Human C3b/C4b receptor (CR1)

Details Human C3b/C4b receptor (CR1) Human C3b/C4b receptor (CR1)

1995-06-05

2001-11-13

Romeo, David S. (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Separation or purification

C530S350000, C530S300000, C530S412000, C530S414000, C530S417000, C530S418000, C435S069100, C435S173300, C435S252300, C514S002600

Reexamination Certificate

active

06316604

ABSTRACT:

TABLE OF CONTENTS
Page
 1.
Introduction
8
 2.
Background of the Invention
8
2.1.
The Complement System
8
2.2.
The C3b/C4b Complement Receptor (CR1)
9
2.3.
Abnormalities of CR1 in Human Disease
12
 3.
Summary of the Invention
14
3.1.
Definitions
15
 4.
Description of the Figures
15
 5.
Detailed Description of the Invention
23
5.1.
Isolation of the CR1 Gene
24
5.2.
Expression of the Cloned CR1 Gene
30
5.3.
Identification and Purification of the
34
Expressed Gene Product
5.4.
Structure of the CR1 Gene and Protein
36
5.4.1.
Genetic Analysis
36
5.4.2.
Protein Analysis
37
5.5.
CR1-Related Derivatives, Analogues, and
39
Peptides
5.6.
Uses of CR1
40
5.6.1.
Assays and Diagnosis
40
5.6.2.
Therapy
42
 6.
Example: The Cloning and Sequencing of the Human
45
C3b/C4b Receptor (CR1)
6.1.
Materials and Methods
46
6.1.1.
Isolation and Sequence of CR1 Tryptic
46
Peptides
6.1.2.
Isolation of cDNA Clones and Genomic
46
Clones
6.1.3.
DNA Sequence Analysis
47
6.2.
Results
48
6.2.1.
Nucleotide Sequence of the CR1 Gene
48
6.2.2.
Analysis of the Nucleotide and Amino
49
Acid Sequence of CR1
6.3.
Discussion
54
 7.
Example: CR1 5′ cDNA Sequences Contain a Fourth
59
Long Homologous Repeat
7.1.
Materials and Methods
59
7.1.1.
Construction of a cDNA Library
59
7.1.2.
Isolation of Clones, Probes, and
60
DNA Sequence Analysis
7.2.
Results
60
7.3.
Discussion
63
 8.
Example: Expression of Recombinant Human CR1
64
8.1.
Construction of pBSABCD Containing the
65
Entire CR1 Coding Sequence
8.2.
Construction and Assay of Plasmid piABCD,
68
a Mammalian Expression Vector Containing the
Entire CR1 Coding Sequence
8.3.
Expression of CR1 Fragments
71
8.3.1.
Construction of Deletion Mutants
71
piBCD, piABD, piACD, piAD, piBD,
piCD and piD
8.3.2.
Construction of Deletion Mutants piP1,
74
piE1, piE2, piE-2, piU1, piU-2 and
piA/D
 9.
Example: Identification of C3b and C4b Binding
76
Domains
9.1.
Assays and Results
76
9.2.
Discussion
79
10.
Example: Demonstration of Factor I Cofactor
81
Activity
11.
Example: Expression of Recombinant Soluble CR1
83
11.1.
Materials and Methods
84
11.1.1.
Enzyme Digestions
84
11.1.2.
DNA Fragment Isolations
84
11.1.3.
Transfection into Mammalian Cells
85
11.1.4.
CHO Transfectant Cell Culture
85
11.1.5.
ELISA for the Detection of
86
CR1 Levels
11.1.5.1.
CR1 Standards
86
11.1.5.2.
CR1 ELISA
86
11.2.
Genetic Modifications of CR1 Coding Sequences
87
11.2.1.
Construction of pBSCR1c
88
11.2.2.
Construction of pBSCR1s
89
11.2.3.
Construction of pBM-CR1c
89
11.2.4.
Construction of Deletion Mutants
90
pT-CR1c1, pT-CR1c2, pT-CR1c3,
pT-CR1c4, and pT-CR1c5
11.2.4.1.
pT-CR1c1
90
11.2.4.2.
pT-CR1c2
91
11.2.4.3.
pT-CR1c3
91
11.2.4.4.
pT-CR1c4
92
11.2.4.5.
pT-CR1c5
92
11.3.
Expression of Soluble CR1
93
11.3.1.
Construction of pTCS Series of
93
Expression Vectors
11.3.1.1.
Construction of pEAXgpt
94
11.3.1.2.
Construction of pMLEgpt
96
11.3.1.3.
Construction of pTCSgpt
96
11.3.1.4.
Construction of pTCSdhfr
97
11.3.1.5.
Construction of pTCSneo
97
11.3.2.
Expression and Assay of Plasmids
97
pBSCR1c, pBSCR1s and pBM-CR1c,
Mammalian Expression Vectors
Containing Soluble CR1 Coding
Sequences
11.3.2.1.
Expression of CR1
98
Constructs Truncated at
Different Positions
Within the CR1 cDNA
11.3.2.2.
Expression of sCR1c in
101
Two Different Expression
Systems
11.3.3.
Expression and Assay of Plasmids
102
pT-CR1c1, pT-CR1c2, pT-CR1c3,
pT-CR1c4, and pT-CR1c5, Mammalian
Expression Vectors Containing
Soluble CR1 Coding Sequences
12.
Example: Production and Purification of Soluble CR1
103
12.1.
Large Scale Production of Soluble CR1
104
12.1.1.
Production of sCR1 in Serum-Free Media
105
12.1.2.
Conclusions
107
12.2.
Purification of Soluble CR1
107
12.2.1.
Antibody Affinity Column
108
Purification
12.2.1.1.
Methods
108
12.2.1.2.
Results
108
12.2.2.
CR1 Purification by HPLC
109
12.2.2.1.
Methods
109
12.2.2.1.1.
Starting Material
12.2.2.1.2.
Cation Exchange
109
HPLC Procedure
12.2.2.1.3.
Anion Exchange
110
HPLC Procedure
12.2.2.1.4.
Western Blot
110
Analysis
12.2.2.2.
Results
110
12.2.2.3.
Characterization of
111
Purified Soluble CR1
12.2.2.4.
Conclusions
112
13.
Example: Demonstration of In Vitro Activity of
112
Soluble CR1
13.1.
Inhibition of the Neutrophil Oxidative Burst
112
13.1.1.
Materials and Methods
113
13.1.1.1.
Materials
113
13.1.1.2.
Preparation of
113
Neutrophils
13.1.1.3.
Preparation of Yeast
114
Particles
13.1.1.4.
Activation of
114
Neutrophils by
Purified C5a
13.1.1.5.
Activation of
114
Neutrophils by
Purified C5a in Human
Serum or Plasma
13.1.1.6.
Activation of
114
Neutrophils by Yeast
Particle-Activated Human
Serum or Plasma
13.1.2.
Results
115
13.1.2.1.
C5a Induces an Oxygen
115
Burst in Human Neutro-
phils Which Can be
Measured Using DCFDA
13.1.2.2.
Human Serum Blocks the
115
Oxygen Burst Effects of
Purified C5a on
Neutrophils
13.1.2.3.
Heparinized Plasma does
115
not Block the Effects of
C5a on Neutrophils
13.1.2.4.
sCR1 Present During
116
Complement Activation
Blocks C5a Generation
13.2.
Inhibition of Complement Mediated Hemolysis
116
13.2.1.
Methods
116
13.2.2.
Results
117
13.3.
Inhibition of C3a and C5a Production
120
13.3.1.
Methods
120
13.3.2.
Results
121
14.
Example: Demonstration of Functional In Vivo
121
Therapeutic Activity of Soluble CR1
14.1.
Soluble CR1 Demonstrates In Vivo Function
121
in a Reversed Passive Arthus Reaction
14.1.1.
Materials and Methods
122
14.1.2.
Results
123
14.1.3.
Effect of Intradermal Administration
123
of Soluble CR1
14.2.
Pharmacokinetics of In Vivo Administered sCR1
124
14.3.
sCR1 Reduces Infarct Size in Rats with
126
Reperfused Infarcted Myocardium
14.3.1.
Methods
126
14.3.1.1.
Induction of Rat
126
Myocardial Infarct
14.3.1.2.
Morphological Analysis of
127
Experimental Infarcts:
Preparation of Hearts
for Study
14.3.2.
Results
128
14.3.3.
Conclusions
128
15.
Deposit of Microorganisms
128
1. INTRODUCTION
The present invention relates to the C3b/C4b receptor (CR1) gene and its encoded protein. The invention also relates to CR1 nucleic acid sequences and fragments thereof comprising 70 nucleotides, and their encoded peptides or proteins comprising 24 amino acids. The invention also provides for the expression of the CR1 protein and fragments thereof. The CR1 nucleic acids and proteins have use in the diagnosis or therapy of disorders involving complement activity, and various inflammatory and immune disorders.
2. BACKGROUND OF THE INVENTION
2.1. The Complement System
The complement system is a group of proteins that constitutes about 10 percent of the globulins in the normal serum of humans (Hood, L. E., et al., 1984, Immunology, 2d Ed., The Benjamin/Cummings Publishing Co., Menlo Park, Calif., p. 339). Complement (C) plays an important role in the mediation of immune and allergic reactions (Rapp, H. J. and Borsos, T, 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts (Meredith), New York). The activation of complement components leads to the generation of a group of factors, including chemotactic peptides that mediate the inflammation associated with complement-dependent diseases. The sequential activation of the complement cascade may occur via the classical pathway involving antigen-antibody complexes, or by an alternative pathway which involves the recognition of certain call wall polysaccharides. The activities mediated by activated complement proteins include lysis of target calls, chemotaxis, opsonization, stimulation of vascular and other smooth muscle cells, and functional aberrations such as dogranulation of mast cells, increased permeability of small blood vessels, directed migration of leukocytes, and activation of B lymphocytes and macrophages (Eisen, H. N., 1974, Immunology, Harper.& Row Publishers, Inc. Hagerstown, Md., p. 512).
During proteolytic cascade steps, biologically active peptide fragments, the anaphylatoxins C3a, C4a, and C5a (See WHO Scientific Group, 1977, WHO Tech. Rep. Ser. 606:5 and references cited therein), are released from the third (C3), fourth (C4), and fifth (C5) native complement components (Hugli, T. E., 1981, CRC Crit. Rev. Immunol. 1:321; Bult, H. and Herman, A. G., 198

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