Semi-synthetic nurse shark VNAR libraries for making and using selective binding compounds

A nucleic acid-containing library whose members each comprise a nucleic acid encoding a polypeptide comprising a VNAR Type 2 backbone amino acid sequence comprising a semi-synthetic Type 2 VNAR framework (FW), hypervariable (HV) and complementary determining region (CDR) regions having a domain structure, from N- to C-terminal:

• FW1-CDR1-FW2-HV2-FW2′-HV4-FW3-CDR3-FW4
• wherein said polypeptide comprises VNAR Type 2 backbone amino acid sequence:
• ARVDQTPX1X2X3(10)TKETGESLTI(20)NCVLRDX4X5CX6(30)LX7X8X9X10WYR X11K(40)SX12SX13NEX14X15IS(50)X16GGRYVETVN(60)SGSKSFSLX17I(70)NDLX18VX19DSGT(80) YRCX20V(85)-[ . . . CDR3 . . . ]-YGX21GTX22VTVNA (SEQ ID NO. 9),
• wherein amino acid compositions at each position X are:
• X1=Q and R, with R being present in more than 4.4% of the polypeptides;
• X2=T and S, with S being present in more than 5.4% of the polypeptides;
• X3=I and V, with V being present in more than 4.4% of the polypeptides;
• X4=S and N
• X5=N and I, with I being present in more than 3.2% of the polypeptides;
• X6=A, D and E, with D being present in more than 3.2% of the polypeptides and E being present in more than 2.2% of the polypeptides;
• X7=S and P, with P being present in more than 2.1% of the polypeptides;
• X8=S, R, T and N, with T being present in more than 1% of the polypeptides, and N being present in more than 5.3% of the polypeptides;
• X9=T and L, with L being present in more than 1.1% of the polypeptides;
• X10=Y, H and L, with H being present in more than 2.1% of the polypeptides and L being present in more than 2.1% of the polypeptides;
• X11=K and T, with T being present in more than 4.4% of the polypeptides;
• X12=G and D, with D being present in more than 4.4% of the polypeptides;
• X13=T and R, with R being present in more than 5.3% of the polypeptides;
• X14=E and A, with A being present in more than 1.1% of the polypeptides;
• X15=S, N and R, with R being present in more than 3.2% of the polypeptides;
• X16=K and L, with L being present in more than 3.2% of the polypeptides;
• X17=R and K, with K being present in more than 3.2% of the polypeptides;
• X18=T and V, with V being present in more than 3.2% of the polypeptides;
• X19=E and K, with K being present in more than 1.1% of the polypeptides;
• X20=N and K;
• X21=G and D;
• and
• X22=V and A;
• and
• wherein CDR3 comprises an amino acid sequence of from 7 to 25 amino acid residues,
• wherein CDR3 comprises at least one cysteine residue,
• wherein the last two amino acid residues of CDR3 are DV, and
• wherein CDR3 is otherwise randomized compared to germline Type 2 VNAR sequences.