Method and composition for altering a B cell mediated pathology

• US 20050238645A1
• Filed: 05/10/2004
• Published: 10/27/2005
• Est. Priority Date: 08/11/2000
• Status: Active Grant

• Current Assignee: Mmrglobal Incorporated
• Original Assignee: Favrille Inc (Mmrglobal Incorporated)
• Inventors: Shopes, Robert J., Gold, Daniel P.
• Granted Patent: US 8,114,404 B2
• Time in Patent Office: Days
• US Class Current: 424/144.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/1774 Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/195 Chemokines, e.g. RANTES
• CPC Class Codes: A61K 39/39541 against normal tissues, cells
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61K 47/646 the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention provides a method for altering a B cell mediated pathology in a patient. This method comprises administering a composition comprising at least one and/or two chimeric proteins. Each chimeric protein comprises at least a portion of either the V_H or V_L region of a immunoglobulin molecule from particular B cells from a patient having a B cell mediated pathology, and an immunoglobulin constant region. The genes encoding V_H and/or V_L regions and the genes encoding immunoglobulin constant regions are isolated and inserted into an expression vector. The chimeric proteins are produced by introducing the expression vectors into insect cell lines. The chimeric proteins are purified using antibody affinity columns, and then chemically conjugated to an immunogenic carrier, keyhole-limpet hemocyanin (KLH). Since the conjugates comprise chimeric proteins made specifically from particular B cells from a patient having B cell mediated pathology, when it is administered to such a patient, with or without a cytokine, such as granulocyte-macrophage-CSF, or a chemokine, it can induce immune responses to alter such a B cell mediated pathology.

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61. A composition comprising a first chimeric protein and a second chimeric protein produced by a process comprising the steps of:

(a) isolating a gene encoding at least a portion of a V_H region associated with a B cell clone of a patient having a B cell mediated pathology;

(b) linking said gene encoding said portion of a V_H region and a gene encoding at least a portion of an immunoglobulin constant region in an expression vector to form a gene encoding said first chimeric protein;

(c) isolating a gene encoding at least a portion of a V_L region associated with a B cell clone of said patient having said B cell mediated pathology;

(d) linking said gene encoding said portion of a V_L region and a gene encoding at least a portion of an immunoglobulin constant region into said expression vector to form a gene encoding said second chimeric protein;

(e) introducing said expression vector into insect cells;

(f) allowing the expression of said first and second chimeric proteins.

63. The composition produced by the process of claim 61 or claim 62, wherein said first chimeric protein is further conjugated to a carrier protein.

64. The composition produced by the process of claim 61 or claim 62, wherein said second chimeric protein is further conjugated to a carrier protein.

65. The composition produced by the process of claim 63 or claim 64 wherein said carrier protein is a keyhole-limpet hemocyanin (KLH).

66. The composition produced by the process of claim 61 or claim 62 wherein said composition is administered to a patient.

67. The composition produced by the process of claim 61 or claim 62 wherein said composition is administered to a patient together with a cytokine or a chemokine.

68. The composition of claim 67 wherein said composition is administered to a patient together with a cytokine wherein said cytokine is granulocyte-macrophage-colony stimulating factor (GM-CSF).

69. The composition produced by the process of claim 61 or claim 62 wherein said gene encoding said chimeric protein comprising a V_H region and a first immunoglobulin constant region is controlled by a p10 promoter in an expression vector, and said gene encoding said chimeric protein comprising a V_L region and a second first immunoglobulin constant region is controlled by a polyhedrin promoter in an expression vector.

71. The composition produced by the process of claim 61 or claim 62 wherein one of said genes encoding said immunoglobulin constant regions is a gene encoding at least a portion of a human κ

or λ

constant region.

72. The composition produced by the process of claim 61 or claim 62 wherein said gene encoding one of said immunoglobulin constant regions is a gene encoding at least a portion of an immunoglobulin constant region selected from the group consisting of a human IgG_γ

1 constant region, a human IgG<sub>γ

2</sub> constant region, a human IgG<sub>γ

3</sub> constant region, a human IgG<sub>γ

4</sub> constant region, a human IgA₁ constant region, a human IgA₂ constant region, a human IgM constant region, a human IgD constant region, a human IgE constant region, a human κ

, chain constant region, and a human λ

chain constant region.

73. The composition produced by the process of claim 72 wherein one of said genes encoding said immunoglobulin constant regions is a gene encoding at least a portion of a human IgG_γ

1 gene.

74. The composition produced by the process of claim 61 or claim 62 wherein said chimeric proteins are isolated using a protein selected from the group consisting of protein A, protein G, protein L, an anti-immunoglobulin antibody, and other proteins being able to bind to an immunoglobulin binding domain.

75. The composition produced by the process of claim 61 or claim 62 wherein said chimeric proteins are analyzed for expression by ELISA.

76. The composition produced by the process of claim 61 or claim 62 wherein said chimeric proteins are used to treat a B cell mediated pathology.

77. The composition produced by the process of claim 61 or claim 62 wherein said chimeric proteins are used to treat a B cell mediated pathology wherein said B cell mediated pathology is selected from the group consisting of refractory low grade lymphoma and follicular B cell lymphoma.

78. The composition produced according to the process of claim 61 wherein said expression vector is a baculovirus expression vector.

80. The composition produced according to the process of claim 61 wherein said expression vector is a baculovirus expression vector that comprises a honey bee melittin secretory signal sequence and a human placental alkaline phosphatase secretory signal sequence.

82. The composition produced according to the process of claim 61 wherein said expression vector is a baculovirus expression vector that comprises a baculovirus p10 promoter and polyhedrin promoter, wherein said p10 promoter controls a honey bee melittin secretory signal sequence, and wherein said polyhedrin promoter controls a human placental alkaline phosphatase secretory signal sequence.

83. The composition produced according to the process of claim 61 wherein said expression vector is a baculovirus expression vector that comprises a baculovirus p10 promoter and polyhedrin promoter, wherein said p10 promoter controls a honey bee melittin secretory signal sequence, and wherein said polyhedrin promoter controls a secretory signal sequence selected from the group consisting of a human placental alkaline phosphatase secretory signal sequence and a honey bee melittin secretory signal sequence.

84. The composition produced by the process of claim 61 wherein said chimeric proteins are produced in insect cells selected from the group consisting of the Trichoplusia ni cell line and the Spodoptera frugiperda cell line.

62. A composition comprising a first chimeric protein and a second chimeric protein produced by a process comprising the steps of:

(a) isolating a gene encoding at least a portion of a V_H region associated with a B cell clone of a patient having a B cell mediated pathology;

(b) linking said gene encoding said portion of a V_H region and a gene encoding at least a portion of an immunoglobulin constant region in an expression vector to form a gene encoding said first chimeric protein;

(c) isolating a gene encoding at least a portion of a V_L region associated with a B cell clone of said patient having said B cell mediated pathology;

(d) linking said gene encoding said portion of a V_L region and a gene encoding at least a portion of an immunoglobulin constant region into an expression vector to form a gene encoding said second chimeric protein;

(e) introducing said expression vectors into insect cells;

(f) allowing the expression of said first and second chimeric proteins.

70. The composition produced by the process of claim 62 wherein said gene comprising said V_H or V_L region and said gene encoding said immunoglobulin constant region in an expression vector is controlled by either a p10 promoter or a polyhedrin promoter.

79. The composition produced according to the process of claim 62 wherein said expression vectors are baculovirus expression vectors.

81. The composition produced according to the process of claim 62 wherein one of said expression vectors is a baculovirus expression vector that comprises a honey bee melittin secretory signal sequence and the other of said expression vectors is a baculovirus expression vector that comprises a human placental alkaline phosphatase secretory signal sequence.

85. A composition produced by expression of a gene encoding a first chimeric protein and by expression of a gene encoding a second chimeric protein from an expression vector in insect cells;

wherein said expression vector comprises both;

(a) a gene encoding said first chimeric protein

wherein said gene encoding said first chimeric protein comprises a gene encoding at least a portion of a V_H region from B cells of a patient having a B cell mediated pathology and a gene encoding at least a portion of an immunoglobulin constant region;

(b) a gene encoding said second chimeric protein

wherein said gene encoding said second chimeric protein comprises a gene encoding at least a portion of a V_L region from B cells of a patient having a B cell mediated pathology and a gene encoding at least a portion of an immunoglobulin constant region.

87. The composition produced according to the process of claim 85 or claim 86 wherein said expression vectors are baculovirus expression vectors.

88. The composition produced according to the process of claim 85 wherein said expression vector is a baculovirus expression vector that comprises a honey bee melittin secretory signal sequence and a human placental alkaline phosphatase secretory signal sequence.

89. The composition produced according to the process of claim 85 wherein said expression vector is a baculovirus expression vector that comprises a baculovirus p10 promoter and polyhedrin promoter, wherein said p10 promoter controls a honey bee melittin secretory signal sequence, and wherein said polyhedrin promoter controls a human placental alkaline phosphatase secretory signal sequence.

90. The composition produced according to the process of claim 85 wherein said expression vector is a baculovirus expression vector that comprises a baculovirus p10 promoter and polyhedrin promoter, wherein said p10 promoter controls a honey bee melittin secretory signal sequence, and wherein said polyhedrin promoter controls a secretory signal sequence selected from the group consisting of a human placental alkaline phosphatase secretory signal sequence and a honey bee melittin secretory signal sequence.

91. The composition produced according to the process of claim 85 or claim 86, wherein said first chimeric protein is conjugated to a carrier protein.

92. The composition produced according to the process of claim 85 or claim 86, wherein said second chimeric protein is conjugated to a carrier protein.

93. The composition of claim 90 or claim 91 wherein said chimeric protein is conjugated to a keyhole-limpet hemocyanin (KLH).

94. The composition produced by the process of claim 85 or claim 86 wherein said composition is administered to a patient.

95. The composition produced by the process of claim 85 or claim 86 wherein said composition is administered to a patient together with a cytokine or a chemokine.

96. The composition of claim 95 wherein said composition is administered to a patient together with a cytokine wherein said cytokine is granulocyte-macrophage-colony stimulating factor (GM-CSF).

97. The composition produced by the process of claim 85 or claim 86 wherein said gene encoding said chimeric protein comprising a V_H region and a first immunoglobulin constant region is controlled by a p10 promoter, and said gene encoding said chimeric protein comprising a V_L region and a second first immunoglobulin constant region is controlled by a polyhedrin promoter.

98. The composition produced by the process of claim 85 or claim 86 wherein said gene comprising said V_H or V_L region and said gene encoding said immunoglobulin constant region is controlled by either a p10 promoter or a polyhedrin promoter.

99. The composition produced by the process of claim 85 or claim 86 wherein one of said genes encoding at least a portion of said immunoglobulin constant regions is a gene encoding at least a portion of a human κ

or λ

constant region.

100. The composition produced by the process of claim 85 or claim 86 wherein said gene encoding at least a portion of said immunoglobulin constant regions is a gene encoding at least a portion of an immunoglobulin constant region selected from the group consisting of a human IgG_γ

1 constant region, a human IgG<sub>γ

2</sub> constant region, a human IgG<sub>γ

3</sub> constant region, a human IgG<sub>γ

4</sub> constant region, a human IgA₁ constant region, a human IgA₂ constant region, a human IgM constant region, a human IgD constant region, a human IgE constant region, a human k chain constant region, and a human 1 chain constant region.

101. The composition produced by the process of claim 102 wherein one of said genes encoding at least a portion of said immunoglobulin constant regions is a gene encoding a human IgG_γ

1.

102. The composition produced by the process of claim 85 or claim 86 wherein said chimeric proteins are produced in insect cells selected from the group consisting of the Trichoplusia ni cell line and the Spodoptera frugiperda cell line.

103. The composition produced by the process of claim 85 or claim 86 wherein said chimeric proteins are isolated using a protein selected from the group consisting of protein A, protein G, protein L, an anti-immunoglobulin antibody, and other proteins being able to bind to an immunoglobulin binding domain.

104. The composition produced by the process of claim 85 or claim 86 wherein said chimeric proteins are analyzed for expression by ELISA.

105. The composition produced by the process of claim 85 or claim 86 wherein said composition is used to treat a B cell mediated pathology.

106. The composition produced by the process of claim 85 or claim 86 wherein said composition is used to treat a B cell mediated pathology wherein said B cell mediated pathology is selected from the group consisting of refractory low grade lymphoma and follicular B cell lymphoma.

86. A composition produced by expression of a gene encoding a first chimeric protein from a first expression vector in insect cells and by expression of a gene encoding a second chimeric protein from a second expression vector in insect cells

(a) wherein said first expression vector comprises:

a gene encoding said first chimeric protein

wherein said gene encoding said first chimeric protein comprises a gene encoding at least

a portion of a V_H region from B cells of a patient having a B cell mediated pathology and

a gene encoding at least a portion of an immunoglobulin constant region;

(b) wherein said second expression vector comprises;

a gene encoding said second chimeric protein

wherein said gene encoding said second chimeric protein comprises a gene encoding at least a portion of a V_L region from B cells of a patient having a B cell mediated pathology and a gene encoding at least a portion of an immunoglobulin constant region.