Method of screening complex protein libraries to identify altered properties

Abstract

The invention provides methods of making designed and constructed protein (e.g., antibody) libraries and libraries resulting from the same.

What is claimed is: 
     
         1 . A method of making a library of proteins, the method comprising the steps of:
 (a) constructing a library of nucleic acid molecules, each encoding a protein or portion thereof, the protein or portion thereof containing a single point mutation in a selected region as compared to a reference protein, wherein the point mutation is selected from the group of all twenty naturally occurring amino acids; and   (b) sequencing the nucleic acid molecules encoding the proteins; thereby identifying the single point mutation in each nucleic acid molecule.   
     
     
         2 . The method of  claim 1 , wherein the protein is an antibody. 
     
     
         3 . The method of  claim 2 , wherein the antibody is selected from the group consisting of IgA, IgG, and IgM. 
     
     
         4 . The method of  claim 2 , wherein the selected region is selected from the group consisting of a heavy chain complementarity determining region (CDR), a light chain CDR, a heavy chain framework (FR) region, a light chain FR, heavy chain constant region, and light chain constant region. 
     
     
         5 . The method of  claim 4 , wherein the heavy chain CDR is selected from the group consisting of CDR1, CDR2, and CDR3. 
     
     
         6 . The method of  claim 4  wherein the light chain CDR is selected from the group consisting of CDR1, CDR2, and CDR3. 
     
     
         7 . The method of  claim 4 , wherein the heavy chain FR is selected from the group consisting of FR1, FR2, FR3, and FR4. 
     
     
         8 . The method of  claim 4 , wherein the light chain FR is selected from the group consisting of FR1, FR2, FR3, and FR4. 
     
     
         9 . The method of  claim 1 , wherein the nucleic acid molecules containing a single point mutation are constructed using NNK codons. 
     
     
         10 . The method of  claim 9 , wherein the NNK codons are optimized such that for each NNK codon, A, T, C and G are each represented in said library at a frequency of 20-35% at each of the first and second position of said NNK codon. 
     
     
         11 . The method of  claim 1 , wherein the protein is displayed on the surface of the cell. 
     
     
         12 . The method of  claim 11 , wherein the cell is a bacteria cell, a yeast cell, or a mammalian cell. 
     
     
         13 . The method of  claim 11 , wherein the protein is displayed on the surface of the cell as a transmembrane domain anchor fusion protein. 
     
     
         14 . The method of  claim 12  is from the B7 or the PDGF receptor protein. 
     
     
         15 . The method of  claim 11 , wherein the cells are sorted using a single selection step. 
     
     
         16 . The method of  claim 11 , wherein the cells are sorted using FACS. 
     
     
         17 . The method of  claim 1 , wherein the cells are sorted using FACS and a fluorescently labeled antigen and a fluorescently labeled anti-IgG antibody. 
     
     
         18 . The method of  claim 1 , wherein the nucleic acid molecule is sequenced using capillary gel electrophoresis or parallel array-based pyrosequencing. 
     
     
         19 . The method of  claim 1 , wherein the binding affinity of at least one member of the library is altered as compared to a reference protein. 
     
     
         20 . The method of  claim 19 , wherein said member's binding affinity is increased as compared to the reference protein. 
     
     
         21 . The method of  claim 19 , wherein said member's binding affinity is decreased as compared to the reference protein. 
     
     
         22 . The method of  claim 19 , wherein said member's binding affinity is similar when compared to the reference protein. 
     
     
         23 . The method of  claim 1 , wherein the expression level of at one member of the library is altered as compared to the reference protein. 
     
     
         24 . The method of  claim 1 , wherein the library has up to 10,000 members that are screened and sequenced at the same time. 
     
     
         25 . The method of  claim 1 , wherein the library has up to 5000 members that are screened and sequence at the same time. 
     
     
         26 . The method of  claim 1 , wherein the library has up to 1000 members that are screened and sequenced at the same time. 
     
     
         27 . The method of  claim 1 , wherein the library has up to 100 members that are screened and sequenced at the same time. 
     
     
         28 . The method of  claim 1 , wherein at least 80% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         29 . The method of  claim 28 , wherein at least 90% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         30 . The method of  claim 29 , wherein at least 95% of possible variants in said protein or portion thereof are represented in said library 
     
     
         31 . A library of nucleic acid molecules encoding proteins or portions thereof, each with a single point mutation in a selected region, wherein the point mutation is selected from the group of all twenty naturally occurring amino acids and wherein the library has less than 10,000 members. 
     
     
         32 . The library of  claim 31 , wherein at least 80% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         33 . The library of  claim 32 , wherein at least 90% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         34 . The method of  claim 33 , wherein at least 95% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         35 . A library of proteins or portions thereof, each with a single point mutation in a selected region, wherein the point mutation is selected from the group of all twenty naturally occurring amino acids and wherein the library has less than 10,000 members. 
     
     
         36 . The library of  claim 35 , wherein at least 80% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         37 . The library of  claim 36 , wherein at least 90% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         38 . The library of  claim 37 , wherein at least 95% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         39 . A cell population displaying on the cell surface a library of proteins or portions thereof, each with a single point mutation in a selected region, wherein the point mutation is selected from the group of all twenty naturally occurring amino acids, and wherein the library has less than 10,000 members. 
     
     
         40 . The cell population of  claim 39 , wherein at least 80% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         41 . The cell population of  claim 40 , wherein at least 90% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         42 . The cell population of  claim 41 , wherein at least 95% of possible variants in said protein or portion thereof are represented in said library. 
     
     
         43 . An information storage medium comprising a listing of the amino acid position and frequency of occurrence of the point mutations generated using the method of  claim 1 . 
     
     
         44 . A method for determining in parallel the effect of a large number of single amino acid substitutions in a polypeptide, the method comprising the steps of
 (a) constructing a library of nucleic acid molecules encoding polypeptide variants the differ by only a single amino acid substitution;   (b) transforming a host cell with the library, such that each cell expresses and displays only a single type of variant on its surface;   (c) contacting the transformed cells with a target molecule and sorting the cells into subpopulations based on the interaction with the target molecule, such that the proportion of each variant is either enriched, depleted, or unaltered in the subpopulation depending on whether the interaction with the target molecule is enhanced, diminished, or unaltered in that variant;   (d) sequencing the sorted subpopulations to determine if the frequency of each variant is enriched, depleted, or unaltered in the subpopulation as compared to the starting population or a different subpopulation; and   (e) analyzing the extent of the depletion or enrichment to determine the magnitude of the altered interaction with the target molecule.   
     
     
         45 . A method of making a library of antibody variable domains each with a single point mutation, the method comprising the steps of:
 (a) constructing a library of less than 10,000 nucleic acid molecules, each encoding an antibody variable domain, the variable domain containing a single point mutation in a selected region as compared to a reference variable domain, wherein the point mutation is selected from the group of all twenty naturally occurring amino acids, and wherein NNK codons are used to construct the point mutations;   (b) subcloning the nucleic acid molecules into vectors comprising an Epstein-Barr virus replication origin and nuclear antigen, and expressing each nucleic acid molecule in a mammalian cell, wherein the variable domain encoded by the nucleic acid is displayed as part of a full length IgG-transmembrane anchor fusion protein on the surface of the cell;   (c) sorting the cells displaying the fusion protein using a fluorescently labeled antigen and a fluorescently labeled anti-IgG antibody; and   (d) sequencing the nucleic acid molecules encoding the variable domains using parallel array-based pyrosequencing; thereby identifying the single point mutation in each nucleic acid molecule.   
     
     
         46 . A nucleic acid population comprising at least 80 different nucleic acid molecules, each of said nucleic acid molecules encoding a different variant of a reference protein, wherein the nucleic acid molecules in said population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said reference protein. 
     
     
         47 . The nucleic acid population of  claim 46 , wherein the population comprises at least 90 different nucleic acid molecules, and wherein said nucleic acid molecules encode at least 18 variants for each amino acid over said region of at least 5 consecutive amino acids. 
     
     
         48 . The nucleic acid population of  claim 47 , wherein the population comprises at least 95 different nucleic acid molecules, and wherein said nucleic acid molecules encode all 19 variants for each amino acid over said region of at least 5 consecutive amino acids. 
     
     
         49 . The nucleic acid population of any one of  claims 46  to  48  wherein the reference protein is an antibody or an antigen-binding portion thereof, and wherein said region is a complementarity determining region (“CDR”). 
     
     
         50 . A host cell population comprising at least 80 different host cells, each host cell comprising a single member of the nucleic acid population of any one of  claims 46  to  48 , in one or more copies, extrachromosomal or chromosomally integrated. 
     
     
         51 . The host cell population of  claim 50 , wherein said reference protein is an antibody or an antigen-binding portion thereof, wherein said region is a CDR, such that the variants encoded by said nucleic acid molecules are CDR variant antibodies or antigen-binding portions thereof. 
     
     
         52 . The host cell population of  claim 51 , wherein each host cell expresses on its surface a CDR variant antibody or antigen-binding portion thereof. 
     
     
         53 . The host cell population of  claim 52 , wherein each host cell is a mammalian host cell. 
     
     
         54 . A nucleic acid library comprising three nucleic acid populations according to  claim 49 , wherein each of said three nucleic acid populations comprises nucleic acid molecules encoding variants of a different CDR of the same antibody chain. 
     
     
         55 . The library of  claim 54 , wherein the antibody chain is an antibody light chain. 
     
     
         56 . The library of  claim 54 , wherein the antibody chain is an antibody heavy chain. 
     
     
         57 . A nucleic acid library comprising six nucleic acid populations according to  claim 49 , wherein each of said six nucleic acid populations comprises nucleic acid molecules encoding variants of a different CDR of the same antibody. 
     
     
         58 . The nucleic acid library of  claim 57  which comprises nucleic acid molecules encoding at least 750 different CDR variants of the same antibody. 
     
     
         59 . The nucleic acid library of  claim 58  which comprises nucleic acid molecules encoding 900 to 1100 CDR variants of the same antibody. 
     
     
         60 . A host cell library comprising at least 500 host cells, each host cell containing a different member of the nucleic acid library of  claim 57 , in one or more copies, extrachromosomal or chromosomally integrated. 
     
     
         61 . The host cell library of  claim 60 , wherein each host cell expresses on its surface a CDR variant antibody or antigen-binding portion thereof. 
     
     
         62 . The host cell library of  claim 61 , wherein each host cell is a mammalian host cell. 
     
     
         63 . A host cell library comprising at least 750 different host cells, each host cell containing a different member of the nucleic acid library of  claim 58 , in one or more copies, extrachromosomal or chromosomally integrated. 
     
     
         64 . The host cell library of  claim 63 , wherein each host cell expresses on its surface a CDR variant antibody or antigen-binding portion thereof. 
     
     
         65 . The host cell library of  claim 64 , wherein each host cell is a mammalian host cell. 
     
     
         66 . A host cell library comprising at 900 to 1100 different host cells, each host cell containing a different member of the nucleic acid library of  claim 59 , in one or more copies, extrachromosomal or chromosomally integrated. 
     
     
         67 . The host cell library of  claim 66 , wherein each host cell expresses on its surface a CDR variant antibody or antigen-binding portion thereof. 
     
     
         68 . The host cell library of  claim 67 , wherein each host cell is a mammalian host cell. 
     
     
         69 . A method for assessing the binding affinity of an antibody variant to a target molecule as compared to a reference antibody:
 (a) contacting the host cell library of  claim 64  with a target molecule recognized by said antibody or antigen-binding fragment;   (b) sorting the host cells into at least two populations:
 (i) a first subpopulation based on antibody expression; and 
 (ii) a second subpopulation based on binding affinity to the target molecule as compared to the reference antibody; 
   (c) identifying a variant that is enriched in the second subpopulation as compared to the first subpopulation, the starting population, or another population containing the first subpopulation;   thereby assessing the binding affinity of an antibody variant to a target molecule as compared to a reference antibody.   
     
     
         70 . The method of  claim 69 , wherein the host cell is sorted into four populations in step (b):
 (i) a first subpopulation based on antibody expression;   (ii) a second subpopulation based on higher binding affinity to the target molecule as compared to the reference antibody;   (iii) a third subpopulation based on lower binding affinity to the target molecule as compared to the reference antibody;   (iv) a fourth subpopulation based on equal binding affinity to the target molecule as compared to the reference antibody,   and wherein the variant is identified as enriched in the second subpopulation as compared to the a population containing the first subpopulation, the second subpopulation the third subpopulation, and the fourth subpopulation, such that a variant with higher binding affinity to the target molecule as compared to the reference antibody is identified.   
     
     
         71 . The method of  claim 69 , wherein the host cell is sorted into four populations in step (b):
 (i) a first subpopulation based on antibody expression;   (ii) a second subpopulation based on lower binding affinity to the target molecule as compared to the reference antibody;   (iii) a third subpopulation based on higher binding affinity to the target molecule as compared to the reference antibody;   (iv) a fourth subpopulation based on equal binding affinity to the target molecule as compared to the reference antibody,   and wherein the variant is identified as enriched in the second subpopulation as compared to the a population containing the first subpopulation, the second subpopulation the third subpopulation, and the fourth subpopulation, such that a variant with lower binding affinity to the target molecule as compared to the reference antibody is identified.   
     
     
         72 . The method of  claim 69 , wherein the host cell is sorted into four populations in step (b):
 (i) a first subpopulation based on antibody expression;   (ii) a second subpopulation based on equal binding affinity to the target molecule as compared to the reference antibody;   (iii) a third subpopulation based on higher binding affinity to the target molecule as compared to the reference antibody;   (iv) a fourth subpopulation based on lower binding affinity to the target molecule as compared to the reference antibody,   and wherein the variant is identified as enriched in the second subpopulation as compared to the a population containing the first subpopulation, the second subpopulation the third subpopulation, and the fourth subpopulation, such that a variant with equal binding affinity to the target molecule as compared to the reference antibody is identified.   
     
     
         73 . A method identifying an antibody variant that is more highly expressed in a host cell as compared to a reference antibody: identifying a host cell within the host cell library of  claim 64  with higher expression of an antibody variant as compared to a reference antibody; thereby identifying an antibody variant that is more highly expressed in a host cell as compared to a reference antibody. 
     
     
         74 . The method of  claim 73 , wherein identifying the host cell comprises contacting the host cell library with a fluorescently labeled anti-immunoglobulin antibody to generate a labeled library, sorting the labeled library into at least two subpopulations based on the level of fluorescence per cell, and identifying at least one host cell that is more prevalent in the higher fluorescence subpopulation. 
     
     
         75 . A method identifying an antibody variant that is more highly expressed in a host cell as compared to a reference antibody and has at least comparable binding to a target molecule as the reference antibody, comprising sorting the host cell library of  claim 64  into at least two subpopulations:
 (i) a first subpopulation based on antibody expression; and 
 (ii) a second subpopulation based on binding affinity to the target molecule as compared to the reference antibody; 
 (b) identifying a variant that is enriched in the first subpopulation as compared to the second subpopulation, the starting population, or another population containing the second subpopulation; 
 thereby identifying an antibody variant that is more highly expressed in a host cell as compared to a reference antibody. 
 
     
     
         76 . The method of  claim 75 , wherein the host cell library is contacted with an anti-immunoglobulin antibody and the target molecule prior to said sorting, wherein the anti-immunoglobulin and target molecule are labeled with distinguishable fluorescent moieties. 
     
     
         77 . A method identifying an antibody variant that is more highly expressed in a host cell as compared to a reference antibody and has at least comparable binding to a target molecule as the reference antibody, comprising (a) sorting the host cell library of  claim 64  into at least a first subpopulation and a second subpopulation based on antibody expression, wherein the first subpopulation displays greater expression than the second subpopulation; (b) identifying a variant that is enriched in the first subpopulation as compared to the second subpopulation, the starting population, or another population containing the second subpopulation; thereby identifying an antibody variant that is more highly expressed in a host cell as compared to a reference antibody. 
     
     
         78 . The method of  claim 77 , wherein the host cell library is contacted with an anti-immunoglobulin antibody labeled with a fluorescent moiety prior to said sorting, and wherein said sorting is based on the level of fluorescence emitted by said fluorescent moiety. 
     
     
         79 . A method of generating the host cell library of CDR variants of a reference antibody chain, comprising:
 (a) generating a first nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a first CDR of a reference antibody chain, wherein the nucleic acid molecules in said first population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said first CDR;   (b) generating a second nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a second CDR of said reference antibody chain, wherein the nucleic acid molecules in said second population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said second CDR;   (c) generating a third nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a third CDR of said reference antibody chain, wherein the nucleic acid molecules in said third population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said third CDR; and   (d) generating a host cell library comprising a population of host cells containing the nucleic acid molecules of said first, second and third populations such that each host cell comprises a different nucleic acid molecule from said first, second and third populations, in one or more copies, extrachromosomal or chromosomally integrated,   thereby generating a host cell library of CDR variants of a reference antibody chain.   
     
     
         80 . The method of  claim 79 , wherein the different nucleic acid molecules in each of said first, second and third populations is generated by NNK codon mutagenesis. 
     
     
         81 . The method of  claim 80 , wherein said NNK codon mutagenesis is optimized such that for each NNK codon, A, T, C and G are each represented in said library at a frequency of 20-35% of each of the first and second position of said NNK codon. 
     
     
         82 . The method of  claim 79 , wherein the host cell library is generated by pooling the nucleic acid molecules of the first, second and third population then transfecting the pooled nucleic acid molecules into a host cell line. 
     
     
         83 . The method of  claim 79 , wherein the host cell library is generated by transfecting the nucleic acid molecules of the first, second and third population into a host cell line then pooling the transfected cells of the host cell line. 
     
     
         84 . The method of  claim 79 , wherein each nucleic acid population comprises nucleic acid molecules encoding at least 80% of possible CDR variants of its respective CDR. 
     
     
         85 . The method of  claim 84 , wherein each nucleic acid population comprises nucleic acid molecules encoding at least 90% of possible CDR variants of its respective CDR. 
     
     
         86 . The method of  claim 85 , wherein each nucleic acid population comprises nucleic acid molecules encoding at least 95% of possible CDR variants of its respective CDR. 
     
     
         87 . The method of  claim 79 , wherein host cells are mammalian host cells and wherein the CDR variants are expressed on the surface of the mammalian host cells. 
     
     
         88 . The method of  claim 79 , wherein the reference antibody chain is a heavy chain, such that the host cell library of CDR variants is a heavy chain CDR variant library. 
     
     
         89 . The method of  claim 79 , wherein the reference antibody chain is a light chain, such that the host cell library of CDR variants is a light chain CDR variant library. 
     
     
         90 . A method of generating a host cell library of CDR variants of a reference antibody, comprising:
 (a) generating a first nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a first heavy chain CDR of a reference antibody, wherein the nucleic acid molecules in said first population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said first heavy chain CDR;   (b) generating a second nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a second heavy chain CDR of said reference antibody, wherein the nucleic acid molecules in said second population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said second heavy chain CDR;   (c) generating a third nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a third heavy chain CDR of said reference antibody, wherein the nucleic acid molecules in said third population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said third heavy chain CDR;   (d) generating a fourth nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a first light chain CDR of a reference antibody, wherein the nucleic acid molecules in said fourth population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said first light chain CDR;   (e) generating a fifth nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a second light chain CDR of said reference antibody, wherein the nucleic acid molecules in said fifth population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said second light chain CDR;   (f) generating a sixth nucleic acid population comprising at least 80 different nucleic acid molecules, each encoding a different variant of a third heavy chain CDR of said reference antibody, wherein the nucleic acid molecules in said sixth population encode at least 16 variants for each amino acid over a region of at least 5 consecutive amino acids within said third heavy chain CDR; and   (g) generating a host cell library comprising a population of host cells containing the nucleic acid molecules of said first, second, third, fourth, fifth and sixth populations such that each host cell comprises a different nucleic acid molecule from said first, second, third, fourth, fifth and sixth populations, in one or more copies, extrachromosomal or chromosomally integrated,   thereby generating a host cell library of CDR variants of a reference antibody.   
     
     
         91 . The method of  claim 90 , wherein the different nucleic acid molecules in each of said first, second, third, fourth, fifth, and sixth populations is generated by NNK codon mutagenesis. 
     
     
         92 . The method of  claim 91 , wherein said NNK codon mutagenesis is optimized such that for each NNK codon, A, T, C and G are each represented in said library at a frequency of 20-35% of each of the first and second position of said NNK codon. 
     
     
         93 . The method of  claim 90 , wherein the host cell library is generated by pooling the nucleic acid molecules of said first, second, third, fourth, fifth and sixth populations then transfecting the pooled nucleic acid molecules into a host cell line. 
     
     
         94 . The method of  claim 90 , wherein the host cell library is generated by transfecting the nucleic acid molecules of said first, second, third, fourth, fifth and sixth populations into a host cell line then pooling the transfected cells of the host cell line. 
     
     
         95 . The method of  claim 90 , wherein each nucleic acid population comprises nucleic acid molecules encoding at least 80% of possible CDR variants of its respective CDR. 
     
     
         96 . The method of  claim 95 , wherein each nucleic acid population comprises nucleic acid molecules encoding at least 90% of possible CDR variants of its respective CDR. 
     
     
         97 . The method of  claim 96 , wherein each nucleic acid population comprises nucleic acid molecules encoding at least 95% of possible CDR variants of its respective CDR. 
     
     
         98 . The method of  claim 90 , wherein host cells are mammalian host cells and wherein the CDR variants are expressed on the surface of the mammalian host cells.