Method for isolation of soluble polypeptides
Polypeptides with biophysical properties such as solubility, stability, high expression, monomericity, binding specificity or non-aggregation, including monomeric human heavy and light chain variable domains (V H s and V L s), are identified using a high throughput method for screening polypeptides, comprising the steps of obtaining a phage display library, allowing infection of a bacterial lawn by the library phage, and identifying phage which form larger than average plaques on the bacterial lawn. Sequences of monomeric human V H s and V L s are identified, which may be useful for immunotherapy or as diagnostic agents. Multimer complexes of human V H s and V L s are also identified. The V H s and V L s identified may be used to create further libraries for identifying additional polypeptides. Further, the V H s and V L s may be subjected to DNA shuffling to select for improved biophysical properties.
1. An antigen-binding V L comprising a FR1 sequence of EIVMTQSPGTLSLSPGDRATLSC (amino acids 1-23 of SEQ ID NO:42), a FR2 sequence of WYQQKPGQAPRLLIY (amino acids 35-49 of SEQ ID NO:42), a FR3 sequence of GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (amino acids 57-88 of SEQ ID NO:42), and a FR4 sequence of FGQGTKVTVL (amino acids 98-107 of SEQ ID NO:42).
2. An antigen-binding V L comprising the FR1, FR2, FR3, and FR4 portion of SEQ ID NO:42 and one or more randomized CDR sequences, wherein one or more of CDR1, CDR2 and CDR3 of SEQ ID NO:42 is replaced, respectively, with a randomized CDR1, CDR2 and CDR3.
3. The antigen-binding V L of claim 1 , wherein the V L is in a multimeric form.
4. The antigen-binding V L of claim 1 , wherein the V L is in a dimeric form.
5. The antigen-binding V L of claim 1 , wherein V L is in a trimeric form.
6. The antigen-binding V L of claim 1 , wherein the V L is in a pentameric form.
7. A display library constructed by preparing nucleic acid sequences coding for the antigen-binding V L of claim 1 or 2 and expressing the V L so as to display the antigen-binding V L sequence of claim 1 or 2 .
8. The display library of claim 7 , wherein the library is a phage display library.
9. The display library of claim 7 , wherein the library is a ribosome display, ARM ribosome display, yeast display, bacterial cell display, or in vitro compartmentalization library.
10. A method for producing a V L library, comprising:
a) providing a nucleotide sequence encoding the antigen-binding V L of claim 1 ;
b) providing oligonucleotide sequences with randomized codons;
c) incorporating the randomized oligonucleotides into the nucleotide sequence encoding the V L , such that one or more than one of the complementary determining regions is randomized, wherein one or more of CDR1, CDR2 and CDR3 of SEQ ID NO.: 42 is replaced, respectively, with a randomized CDR1, CDR2 and CDR3;
d) expressing the nucleotide sequences produced in step c); and
e) screening the expressed sequences for binding to a target polypeptide.
11. The method of claim 10 , wherein the screening comprises panning against a target molecule.
12. A method of screening the display library of claim 7 , comprising panning the displayed V L against a target molecule.
13. The antigen-binding V L of claim 2 , wherein CDR3 of SEQ ID NO:42 is replaced with a randomized CDR3, and the randomized CDR3 comprises 9 amino acid residues.
14. The antigen-binding V L of claim 1 , which is aggregation resistant.