Antibody libraries
First Claim
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1. A method of making a library comprising synthetic polynucleotides that encode an antibody heavy chain containing at least about 104 unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences has an antibody CDRH3 amino acid sequence represented by the following formula:
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[TN1]-[DH]-[N2]-[H3-JH],the method comprising;
(a) providing a theoretical segment pool containing TN1, DH, N2, and H3-JH segments, wherein, the DH segments are provided according to the following steps;
(i) obtaining a set of polynucleotide sequences of human IGHD genes and alleles;
(ii) progressively deleting one or more nucleotide bases from the polynucleotide sequences of (i) on either or both of the 5′ and
3′
ends;
(iii) systematically adding 0, 1 and/or 2 or more nucleotides to the 5′ and
/or 3′
ends of the polynucleotide sequences from (ii);
(iv) translating the polynucleotide sequences of (iii) and selecting unique amino acid sequences to thereby provide the DH theoretical segment pool;
(b) providing a reference set of preimmune CDRH3 sequences having sequence diversities and length diversities similar to naturally occurring human antibody sequences before these sequences have undergone negative selection and/or hypermutation;
(c) utilizing combinations of the TN1, DH, N2, and H3-JH segments contained in the theoretical segment pool of (a) to identify the closest match(es) to each CDRH3 sequence in the reference set of (b);
(d) selecting segments from the closest match(es) identified in step (c) for inclusion in a library comprising synthetic polynucleotides that encode an antibody heavy chain; and
(e) synthesizing the synthetic polynucleotides that encode an antibody heavy chain;
wherein the diversity of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences is created by polynucleotides having CDRH3 sequences that are different from the CDRH3 sequences of other polynucleotides; and
wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4.
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Abstract
The present invention overcomes the inadequacies inherent in the known methods for generating libraries of antibody-encoding polynucleotides by specifically designing the libraries with directed sequence and length diversity.
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Citations
17 Claims
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1. A method of making a library comprising synthetic polynucleotides that encode an antibody heavy chain containing at least about 104 unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences has an antibody CDRH3 amino acid sequence represented by the following formula:
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[TN1]-[DH]-[N2]-[H3-JH],the method comprising; (a) providing a theoretical segment pool containing TN1, DH, N2, and H3-JH segments, wherein, the DH segments are provided according to the following steps; (i) obtaining a set of polynucleotide sequences of human IGHD genes and alleles; (ii) progressively deleting one or more nucleotide bases from the polynucleotide sequences of (i) on either or both of the 5′ and
3′
ends;(iii) systematically adding 0, 1 and/or 2 or more nucleotides to the 5′ and
/or 3′
ends of the polynucleotide sequences from (ii);(iv) translating the polynucleotide sequences of (iii) and selecting unique amino acid sequences to thereby provide the DH theoretical segment pool; (b) providing a reference set of preimmune CDRH3 sequences having sequence diversities and length diversities similar to naturally occurring human antibody sequences before these sequences have undergone negative selection and/or hypermutation; (c) utilizing combinations of the TN1, DH, N2, and H3-JH segments contained in the theoretical segment pool of (a) to identify the closest match(es) to each CDRH3 sequence in the reference set of (b); (d) selecting segments from the closest match(es) identified in step (c) for inclusion in a library comprising synthetic polynucleotides that encode an antibody heavy chain; and (e) synthesizing the synthetic polynucleotides that encode an antibody heavy chain; wherein the diversity of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences is created by polynucleotides having CDRH3 sequences that are different from the CDRH3 sequences of other polynucleotides; and wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of making a library comprising synthetic polynucleotides that encode an antibody heavy chain containing at least about 104 unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences has an antibody CDRH3 amino acid sequence represented by the following formula:
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[TN1]-[DH]-[N2]-[H3-JH],the method comprising; (a) providing a theoretical segment pool containing TN1, DH, N2, and H3-JH segments, wherein the DH segments are provided according to the following steps; (i) obtaining a set of polynucleotide sequences of human IGHD genes and alleles; (ii) translating the polynucleotide sequences of (i), wherein translation of a partial codon containing two untranslated bases is completed to produce a full codon if the two bases encode a single amino acid regardless of the third position; (iii) progressively deleting amino acid sequences of (ii) at their N- and/or C-termini until at least two amino acids remain; and (iv) collecting unique sequences from (iii) that contain 2 or more amino acids to thereby provide the DH theoretical segment pool; (b) providing a reference set of preimmune CDRH3 sequences having sequence diversities and length diversities similar to naturally occurring human antibody sequences before these sequences have undergone negative selection and/or hypermutation; (c) utilizing combinations of the TN1, DH, N2, and H3-JH segments contained in the theoretical segment pool of (a) to identify the closest match(es) to each CDRH3 sequence in the reference set of (b); (d) selecting segments from the closest match(es) identified in step (c) for inclusion in a library comprising synthetic polynucleotides that encode an antibody heavy chain; and (e) synthesizing the synthetic polynucleotides that encode an antibody heavy chain; wherein the diversity of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences is created by polynucleotides having CDRH3 sequences that are different from the CDRH3 sequences of other polynucleotides; and wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4. - View Dependent Claims (15, 16)
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17. A method of making a library comprising synthetic polynucleotides that encode an antibody heavy chain containing at least about 104 unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences has an antibody CDRH3 amino acid sequence represented by the following formula:
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[TN1]-[DH]-[N2]-[H3-JH],the method comprising; (a) providing a theoretical segment pool containing TN1, DH, N2, and H3-JH segments, wherein DH is a polypeptide corresponding to any of the DH polypeptides of Tables 11, 17-25 and 28, or a polypeptide produced by translation of any of the DH-encoding polynucleotides of Tables 16, 25 and 27; (b) providing a reference set of preimmune CDRH3 sequences having sequence diversities and length diversities similar to naturally occurring human antibody sequences before these sequences have undergone negative selection and/or hypermutation; (c) utilizing combinations of the TN1, DH, N2, and H3-JH segments contained in the theoretical segment pool of (a) to identify the closest match(es) to each CDRH3 sequence in the reference set of (b); (d) selecting segments from the closest match(es) identified in step (c) for inclusion in a library comprising synthetic polynucleotides that encode an antibody heavy chain; and (e) synthesizing the synthetic polynucleotides that encode an antibody heavy chain; wherein the diversity of the polynucleotides encoding the at least about 104 unique antibody CDRH3 amino acid sequences is created by polynucleotides having CDRH3 sequences that are different from the CDRH3 sequences of other polynucleotides; and wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4.
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Specification