The present disclosure relates to multifunctional molecules, including molecules according to formula (I):

([(B.sub.1).sub.M-D-L.sub.1].sub.Y—H.sub.1).sub.O-G-(H.sub.2-[L.sub.2-E-(B.sub.2).sub.K].sub.W).sub.P,   (I)

wherein G, H.sub.1, H.sub.2, D, E, B.sub.1, B.sub.2, M, K, L.sub.1, L.sub.2, O, P, Y, and W are defined herein. The present disclosure also relates to methods of preparing and using such multifunctional molecules to identify encoded molecules capable of binding target molecules.

The invention provides efficient methods for combining single-substitution libraries of nucleic acids that span and encode proteins of interest and for selecting resultant mutant proteins after expression which have improved properties or characteristics.

The present disclosure relates to multifunctional molecules, including molecules according to formula (I):
([(B.sub.1).sub.M-D-L.sub.1].sub.Y-H.sub.1).sub.O-G-(H.sub.2-[L.sub.2-E-(B.sub.2).sub.K].sub.W).sub.P,   (I)
wherein G, H.sub.1, H.sub.2, D, E, B.sub.1, B.sub.2, M, K, L.sub.1, L.sub.2, O, P, Y, and W are defined herein. The present disclosure also relates to methods of preparing and using such multifunctional molecules to identify encoded molecules capable of binding target molecules.

The present disclosure relates to multifunctional molecules, including molecules according to formula (I):
([(B.sub.1).sub.M-D-L.sub.1].sub.Y-H.sub.1).sub.O-G-(H.sub.2-[L.sub.2-E-(B.sub.2).sub.K].sub.W).sub.P,   (I)
wherein G, H.sub.1, H.sub.2, D, E, B.sub.1, B.sub.2, M, K, L.sub.1, L.sub.2, O, P, Y, and W are defined herein. The present disclosure also relates to methods of preparing and using such multifunctional molecules to identify encoded molecules capable of binding target molecules.

The present invention is directed to a method for generating a library of antigen binding molecules for screening for binding to an epitope of interest, said method comprising: a. selecting a template antigen-binding molecule from a set of possible template antigen binding molecules wherein said selected template does not specifically bind the epitope of interest but is known to specifically bind another epitope; b. selecting at least one residue position in said template antigen-binding molecule for mutation; and c. selecting at least one variant residue to substitute at the at least one residue position selected in b; such that a library containing a plurality of variants of said template is generated.

The present invention is directed to a method for generating a library of antigen binding molecules for screening for binding to an epitope of interest, said method comprising: a. selecting a template antigen-binding molecule from a set of possible template antigen binding molecules wherein said selected template does not specifically bind the epitope of interest but is known to specifically bind another epitope; b. selecting at least one residue position in said template antigen-binding molecule for mutation; and c. selecting at least one variant residue to substitute at the at least one residue position selected in b; such that a library containing a plurality of variants of said template is generated.

According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.