peptides derived using soluble divalent TcR. A distinct advantage of soluble high affinity TCR / Ig chimeras is that even in the absence of any a priori knowledge about their ligands, they may be useful in defining the specific peptide / MHC ligands recognized by uncharacterized tumor-specific T cells and T cells involved in autoimmune responses. Not only are soluble divalent TCR / IG molecules efficient probes for the quantitative detection of specific peptide / MHC complexes, but due to their strong affinity for the target molecule, they will consequently play an important role in the purification of such complexes and facilitate their characterization.

Problems solved by technology

It is this regulatory function that often goes awry in the development of autoimmune diseases.

These cells are not well understood; their antigen / MHC specificity is poorly defined and in most cases their ligands are completely unknown.

However, simply making soluble monovalent analogs of either T cell receptors or peptide / MHC complexes has not proven to be effective at regulating immune responses with the required specificity and avidity.

Previously, replacement of two transmembrane domains in the generation of multivalent analogs has not been achieved.

The challenge of generating these molecules lies in achieving the proper folding and expression of two polypeptides, both of which ordinarily require transmembrane domains (FIG. 1).

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