Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-07-10
2002-12-17
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C424S192100
Reexamination Certificate
active
06495347
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to fusion proteins and methods of stimulating a Th1-like response in vitro.
BACKGROUND
T lymphocytes can generally be divided into two classes based upon expression of the CD4 and CD8 antigens. The immune response mediated by CD4+ T cells is restricted by class II major histocompatibility complex (MHC) molecules. CD4+ T cells, also known as helper T lymphocytes, carry out their helper functions via the secretion of lymphokines. The immune response mediated by CD8+ T cells is restricted by class I MHC molecules. CD8+ T cells, also known as cytolytic T lymphocytes (CTLs), carry out cell mediated cytotoxicity and also secrete some lymphokines upon activation.
CD4+ T cells can be further divided into Th1 and Th2 subsets. Th1 cells participate in cell mediated immunity by producing lymphokines, such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-beta, that activate cell mediated immunity. Th2 cells provide help for humoral immunity by secreting lymphokines that stimulate B cells, such as IL-4 and IL-5. Antigenic stimuli that activate either the Th1 or Th2 pathway can inhibit the development of the other. For example, IFN-gamma produced by a stimulated Th1 cell can inhibit the formation of Th2 cells, and IL-4 produced by a stimulated Th2 cell can inhibit the formation of Th1 cells.
Certain disease conditions, such as cancer, allergy, and parasitic infections, are characterized by a predominantly Th2 response. Under certain circumstances, the induction of the Th1 response, typified by the production of IFN-gamma, may ameliorate these conditions.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a cell sample containing naive lymphocytes can be stimulated in vitro to exhibit a Th1-like response.
Accordingly, the invention features a method of determining whether a fusion protein stimulates a Th1-like response by: (a) providing a cell sample containing naive lymphocytes in vitro; (b) providing a fusion protein containing (i) a heat shock protein (Hsp) or a fragment thereof at least eight amino acid residues in length, fused to (ii) a heterologous polypeptide at least eight amino acid residues in length; (c) contacting the cell sample with the fusion protein; and (d) determining whether the fusion protein stimulates a Th1-like response in the cell sample.
“Naive lymphocytes” are lymphocytes that have not been exposed to the fusion protein (in vivo or in vitro) prior to their use in a method the invention. An “Hsp” is a polypeptide consisting of a sequence that is at least 40% identical to that of a protein whose expression is induced or enhanced in a cell exposed to stress, e.g., heat shock. A “fusion protein” is a non-naturally occurring polypeptide containing amino acid sequences derived from at least two different proteins.
The Hsp used in the method can be selected from the group consisting of Hsp65, Hsp40, Hsp10, Hsp60, and Hsp71. Additionally, the fusion protein can contain the full amino acid sequence of any of Hsp65, Hsp40, Hsp10, Hsp60, or Hsp71. In some embodiments, the fusion protein contains a fragment of an Hsp, e.g., amino acids 1-200 of Hsp65 of
Mycobacterium bovis.
The heterologous polypeptide can contain a sequence identical to at least eight consecutive amino acids of (i) a protein of a human pathogen, e.g., a virus, or (ii) a tumor associated antigen. Examples of viruses include human papilloma virus (HPV), herpes simplex virus (HSV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza virus, measles virus, and human immunodeficiency virus (HIV). The heterologous polypeptide can contain an HPV E6 antigen, e.g., HPV16 E6, an HPV E7 antigen, e.g., HPV16 E7, or a fragment of any of these antigens that is at least eight amino acid residues in length.
In one example, the fusion protein contains
Mycobacterium bovis
BCG Hsp65 and HPV16 E7.
The cell sample used in the methods of the invention can contain cells derived from a spleen, lymph node, peripheral blood, bone marrow, thymus, lung, respiratory tract, or anogenital mucosa. In preferred embodiments, the cells are splenocytes or lymph node cells.
The stimulation of a Th1-like response can be determined by detecting the presence of a lymphokine produced by the cell sample, e.g. IFN-gamma or TNF-beta.
In one embodiment, the method also includes the steps of: (e) providing a second cell sample containing naive lymphocytes; (f) contacting the second cell sample with a second fusion protein; and (g) determining whether the second fusion protein stimulates a Th1-like response in the second cell sample. In this example, the first fusion protein contains the sequence of a full-length, naturally occurring Hsp, and the second fusion protein contains at least eight amino acids but less than all of the sequence of a naturally occurring Hsp.
In another aspect, the invention features a method of screening a compound by: (a) providing a cell sample containing naive lymphocytes in vitro; (b) providing a fusion protein containing (i) a Hsp or a fragment thereof at least eight amino acid residues in length, fused to (ii) a heterologous polypeptide at least eight amino acid residues in length; (c) contacting the cell sample with the compound and the fusion protein; and (d) determining whether the cell sample exhibits a Th1-like response following the contacting step. In this method, a decrease in the Th1-like response in the presence of the compound compared to in the absence of the compound indicates that the compound inhibits a Th1-like response by the cell sample.
The invention also includes a method of screening a compound by: (a) providing a cell sample containing naive lymphocytes in vitro; (b) providing a fusion protein containing (i) a Hsp or a fragment thereof at least eight amino acid residues in length, fused to (ii) a heterologous polypeptide at least eight amino acid residues in length; (c) contacting the cell sample with the compound and the fusion protein; and (d) determining whether the cell sample exhibits a Th1-like response following the contacting step. In this method, an increase in the Th1-like response in the presence of the compound compared to in the absence of the compound indicates that the compound promotes a Th1-like response by the cell sample.
In another aspect, the invention features a method of determining whether a hybrid compound stimulates a Th1-like response by: (a) providing a cell sample containing naive lymphocytes in vitro; (b) providing a hybrid compound that is non-naturally occurring and contains (i) a non-peptide compound having a molecular weight of less than 1,500, covalently linked to (ii) a polypeptide of at least eight amino acids in length, wherein the hybrid compound is made by covalently linking the non-peptide compound to the polypeptide; (c) contacting the cell sample with the hybrid compound; and (d) determining whether the hybrid compound stimulates a Th1-like response in the cell sample. In one embodiment, the non-peptide compound has a molecular weight of at least 100.
In another aspect, the invention features a method of determining whether a hybrid compound stimulates a Th1-like response by: (a) producing a hybrid compound by covalently linking a non-peptide compound to a polypeptide of at least eight amino acids in length; (b) providing a cell sample containing naive lymphocytes in vitro; (c) contacting the cell sample with the hybrid compound; and (d) determining whether the hybrid compound stimulates a Th1-like response in the cell sample. In one embodiment, the non-peptide compound has a molecular weight between 100 and 1,500.
In another aspect, the invention features a method of determining whether a fusion protein stimulates a Th1-like response by: (a) providing a cell sample containing naive lymphocytes in vitro; (b) providing a fusion protein comprising (i) a first polypeptide at least eight amino acids in length, fused to (ii) a second polypeptide at least eight amino acids in length; (c) contacting the cell sam
Chu N. Randall
Mizzen Lee A.
Siegel Marvin
Fish & Richardson P.C.
Park Hankyel T.
Stressgen Biotechnologies Corporation
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