Method for tapping the immunological repertiore
First Claim
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1. A method of producing an isolated coexpression vector capable of expressing a VH and VL containing molecule of predetermined specificity, which method comprises the steps:
- (a) synthesizing a VH-coding gene library containing a plurality of different VH-coding DNA sequences by a method comprising the steps;
(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different VH-coding sequences (ii) amplifying a plurality of VH-coding sequences in said polynucleotide containing composition, using amplification primers (b) synthesizing a VL-coding gene library containing a plurality of different VL-coding DNA sequences by a method comprising the steps;
(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different VL-coding sequences (ii) amplifying a plurality of VL-coding sequences in said polynucleotide containing composition, using amplification primers (c) joining in operable combination VH-coding sequences from said VH-coding gene library with VL-coding sequences from said VL-coding gene library into expression vectors to form cexpression vectors so as to be able to coexpress a VH-coding sequence and a VL-coding sequence from each vector, whereby a diverse library is formed, (d) selecting from said diverse library at least one coexpression vector capable of producing VH and VL containing molecules having the desired specificity.
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Abstract
The present invention relates to a method for isolating from the immunological gene repertoire a gene coding for a receptor having the ability to bind a preselected ligand. Receptors produced by the gene isolated by the method, particularly catalytic receptors, are also contemplated.
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Citations
58 Claims
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1. A method of producing an isolated coexpression vector capable of expressing a VH and VL containing molecule of predetermined specificity, which method comprises the steps:
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(a) synthesizing a VH-coding gene library containing a plurality of different VH-coding DNA sequences by a method comprising the steps;
(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different VH-coding sequences (ii) amplifying a plurality of VH-coding sequences in said polynucleotide containing composition, using amplification primers (b) synthesizing a VL-coding gene library containing a plurality of different VL-coding DNA sequences by a method comprising the steps;
(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different VL-coding sequences (ii) amplifying a plurality of VL-coding sequences in said polynucleotide containing composition, using amplification primers (c) joining in operable combination VH-coding sequences from said VH-coding gene library with VL-coding sequences from said VL-coding gene library into expression vectors to form cexpression vectors so as to be able to coexpress a VH-coding sequence and a VL-coding sequence from each vector, whereby a diverse library is formed, (d) selecting from said diverse library at least one coexpression vector capable of producing VH and VL containing molecules having the desired specificity. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
isolating a coexpression vector according to claim 1, transforming a host cell with said expression vector, isolating a VH and VL containing molecule encoded by said vector from said host cell.
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10. A method according to claim 1, wherein said VH and VL containing molecule of predetermined specificity is a catalytic antibody and said selecting step comprises the step of adding a substrate for a reaction catalyzed by said catalytic antibody.
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11. A method of producing an isolated coexpression vector capable of expressing a VH and VL containing molecule of predetermined specificity, which method comprises the steps:
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(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of VH-coding sequences (ii) amplifying a plurality of VH-coding sequences from said polynucleotide containing composition by a method of amplification comprising the step of adding primer sequences capable of hybridizing upstream and downstream from a plurality of said VH-coding sequences under conditions permitting hybridization to occur, wherein the number of primer sequences is less than the number of unique VH-coding sequences present in the polynucleotide containing composition, (iii) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of VL-coding sequences, (iv) amplifying a plurality of VL-coding sequences from said polynucleotide containing composition by a method of amplification comprising the step of adding primer sequences capable of hybridizing upstream and downstream from a plurality of said VL-coding sequences under conditions permitting hybridization to occur, wherein the number of primer sequences is less than the number of unique VL-coding sequences present in the polynucleotide containig composition, (v) joining in operable combination VH-coding sequences from said VH-coding library with VL-coding sequences from said VL-coding library into expression vectors so as to be able to coexpress a VH-coding sequence and a VL-coding sequence from each vector, whereby a diverse library is formed, and (vi) selecting from said diverse library at least one coexpression vector capable of producing VH and VL containing molecules having the desired specificity. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
isolating a coexpression vector according to claim 11, transforming a host cell with said expression vector, isolating a VH and VL containing molecule encoded by said vector from said host cell.
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20. A method according to claim 11, wherein said a VH and VL containing molecule of predetermined specificity is a catalytic antibody and said selecting step comprises the step of adding a substrate for a reaction catalyzed by said catalytic antibody.
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21. A method of producing an isolated coexpression vector capable of expressing a VH and VL containing molecule of predetermined specificity, which method comprises the steps:
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(a) producing a VH-coding library and a VL-coding library, by a method comprising the steps, (i) adding a first primer, wherein said first primer is capable of hybridizing to a primer binding site adjacent to a plurality of VH and VL coding sequences, and said coding sequences are present in a polynucleotide containing composition that comprises a plurality of different VH-coding sequences and VL-coding sequences, (ii) adding a second primer to said nucleotide containing composition, wherein said second primer is capable of hybridizing to a second primer binding site adjacent to a plurality of VH and VL coding sequences and said second primer binding site is not adjacent to said first primer binding site (iii) amplifying said plurality of VH and VL coding sequences (b) joining in operable combination VH-coding sequences from said VH-coding gene library with VL-coding sequences from said VL-coding gene library into expression vectors so as to be able to coexpress a VH-coding sequence and a VL-coding sequence from each vector, whereby a diverse library is formed, (c) selecting from said diverse library at least one coexpression vector capable of producing VH and VL containing molecules having the desired specificity. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30)
isolating a coexpression vector according to claim 21, transforming a host cell with said expression vector, isolating a VH and VL containing molecule encoded by said vector from said host cell.
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30. A method according to claim 21, wherein said VH and VL containing molecule of predetermined specificity is a catalytic antibody and said selecting step comprises the step of adding a substrate for a reaction catalyzed by said catalytic antibody.
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31. A method of producing a genetic library encoding a plurality of diverse VH encoding polynucleotide sequence and VL encoding polynucleotide sequence, which method comprises the steps:
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(a) synthesizing a VH-coding gene library containing a plurality of different VH-coding DNA sequences by a method comprising the steps;
(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different VH-coding sequences (ii) amplifying a plurality of VH-coding sequences in said polynucleotide containing composition, using amplification primers (b) synthesizing a VL-coding gene library containing a plurality of different VL-coding DNA sequences by a method comprising the steps;
(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different VL-coding sequences (ii) amplifying a plurality of VL-coding sequences in said polynucleotide containing composition, using amplification primers (c) joining in operable combination VH-coding sequences from said VH-coding gene library with VL-coding sequences from said VL-coding gene library into expression vectors so as to be able to coexpress a VH-coding sequence and a VL-coding sequence from a single each vector is selected from the group consisting of lambda and lambda zap II phage, whereby a diverse library is formed. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38)
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39. A method of producing a genetic library encoding a plurality of diverse VH and VL sequences, said method comprising the steps:
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(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of VH-coding sequences (ii) amplifying a plurality of VH-coding sequences from said polynucleotide containing composition by a method of amplification comprising the step of adding primer sequences capable of hybridizing upstream and downstream from a plurality of said VH-coding sequences under conditions permitting hybridization to occur, whereby a plurality of amplified VH-coding sequences are produced and said amplified VH-coding sequences form a VH-coding library, (iii) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of VL-coding sequences, and (iv) amplifying a plurality of VL-coding sequences from said polynucleotide containing composition by a method of amplification comprising the step of adding primer sequences capable of hybridizing upstream and downstream from a plurality of said VL-coding sequences under conditions permitting hybridization to occur, whereby a plurality of amplified VL-coding sequences are produced and said amplified VL-coding sequences form a VL-coding library, (v) joining in operable combination VH-coding sequences from said VH-coding gene library with VL-coding sequences from said VL-coding gene library into expression vectors so as to be able to coexpress a VH-coding sequence and a VL-coding sequence from a single vector, whereby a diverse library is formed. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47)
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48. A method of producing a genetic library encoding a plurality of diverse VH and VL sequences, said method comprising the steps:
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adding a first primer, wherein said first primer is capable of hybridizing to a first primer binding site adjacent to a plurality of VH and VL coding sequences, adding a second primer, wherein said second primer is capable of hybridizing to a second Primer biding site adjacent to a plurality of VH and VL coding sequences and said second conserved nucleotide sequence is not adjacent to said first conserved nucleotide sequence, amplifying a plurality of VH and VL coding sequences by a method of amplification employing said first and second primers, whereby a VH-coding library and a VL-coding library are formed, joining in operable combination VH-coding sequences from said VH-coding gene library with VL-coding sequences from said VL-coding gene library into expression vectors so as to be able to coexpress a VH-coding sequence and a VL-coding sequence from each vector, whereby genetic libray encoding a plurality of diverse VH and VL sequences is formed. - View Dependent Claims (49, 50, 51, 52, 53, 54, 58)
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- 55. An expression library comprising a plurality of nucleic acid sequences for expressing a plurality of at least 104 proteins each comprising a functional portion of an immunoglobulin heavy chain variable domain (VH domain) and a functional portion of an immunoglobulin light chain variable domain (VL domain).
Specification