Patent ID: 8716196

Claim:
A method for producing a collection of nucleic acids, wherein each nucleic acid encodes a human immunoglobulin variable domain comprising a plurality of complementarity determining region 3 (CDR3) sequences isolated separately from the immunoglobulin variable domain repertoire from a mammalian species, the method comprising: (a) providing a plurality of Acceptor Framework nucleic acid sequences encoding distinct human immunoglobulin variable domains, each Acceptor Framework nucleic acid sequence comprising a first framework region (FR1), a second framework region (FR2), a third framework region (FR3), and a fourth framework region (FR4), wherein the FR1 and FR2 regions are interspaced by a complementarity determining region 1 (CDR1), the FR2 and FR3 regions are interspaced by a complementarity determining region 2 (CDR2), and the FR3 and FR4 regions are interspaced by a stuffer nucleic acid sequence comprising at least two Type IIs restriction enzyme recognition sites interspaced by a random nucleic acid sequence encodes a polypeptide that performs the function of a variable immunoglobulin CDR3; (b) providing a plurality of diversified nucleic acid sequences encoding complementarity determining region 3 (CDR3) sequences isolated from the mammalian species immunoglobulin repertoire wherein each of the plurality of diversified nucleic acid sequences comprises a Type IIs restriction enzyme recognition site at each extremity and wherein each of the plurality of diversified nucleic acid sequences encodes a complete CDR3; (c) digesting each of the plurality of nucleic acid sequences encoding the complete CDR3 using a Type IIs restriction enzyme that binds to the Type IIs restriction enzyme recognition site of step (b) and digesting the stuffer nucleic acid sequence of step (a) from the Acceptor Framework using a Type IIs restriction enzyme that binds to the Type IIs restriction enzyme recognition site of step (a); and (d) ligating the digested nucleic acid sequences encoding the complete CDR3 of step (c) into the digested Acceptor Framework of step (c) such that the FR3 and FR4 regions are interspaced by the nucleic acid sequences encoding the complete CDR3 and a wherein the complete immunoglobulin variable domain encoding sequences do not contain the Type IIs restriction enzyme recognition sites of steps (a) and (b).