Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-01-03
2001-11-13
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S326000, C435S328000, C435S329000
Reexamination Certificate
active
06316256
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel methods for transferring one or more proteins to a cell. In addition to other applications, the methodology is useful in the treatment of cancer and autoimmune diseases, and for determining costimulator activation thresholds and cooperative interactions among costimulators.
BACKGROUND INFORMATION
T-cells, including cytotoxic T-lymphocytes (CTLs), are a critical component of effective human immune responses to tumors, viral infections and other infectious diseases. T-cells destroy neoplastic or virally infected cells through recognition of antigenic peptides presented by MHC class I molecules on the surfaces of target cells. Activation of T-cells is dependent upon coordinate signaling through antigen receptors and costimulator receptors on T-cell surfaces. Many mechanisms contribute to the escape of tumor cells and virally infected cells from immune surveillance. One of the mechanisms is that these cells lack the costimulatory molecules required for T-cell activation. Immunotherapeutic strategies have been developed that are predicated upon expressing costimulators on tumor cell, and other antigen-presenting cell, surfaces.
Professional antigen-presenting cells (APCs), by virtue of the surface costimulatory molecules, are geared towards potent T-cell activation. APCs can be converted into deletional APCs, or “artificial veto cells”, by expressing coinhibitors at their surfaces. This is discussed, for example, in U.S. Pat. Nos. 5,242,687; 5,601,828; and 5,623,056. Such coinhibitors bind to coinhibitor receptors on cells, leading to T-cell inactivation.
One approach for expressing costimulators and coinhibitors on APCs, such as tumor cells, is gene transfer. When used for APC and tumor cell engineering, gene transfer techniques have shortcomings. For example, APCs, including tumor cells, are often poorly transfectable. In addition, transfection proceedings are cumbersome and time-consuming. Furthermore, expressing more than a costimulator (or coinhibitor) is difficult. These and other issues have impeded the widespread application of gene therapy for APC and tumor cell engineering.
Protein transfer offers a number of advantages over gene transfer for engineering APCs and other cells. These advantages include the ability to modify poorly transfectable cells (for example, biopsy-derived tumor cells), the simplicity of expressing multiple proteins on the same cell surface, and the relative ease and rapidity of the procedure. The successful use of recombinant GPI-modified costimulator and MHC protein derivatives for protein transfer has been reported. (See, Brunschwig, et al.
J. Immunol.,
155:5498 (1995); McHugh, et al;
Proc. Natl. Acad. Sci. USA,
92:8059 (1995); and McHugh, et al.
Cancer Res.,
59:2433 (1999)). A shortcoming of the GPI protein transfer strategy, however, resides in scaling up the purification of GPI proteins from membranes of transfected cells.
Kim and Peacock,
J. Immunol. Methods,
158:57 (1993), report the use of palmitate-conjugated protein A for coating cells with artificial receptors which facilitate intercellular interactions. More specifically, a method is reported for attaching an antibody onto the surface of a cell using palmitated protein A. The article does not teach use of a lipidated protein for attachment of anything other than an antibody to a cell. As such, their modified cells serve only as artificial receptors for antigens.
Phillips et al.,
Immunity,
5:163-172 (August, 1996) report the preparation of a fusion protein using a CD8 leader segment, the Fc domain, of immunoglobin and the ectodomain of a type II membrane protein, CD94. The present transfer methods are applicable to both type I and type II proteins and are neither taught nor suggested in the article.
Darling, et al.,
Gene Therapy,
4(12):1350-60 (December, 1997) report the use of a biotin/avidin-based system for protein transfer. This method involves biotinylation of the target cell, attachment of an avidin group to the protein to be transferred, and combining the biotinylated target cell and the avidin-tagged protein. This method has significant limitations, including its dependence on covalent modifications that could perturb multiple proteins on cell surfaces.
There remains a need, therefore, for methods of efficient and quantitative transfer of proteins and peptides to cells. A further need is to provide such methods in which immunoregulatory molecules that retain their function can be attached to cells of interest.
SUMMARY OF THE INVENTION
The present invention has met the above needs, by providing methods for quantitative transfer of a domain having trans-signaling and/or adhesion function onto a cell surface. Typically, the domain will be the extracellular domain having one or both of these functions. In a preferred embodiment, the extracellular domain of an immuno-regulatory molecule is used. More specifically, the present methods provide a two-step protein transfer approach, which permits delivery of graded amounts of proteins to a cell surface. The methods utilize a fusion protein comprised of at least two domains, one of which preferably encodes a molecule having immunoregulatory function. By adding the fusion protein to cells coated with a lipidated protein, fine titration of, for example, the immunoregulatory molecule's extracellular domain is achieved.
The present protein transfer methods have wide application. For example, the methods have been used to establish that costimulator thresholds exist, and that the levels of surface costimulator on APCs can dictate both the magnitude and the quality of evoked T-cell responses. The present methods are also applicable to the generation of cancer vaccines; these vaccines show significant anti-tumor effects in vivo. Furthermore, the methods can be used to generate artificial veto cells, expressing one or more coinhibitors, that can be used to delete pathogenic T-cells. Cells produced according to the present methods are therefore useful in the treatment of cancer and also in the treatment of autoimmune diseases. The methodologies described herein can also be used in the establishment of animal models and for the study of immunological issues regarding, for example, T-cell activation, use of costimulators to override apoptotic signals, function of coinhibitors versus costimulators, synergy of costimulators used in the treatment of cancer, and use of coinhibitors in the treatment of autoimmune diseases.
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Brunschwig et al.,J. Immunolo.,155:5498 (1995).
McHugh et al.,Proc. Natl. Acad. Sci. USA, 92:8059 (1995).
McHugh et al.,Cancer Res.,59:2433 (1999).
Kim and Peacock,J. Immunol. Methods,158:57 (1993).
Phillips et al., “CD94 and a Novel Associated Protein (94AP) Form a NK Cell Receptor Involved in the Recognition of HLA-A, HLA-B, and HLA-C Allotypes,”Immunity,vol. 5:163-172 (Aug., 1996).
Darling et al., “In Vitro immune modulation by antibodies coupled to tumor cells”,Gene Therapy, 4(12):1350-60 (Dec. 1997).
Londo et al., “Lateral Diffusion of Antigen Receptors Artificially Incorporated Onto B Lymphocytes,”The Journal of Immunology, vol. 137, 1924-1931, No. 6 (Sep. 15, 1986).
Peacock et al., “Biologic Activity of Antigen Receptors Artificially Incorporated Onto B Lymphocytes”,The Journal of Immunology, vol. 137, 1916-1923, No. 6 (Sep. 15, 1986).
Colsky et al., “Surrogate Receptor-Mediated Cellular Cytotoxicity”,The Journal of Immunology, vol. 140, 2515-2519, No. 8, (Apr. 15, 1988).
Colsky and Peacock, “Palmitate-derivatized antibodies can function as surrogate receptors for mediating specific cell-cell interactions”,Journal of Immunological Methods, vol. 124, 179-187 (1989).
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Chen Aoshuang
Tykocinski Mark L.
Zheng Guoxing
Anderson Debra Z.
Eckert Seamans Cherin & Mellott , LLC
Harris Alana M.
Huff Sheela
Meyers Diane R.
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