Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1996-02-22
2002-06-11
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S388240
Reexamination Certificate
active
06403769
ABSTRACT:
BACKGROUND OF THE INVENTION
Immunoglobulin (Ig) molecules have been the focus of ongoing research because they react with a diverse range of antigens, possess different effector functions, and are important biologically. Of particular interest is the principal serum immunoglobulin in mammals, IgG, which is the major constituent of the secondary immunological response to most antigens.
Immunoglobulin G is a tetramer composed of two identical light (L) chains and two identical heavy (H) chains joined, respectively, by disulfide linkages. The L chains fold into two functional domains, while the H chains fold into four or five. Each domain consists of about 100 to 120 amino acid residues.
The H- and L-chain halfmers are covalently bonded by disulfide bonds in the H-chain “hinge” region, as shown in FIG.
1
. The number of hinge disulfide bonds is variable and depends on the H chain isotype. The hinge region is flexible and susceptible to proteolytic digestion.
So-called “variable” regions, formed by the N-terminal domains for each chain, differ from antibody to antibody in amino acid sequence and define antigen-binding sites of unique specificity and affinity. The other IgG “constant” (C) domains, CH
1
, CH
2
and CH
3
, have the same amino acid sequence for a given antibody chain of the same isotype, except for single-residue differences at a few positions, and contribute to the activation of host effector mechanisms to eliminate antigen.
The V domains of the IgG molecule thus are responsible for antigen recognition and the binding of antigens, while the C domains mediate binding of the immunoglobulin to host cells, including various cells of the immune system and some phagocytic cells, and to C
1
q, the first component of the classic complement system. More specifically, C
1
q interacts with the CH
2
domain of IgG. Among four recognized IgG subtypes, two (IgG1 and IgG3) possess higher complement fixation activity than the others (IgG2 and IgG4).
The domain structure of IgG and other antibodies recommend them as targets for protein engineering. See Rothwell,
Nature
342: 99 (1989). Past efforts in this regard, as reviewed, for example, by Wright et al.,
Crit. Rev. Immunol
. 12: 125 (1992), focussed on creating potentially valuable agents for treatment of human disease. Much of this “antibody engineering” involved maintaining the original specificity of the V region for a given Ig molecule while altering the remainder of the molecule, for example, by attaching an enzyme, toxin or growth factor to all or part of the molecule. See, for example, Shin & Morrison,
Proc. Nat'l Acad. Sci
. USA 87: 5322 (1990) (insulin-like growth factor 1 replaces constant region of mouse human IgG3 anti-dansyl antibody). Conversely, chimeric molecules were produced in which the V region was replaced with all or part of another molecule, including receptor molecules such as CD4 [Capon et al.,
Nature
: 525 (1989)], human natriuretic peptide receptor [Bennett et al.,
J. Biol. Chem
. 266: 23060 (1991)] and human hepatocyte growth factor receptor [Mark et al.,
ibid
. 267: 26166 (1992)], and the cytokine interleukin-2 [Landolfi,
J. Immunol
. 146: 915 (1992)].
These IgG-containing fusion proteins demonstrated the feasibility of maintaining Ig effector function upon replacement of the variable region not only with a nonantibody component like CD4, which is associated with the so-called “immunoglobulin superfamily” and, hence, folds in a manner compatible with the IgG constant region, but also by a nonantibody domain like IL-2, which is structurally disparate to those of the Ig superfamily. For example, Landolfi (1992) took note of a “potential to create a variety of immunoligands in which the binding specificity is non-Ig in nature (e.g., hormone, lectin, peptide, or other ligand),” and he speculated that such “agents could have therapeutic potential if their binding specificity is unique to a neoplasia or other tissue characteristic of a disease state.” Id. at 918.
Nevertheless, practical applications of such IgG-based chimeras actually has been slow to emerge. This is due in part to the fact that H and L chains of IgG are poorly secreted and rapidly degraded in the absence of partner L and H chains, respectively, a situation that applies to the chimeric molecules in question. There also is relatively little specific information or predictive theory to illuminate the biological properties of different categories of chimeric molecules.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide IgG
on-IgG fusion proteins which, upon heterologous expression in transfected mammalian cells, are potently secreted in stable form, and which display effector properties characteristic of antibody and nonantibody predecessor molecules, respectively.
It is another object of the present invention to provide an approach for producing Fc-containing chimeric molecules in a form that is readily useable in applications conventionally associated with monoclonal antibodies, including flow cytometry, immunohisto-chemistry and immunoprecipitation.
In accomplishing these and other objects, there has been provided, in accordance with one aspect of the present invention, a fusion protein comprising (A) an IgG sequence, (B) a nonantibody sequence covalently joined to the aminoterminal end of the IgG sequence and (C) a heterologous signal peptide that is covalently joined to the aminoterminal of the nonantibody sequence, wherein
(i) the IgG sequence consists essentially of a hinge region, a CH
2
domain and a CH
3
domain, in that order, the IgG sequence lacking a CH
1
domain,
(ii) the nonantibody sequence comprises an effector domain of a molecule, and
(iii) the effector domain displays an activity that is characteristic of the effector domain in the molecule.
In accordance with another embodiment of the present invention, there has been provided a fusion protein comprising a nonantibody sequence covalently joined to the aminoterminal end of an IgG sequence that consists essentially of a hinge region, a CH
2
domain and a CH
3
domain, in that order, the IgG sequence lacking a CH
1
domain, wherein the nonantibody sequence comprises an effector domain of a growth factor molecule that in nature binds a single-unit receptor, such that the fusion protein induces DNA synthesis, as measured by uptake of
3
H-thymidine, in a target cell.
In accordance with a further embodiment of the present invention, there has been provided a method for detecting a pathological condition associated with overexpression of a molecule that participates in a binding interaction, comprising the steps of
(A) providing a fusion protein comprising a nonantibody sequence covalently joined to the aminoterminal end of an IgG sequence that consists essentially of a hinge region, a CH
2
domain and a CH
3
domain, in that order, the IgG sequence lacking a CH
1
domain, wherein the nonantibody sequence comprises an effector domain of a molecule, and wherein the effector domain displays an activity that is characteristic of the effector domain in the molecule;
(B) bringing the fusion protein into contact with a biological sample which contains a binding partner for the effector domain; and
(C) monitoring binding of the effector domain by the binding partner in the sample to detect overexpression, relative to a control, of the binding partner.
In accordance with a further embodiment of the present invention, there has been provided a method for identifying agonists and antagonists that interfere with a binding interaction, comprising the steps of
(A) providing a fusion protein comprising a nonantibody sequence covalently joined to the aminoterminal end of an IgG sequence that consists essentially of a hinge region, a CH
2
domain and a CH
3
domain, in that order, the IgG sequence lacking a CH
1
domain, wherein the nonantibody sequence comprises an effector domain of a molecule, and wherein the effector domain displays an activity that is characteristic of the effector domain in the molec
Aaronson Stuart A.
Dirsch Olaf
Larochelle William J.
Caputa Anthony C.
Needle & Rosenberg P.C.
Nickol Gary B.
The United States of America as represented by the Department of
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