Novel antigen binding molecules for therapeutic, diagnostic, prophylactic, enzymatic, industrial, and agricultural applications, and methods for generating and screening thereof
First Claim
1. A method for producing a library of nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, the method comprising:
- (a) providing a first nucleic acid encoding a first antigen binding site;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
, (c) using the set of mutagenic oligonucleotides to generate a set of antigen binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified antigen binding sites.
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Abstract
The invention is directed to methods for generating sets, or libraries, of nucleic acids encoding antigen-binding sites, such as antibodies, antibody domains or other fragments, including single and double stranded antibodies, major histocompatibility complex (MHC) molecules, T cell receptors (TCRs), and the like. This invention provides methods for generating variant antigen binding sites, e.g., antibodies and specific domains or fragments of antibodies (e.g., Fab or Fc domains), by altering template nucleic acids including by saturation mutagenesis, synthetic ligation reassembly, or a combination thereof. In one aspect, invention provides methods for generating all human or humanized antibodies and evolving them to achieve optimized properties related to stability, duration, expression, production, enzymatic activity, affinity, avidity, localization, and other immunological properties. Polypeptides generated by these methods can be analyzed using a novel capillary array platform, which provides unprecedented ultra-high throughput screening.
103 Citations
102 Claims
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1. A method for producing a library of nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, the method comprising:
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(a) providing a first nucleic acid encoding a first antigen binding site;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,(c) using the set of mutagenic oligonucleotides to generate a set of antigen binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified antigen binding sites. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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45. A library of nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, made by a method comprising the following steps:
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(a) providing a first nucleic acid encoding a first antigen binding site;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,(c) using the set of mutagenic oligonucleotides to generate a set of antigen binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified antigen binding sites.
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46. A method for producing from a library of variant antibodies from a template antibody, the method comprising:
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(a) providing a first nucleic acid encoding the template antibody;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,c) using the set of mutagenic oligonucleotides to generate a set of antibody-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant antibodies. - View Dependent Claims (47, 48, 49, 50)
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51. A library of variant antibodies derived from a template antibody made by a method comprising the following steps:
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(a) providing a first nucleic acid encoding the template antibody;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,c) using the set of mutagenic oligonucleotides to generate a set of antibody-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant antibodies.
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52. A method for producing from a library of variant T cell receptors (TCRs) from a template T cell receptor (TCR), the method comprising:
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(a) providing a first nucleic acid encoding the template T cell receptor;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,c) using the set of mutagenic oligonucleotides to generate a set of T cell receptor (TCR)-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant T cell receptors (TCRs).
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53. A library of variant T cell receptors (TCRs) derived from a template T cell receptor (TCR) made by a method comprising the following steps:
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(a) providing a first nucleic acid encoding the template T cell receptor;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,c) using the set of mutagenic oligonucleotides to generate a set of T cell receptor (TCR)-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant T cell receptors (TCRs).
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54. A method for producing from a library of variant major histocompatibility complex (MHC) molecules from a template major histocompatibility complex (MHC) molecule, the method comprising:
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(a) providing a first nucleic acid encoding the template major histocompatibility complex (MHC) molecule;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,c) using the set of mutagenic oligonucleotides to generate a set of major histocompatibility complex (MHC) molecule-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant major histocompatibility complex (MHC) molecules.
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55. A library of variant major histocompatibility complex (MHC) molecules derived from a template major histocompatibility complex (MHC) molecule made by a method comprising the following steps:
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(a) providing a first nucleic acid encoding the template major histocompatibility complex (MIC) molecule;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
,c) using the set of mutagenic oligonucleotides to generate a set of major histocompatibility complex (MHC) molecule-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant major histocompatibility complex (MHC) molecules.
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56. A method of making a set of nucleic acids encoding a set of antigen binding site variants comprising the steps of:
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(a) providing a template nucleic acid encoding an antigen-binding polypeptide;
(b) providing a plurality of oligonucleotides that encode all nineteen naturally-occurring amino acid variants at a single amino acid residue of the antigen-binding polypeptide; and
,(c) generating a set of progeny antigen binding site-encoding variant nucleic acids encoding a non-stochastic range of single amino acid substitutions at each amino acid codon that was mutagenized, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby making a set of nucleic acids encoding a set of antigen binding site variants. - View Dependent Claims (57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86)
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87. A method of making a set of antibody variants comprising the steps of:
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(a) providing a nucleic acid encoding an antibody;
(b) providing a plurality of oligonucleotides;
(c) generating a non-stochastic range of single amino acid substitutions at each amino acid codon, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby generating a set of variant nucleic acids; and
,(d) expressing the set of variant nucleic acids such that the antibody variants encoded by the variant nucleic acids are expressed. - View Dependent Claims (88, 89, 90)
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91. A method of identifying a variant of an antigen binding site comprising the steps of:
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(a) providing a nucleic acid encoding an antigen binding site;
(b) providing a set of oligonucleotides that encode all nineteen naturally-occurring amino acid variants at all residues of the antigen-binding site;
(c) incorporating the sequence of the oligonucleotides of step (b) into the nucleic acid of step (a) to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at each residue of the antigen binding site;
(d) expressing each of the variant nucleic acids as polypeptides and measuring the variant'"'"'s affinity to the antigen; and
,(e) identifying a variant of the antigen binding site by its increased or decreased antigen binding specificity as compared to the antigen binding affinity of the antigen binding site encoded by the nucleic acid of step (a). - View Dependent Claims (92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102)
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Specification