Humanized antibodies

Abstract

The present disclosure provides humanized antibodies, including antibodies comprising an ultralong CDR3 and uses thereof.

1 . A polynucleotide encoding a humanized antibody or binding fragment thereof, wherein the humanized antibody or binding fragment thereof comprises a variable heavy chain comprising a human acceptor framework comprising a CDR3 that is an ultralong CDR3 in which the CDR3 of the human acceptor framework is replaced by a CDR3 scaffold which is 35 amino acids in length or longer and comprises a X 1 X 2 X 3 X 4 X 5 X n (X a X b ) z  motif and at least a portion of a human JH sequence or derivative thereof,
 wherein the X 1 X 2 X 3 X 4 X 5  motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154),   wherein, for X n , X is any amino acid and n is 27-54, and   wherein the (X a X b ) z  motif is YXYXYX with X being any amino acid.   
     
     
         2 . The polynucleotide of  claim 1 , wherein the ultralong CDR3 is 45 amino acids in length or longer. 
     
     
         3 . The polynucleotide of  claim 1 , wherein the ultralong CDR3 is 50 amino acids in length or longer. 
     
     
         4 . The polynucleotide of  claim 1 , wherein the human acceptor framework is from a human heavy chain germline sequence that is a VH4 family member germline sequence. 
     
     
         5 . The polynucleotide of  claim 1 , wherein the human acceptor framework is from a VH4-34 germline sequence or is derived from a VH4-34 germline sequence. 
     
     
         6 . The polynucleotide of  claim 5 , wherein the VH4-34 germline sequence comprises a CDR1 comprising a Gly31Asp and Tyr32Lys substitution based on Kabat numbering and/or a CDR2 comprising a Glu50Ser substitution based on Kabat numbering. 
     
     
         7 . The polynucleotide of  claim 6 , wherein the VH4-34 germline sequence comprises a CDR1 comprising Ala23Thr, Val24Ala, Tyr25Ser, Gly27Phe, Phe29Leu, Gly31Asp, Tyr32Lys, Tyr33Ala, Trp34Val, and Ser35Gly substitutions, and a CDR2 comprising Ile48Leu, Glu50Ser, Asn52Asp, His53Thr, Ser54Gly, Ser56Asn, and Asn58Gly substitutions, based on Kabat numbering. 
     
     
         8 . The polynucleotide of  claim 1 , wherein the humanized antibody or antigen binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 460 and the ultralong CDR3. 
     
     
         9 . The polynucleotide of  claim 1 , wherein the humanized antibody or antigen binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 461, 462, 463, 464, 465, 466, 467, or 468 and the ultralong CDR3. 
     
     
         10 . The polynucleotide of  claim 1 , wherein the ultralong CDR3 comprises a non-bovine sequence or a non-antibody sequence. 
     
     
         11 . The polynucleotide of  claim 11 , wherein ultralong CDR3 comprises a non-antibody sequence that is a cytokine sequence, a lymphokine sequence, a chemokine sequence, a growth factor sequence, a hormone sequence, or a toxin sequence. 
     
     
         12 . The polynucleotide of  claim 1 , wherein the at least a portion of a human JH sequence or derivative thereof comprises the amino acid sequence set forth in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. 
     
     
         13 . The polynucleotide of  claim 1 , further comprising a variable light chain. 
     
     
         14 . The polynucleotide of  claim 13 , wherein the variable heavy chain and the variable light chain are encoded on the same vector. 
     
     
         15 . A vector comprising the polynucleotide of  claim 1 . 
     
     
         16 . A vector comprising the polynucleotide of  claim 13 . 
     
     
         17 . A host cell comprising the vector of  claim 15 . 
     
     
         18 . A host cell comprising the vector of  claim 16 . 
     
     
         19 . A nucleic acid library comprising a plurality of polynucleotides comprising sequences coding for humanized antibodies or binding fragments thereof of  claim 1 . 
     
     
         20 . A nucleic acid library comprising a plurality of polynucleotides comprising sequences coding for humanized antibodies or binding fragments thereof of  claim 13 .