Humanized antibodies with extremely long complementarity determining regions

Abstract

Provided are novel antibodies, methods for making same, and uses thereof, which are useful for use in treating or diagnosing disease states or for biological research. [Solution] A humanized antibody or binding fragment thereof comprising: (a) a specific amino acid sequence; and (b) a heavy chain variable region comprising an extremely long CDR3.

Description

[Technical field]

[0001] Field The present disclosure relates to humanized antibodies, including antibodies that contain extremely long CDR3s.

[0002] cross reference This application claims the benefit of priority to U.S. Provisional Application No. 61 / 856,010, filed July 18, 2013, the entire contents of which are incorporated herein by reference.

[0003] This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on July 18, 2014, is named 13379-006-228_SequenceListing.txt and is 686,375 bytes in size. [Background technology]

[0004] background Antibodies are natural proteins that the immune system of vertebrates forms in response to foreign substances (antigens), primarily to protect against infection. For over a century, antibodies have been induced in animals under artificial conditions and harvested for use in treating or diagnosing disease states or for biological research. Although each individual antibody-producing cell produces one type of antibody with a chemically defined composition, antibodies obtained directly from animal serum in response to antigen challenge actually contain a population of non-identical molecules (e.g., polyclonal antibodies) made from an ensemble of individual antibody-producing cells.

[0005] Typically, some bovine antibodies have long VH CDR3 sequences compared to other vertebrates. For example, about 10% of IgM contain "extremely long" CDR3 sequences that can be up to 61 amino acids long. Often, these unusual CDR3s have a large number of cysteines. Functional VH genes arise through a process called V(D)J recombination, with the D region encoding a significant proportion of the CDR3. Unique D regions encoding extremely long sequences have been identified in bovines. Extremely long CDR3s are partially encoded in the bovine genome, providing an antibody repertoire with unique characteristics compared to humans. Kaushik et al. (U.S. Patent Nos. 6,740,747 and 7,196,185) disclose several bovine germline D gene sequences unique to bovines that are described as useful as probes, and bovine VDJ cassettes that are described as useful as vaccine vectors. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 6,740,747 [Patent Document 2] U.S. Patent No. 7,196,185 Summary of the Invention

[0007] overview The present disclosure provides humanized antibodies, including antibodies containing extremely long CDR3s, methods of making same, and uses thereof.

[0008] The disclosure provides a humanized antibody or binding fragment thereof comprising: (a) an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3.

[0009] In some embodiments, a humanized antibody or binding fragment thereof comprises one or more human variable region framework sequences.

[0010] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length.

[0011] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 is 35 amino acids in length or longer.

[0012] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises three or more cysteine ​​residues, four or more cysteine ​​residues, five or more cysteine ​​residues, six or more cysteine ​​residues, seven or more cysteine ​​residues, eight or more cysteine ​​residues, nine or more cysteine ​​residues, ten or more cysteine ​​residues, eleven or more cysteine ​​residues, or twelve or more cysteine ​​residues.

[0013] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises three or more cysteine ​​residues.

[0014] In some embodiments of each or any of the foregoing or following aspects, the antibody or binding fragment thereof comprises a cysteine ​​motif.

[0015] In some embodiments of each or any of the aforementioned or following embodiments, the cysteine ​​motif is TIFF0007680480000001.tif186157.

[0016] In some embodiments of each or any of the aforementioned or following embodiments, the cysteine ​​motif is TIFF0007680480000002.tif212158.

[0017] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises from 2 to 6 disulfide bonds.

[0018] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises SEQ ID NO: 40, or a derivative thereof.

[0019] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises amino acid residues 3-6 of any one of SEQ ID NOs: 1-4.

[0020] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises a non-human DH or a derivative thereof.

[0021] In some embodiments of each or any of the foregoing or following embodiments, the non-human DH is SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0022] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises a JH sequence or a derivative thereof.

[0023] In some embodiments of each or any of the foregoing or following embodiments, the JH sequence comprises amino acids 5 to 15 of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0024] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises a sequence derived from a non-human or human VH sequence (e.g. a germline VH) or a derivative thereof; a sequence derived from a non-human D sequence or a derivative thereof; and / or a sequence derived from a JH sequence or a derivative thereof.

[0025] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises an additional amino acid sequence comprising 2 to 6 or more amino acid residues located between the VH and DH sequences.

[0026] In some embodiments of each or any of the above or below embodiments, the additional amino acid sequence is selected from the group consisting of IR, IF, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0027] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises a sequence derived from or based on SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

[0028] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises a bovine sequence, a non-bovine sequence, an antibody sequence, or a non-antibody sequence.

[0029] In some embodiments of each or any of the foregoing or following aspects, the non-antibody sequence is a synthetic sequence.

[0030] In some embodiments of each or any of the preceding or following embodiments, the non-antibody sequence is a cytokine sequence, a lymphokine sequence, a chemokine sequence, a growth factor sequence, a hormone sequence, or a toxin sequence.

[0031] In some embodiments of each or any of the foregoing or following embodiments, the non-antibody sequence is an IL-8 sequence, an IL-21 sequence, an IL-1 sequence, an IL-2 sequence, an IL-4 sequence, an IL-10 sequence, an IL-17 sequence, a GLP-1 sequence, an SDF-1(α) sequence, a somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence, a ziconotide sequence, an ADWX-1 sequence, an HsTx1 sequence, an OSK1 sequence, a Pi2 sequence, a Hongotoxin sequence, a xin (HgTX) sequence, Margatoxin sequence, Agitoxin-2 sequence, Pi3 sequence, Kaliotoxin sequence, Anuroctoxin sequence, Charybdotoxin sequence, Tityustoxin-K-α sequence, Maurotoxin sequence, Ceratotoxin 1 (CcoTx1) sequence, CcoTx 2 sequence, CcoTx3 sequence, PaurTx3 sequence, Hanatoxin 1 sequence, Flixotoxin 1 sequence, Huentoxin-IV sequence, α-conotoxin ImI sequence, α-conotoxin EpI sequence, α-conotoxin PnIA sequence, α-conotoxin PnIB sequence, α-conotoxin MII sequence, α-conotoxin AuIA sequence, α-conotoxin AuIB sequence, α-conotoxin AuIC sequence, Conotoxin κ-PVI sequence IA sequence, charybdotoxin sequence, neurotoxin B-IV sequence, crotamine sequence, ω-GVIA (conotoxin) sequence, κ-hefutoxin 1 sequence, Css4 sequence, Bj-xtrIT sequence, BcIV sequence, Hm-1 sequence, Hm-2 sequence, GsAF-I (β-theraphotoxin-Gr1b) sequence, protoxin I (ProTx-I sequence, β-theraphotoxin-Tp1a) sequence, protoxin II (ProTxII) sequence, Huentoxin I sequence, μ-Conotoxin PIIIA sequence, Jinzaotoxin-III (β-TRTX-Cj1α) sequence, GsAF-II (κ-Terafotoxin-Gr2c) sequence, ShK (Stichodactyla toxin) sequence, HsTx1 sequence, Guangxitoxin 1E (GxTx-1E) sequence, Maurotoxin sequence, Charybdotoxin (ChTX) sequence, Iberiotoxin (IbTx) sequence, Leiurotoxin 1 (Scyllatoxin) sequence, ) sequence, Tamapin sequence, Karyotoxin-1 (KTX) sequence, Purotoxin 1 (PT-1) sequence, or GpTx-1 sequence, MOKA toxin sequence, OSK1(P12, K16, D20) sequence, OSK1(K16, D20) sequence, HmK sequence, ShK(K16, Y26, K29) sequence, ShK(K16) sequence, ShK-A(K16) sequence, ShK(K16, E30) sequence, ShK(Q21) sequence, ShK(L21) sequence, ShK(F21) sequence, ShK(I21) sequence, or ShK(A21) sequence.

[0032] In some embodiments of each or any of the foregoing or following embodiments, the non-antibody sequence is any one of SEQ ID NOs: 475-481, 599-655, 666-698, 727-733, 808-810, and 831-835.

[0033] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises an antibody heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 770-779, 784-791, 903-922, and 925-955.

[0034] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises a light chain variable region comprising the amino acid sequence SEQ ID NO: 780 or 807.

[0035] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises an antibody heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 770-779, 784-791, 903-922, and 925-955.

[0036] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 956 or 959.

[0037] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 770-779, 784-791, 903-922, and 925-955, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 959. In some aspects, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 941, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 959.

[0038] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 770-779, 784-791, 903-922, and 925-955, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 956.

[0039] In some embodiments of each or any of the preceding or following embodiments, the non-antibody sequence replaces at least a portion of the very long CDR3.

[0040] In some embodiments of each or any of the foregoing or following embodiments, a non-antibody sequence (e.g., a non-antibody human sequence) is inserted into the CDR3, optionally including cases where a portion of the CDR3 (e.g., one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g., all or substantially all of the amino acids of the CDR3) is removed.

[0041] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises 1 X 2 X 3 X 4 X 5 Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q).

[0042] In some embodiments of each or any of the preceding or following embodiments, X 1 X 2 X 3 X 4 X 5 The motif is The file is TIFF0007680480000003.tif72158.

[0043] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 1 X 2 X 3 X 4 X 5 Contains motifs.

[0044] In some embodiments of each or any of the preceding or following embodiments, CX 1 X 2 X 3 X 4 X 5 The motif is The file is TIFF0007680480000004.tif79158.

[0045] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4.

[0046] In some embodiments of each or any of the preceding or following embodiments, (X a X b ) z The motif is The file is TIFF0007680480000005.tif132158.

[0047] In some embodiments of each or any of the preceding or following embodiments, (X a X b ) z The motif is YXYXYX.

[0048] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises 1 X 2 X 3 X 4 X 5 X n Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and n is 27 to 54).

[0049] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises n (X a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4.

[0050] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises 1 X 2 X 3 X 4 X 5 X n (X a X b ) z Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and X is a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4.

[0051] In some embodiments of each or any of the preceding or following embodiments, X 1 X 2 X 3 X 4 X 5 The motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and (X a X b ) z The motif is YXYXYX.

[0052] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 1 X 2 X 3 X 4 X 5 Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q); TIFF0007680480000006.tif186159; and (X a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4.

[0053] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 1 X 2 X 3 X 4 X 5 Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q); TIFF0007680480000007.tif213158; and (X a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4.

[0054] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises an additional sequence which is a linker.

[0055] In some embodiments of each or any of the foregoing or following embodiments, the linker is linked to the C-terminus, the N-terminus, or both the C-terminus and the N-terminus of the non-antibody sequence.

[0056] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 is a ruminant CDR3.

[0057] In some embodiments of each or any of the foregoing or following embodiments, the ruminant is a bovine.

[0058] In some embodiments of each or any of the foregoing or following aspects, the humanized antibody or binding fragment thereof comprises human heavy chain variable region framework sequences.

[0059] In some embodiments of each or any of the foregoing or following aspects, the humanized antibody or binding fragment thereof comprises or is derived from a human heavy chain germline sequence.

[0060] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:735.

[0061] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:737.

[0062] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:739.

[0063] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:741.

[0064] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:743.

[0065] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:745.

[0066] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:747.

[0067] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:749.

[0068] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000008.tif106165.

[0069] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000009.tif119164.

[0070] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000010.tif226165.

[0071] In some embodiments of each or any of the foregoing or following aspects, the humanized antibody or binding fragment thereof comprises a human light chain variable region framework sequence.

[0072] In some embodiments of each or any of the aforementioned or following embodiments, the humanized antibody or binding fragment thereof comprises a light chain variable region comprising a sequence selected from the group of: (i) SEQ ID NO: 750, (ii) SEQ ID NO: 751, (iii) SEQ ID NO: 752, and (iv) SEQ ID NO: 753.

[0073] In some embodiments of each or any of the preceding or following embodiments, the light chain variable comprises or is derived from a lambda light chain variable region sequence.

[0074] In some embodiments of each or any of the preceding or following embodiments, the light chain variable region sequence comprises or is derived from a human λ light chain variable region sequence.

[0075] In some embodiments of each or any of the foregoing or following embodiments, the light chain variable region comprises a VL1-51 germline sequence.

[0076] In some embodiments of each or any of the foregoing or following embodiments, the light chain variable region is derived from the VL1-51 germline sequence.

[0077] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises a CDR1 that comprises an Ile29Val substitution and an Asn32Gly substitution based on Kabat numbering.

[0078] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises a CDR2 comprising a DNN to GDT substitution.

[0079] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises a CDR2 that comprises a DNNKRP (SEQ ID NO: 471) to GDTSRA (SEQ ID NO: 472) substitution.

[0080] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence includes an S2A substitution, a T5N substitution, a P8S substitution, an A12G substitution, an A13S substitution, and a P14L substitution based on Kabat numbering.

[0081] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises an S2A substitution, a T5N substitution, a P8S substitution, an A12G substitution, an A13S substitution, and a P14L substitution based on Kabat numbering, and a CDR2 that comprises a DNN to GDT substitution.

[0082] In some embodiments of each or any of the above or below embodiments, a humanized antibody or binding fragment thereof comprising: (a) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, and SEQ ID NO:743; and (b) a light chain variable region comprising SEQ ID NO:750.

[0083] The present disclosure also provides a polynucleotide encoding the heavy chain variable region of the humanized antibody or binding fragment thereof disclosed herein.

[0084] The present disclosure also provides a polynucleotide encoding the light chain variable region of the humanized antibody or binding fragment thereof disclosed herein.

[0085] The disclosure also provides a polynucleotide encoding a heavy chain variable region comprising a very long CDR3, the polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 494, SEQ ID NO: 495, SEQ ID NO: 496, and SEQ ID NO: 497.

[0086] The present disclosure also provides vectors comprising the polynucleotides disclosed herein.

[0087] The present disclosure also provides a host cell comprising the vectors disclosed herein.

[0088] The disclosure also provides a nucleic acid library comprising a plurality of polynucleotides constituting a sequence encoding a humanized antibody or binding fragment thereof, wherein the antibody or binding fragment thereof comprises: (a) an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3.

[0089] The present disclosure also provides a library of humanized antibodies or binding fragments thereof, wherein the antibodies or binding fragments thereof comprise: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) an extra long CDR3.

[0090] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length.

[0091] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 is 35 amino acids in length or longer.

[0092] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises three or more cysteine ​​residues, four or more cysteine ​​residues, five or more cysteine ​​residues, six or more cysteine ​​residues, seven or more cysteine ​​residues, eight or more cysteine ​​residues, nine or more cysteine ​​residues, ten or more cysteine ​​residues, eleven or more cysteine ​​residues, or twelve or more cysteine ​​residues.

[0093] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises three or more cysteine ​​residues.

[0094] In some embodiments of each or any of the foregoing or following aspects, the antibody or binding fragment thereof comprises a cysteine ​​motif.

[0095] In some embodiments of each or any of the aforementioned or following embodiments, the cysteine ​​motif is TIFF0007680480000011.tif185157.

[0096] In some embodiments of each or any of the aforementioned or following embodiments, the cysteine ​​motif is TIFF0007680480000012.tif213158.

[0097] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises from 2 to 6 disulfide bonds.

[0098] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises SEQ ID NO: 40, or a derivative thereof.

[0099] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises amino acid residues 3-6 of any one of SEQ ID NOs: 1-4.

[0100] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises a non-human DH or a derivative thereof.

[0101] In some embodiments of each or any of the foregoing or following embodiments, the non-human DH is SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0102] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises a JH sequence or a derivative thereof.

[0103] In some embodiments of each or any of the foregoing or following embodiments, the JH sequence comprises amino acids 5 to 15 of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0104] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises a sequence derived from a non-human VH sequence or a derivative thereof; a sequence derived from a non-human DH sequence or a derivative thereof; and / or a sequence derived from a JH sequence or a derivative thereof.

[0105] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises an additional amino acid sequence comprising 2 to 6 or more amino acid residues located between the VH and DH sequences.

[0106] In some embodiments of each or any of the above or below embodiments, the additional amino acid sequence is selected from the group consisting of IR, IF, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0107] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 comprises a sequence derived from or based on SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

[0108] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises a non-bovine sequence or a non-antibody sequence, for example, a non-antibody sequence (e.g., a non-antibody human sequence) is inserted into the CDR3, optionally including cases where a portion of the CDR3 (e.g., one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g., all or substantially all of the amino acids of the CDR3) is removed.

[0109] In some embodiments of each or any of the foregoing or following aspects, the non-antibody sequence is a synthetic sequence.

[0110] In some embodiments of each or any of the preceding or following embodiments, the non-antibody sequence is a cytokine sequence, a lymphokine sequence, a chemokine sequence, a growth factor sequence, a hormone sequence, or a toxin sequence.

[0111] In some embodiments of each or any of the foregoing or following embodiments, the non-antibody sequence is an IL-8 sequence, an IL-21 sequence, an SDF-1(α) sequence, a somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence, a ziconotide sequence, an ADWX-1 sequence, an HsTx1 sequence, an OSK1 sequence, a Pi2 sequence, a Hongotoxin (HgTX) sequence, a margatoxin sequence, an agitoxin-2 sequence, a Pi3 sequence, a karyotoxin sequence, an anuloctoxin sequence, a charybdotoxin sequence, a chitiustoxin-K-α sequence, a maurotoxin sequence, a ceratotoxin 1 (CcoTx1) sequence, a CcoTx2 sequence, a CcoTx3 sequence, a flixotoxin 3 (PaurTx3) sequence, a hanatoxin 1 sequence, a flixotoxin 1 sequence, a Hue Conotoxin-IV sequence, α-conotoxin ImI sequence, α-conotoxin EpI sequence, α-conotoxin PnIA sequence, α-conotoxin PnIB sequence, α-conotoxin MII sequence, α-conotoxin AuIA sequence, α-conotoxin AuIB sequence, α-conotoxin AuIC sequence, conotoxin κ-PVIIA sequence, charybdotoxin sequence, neurotoxin B-IV sequence, crotamine sequence, ω-GVIA (conotoxin) sequence, κ-heftoxin 1 sequence, Css4 sequence, Bj-xtrIT sequence, BcIV sequence, Hm-1 sequence, Hm-2 sequence, GsAF-I (β-teraphotoxin-Gr1b) sequence, protoxin I (ProTx-I sequence, β-teraphotoxin-Tp1a) sequence, protoxin II (ProTxII) sequence, Huentoxin I sequence, μ-conotoxin PIIIA sequence, Jinzaotoxin-III (β-TRTX-Cj1α) sequence, GsAF-II (κ-teraphotoxin-Gr2c) sequence, ShK (Stichodactyla toxin) sequence, HsTx1 sequence, Gangicitoxin 1E (GxTx-1E) sequence, Maurotoxin sequence, Charybdotoxin (ChTX) sequence, Iberiotoxin (IbTx) sequence, Leiurotoxin 1 (Scyllatoxin) sequence, Tamapin sequence, and Karyotoxin (Kr) sequence. the KTX sequence, the purotoxin 1 (PT-1) sequence, or the GpTx-1 sequence, the MOKA toxin sequence, the OSK1(P12, K16, D20) sequence, the OSK1(K16, D20) sequence, the HmK sequence, the ShK(K16, Y26, K29) sequence, the ShK(K16) sequence, the ShK-A(K16) sequence, the ShK(K16, E30) sequence, the ShK(Q21) sequence, the ShK(L21) sequence, the ShK(F21) sequence, the ShK(I21) sequence, or the ShK(A21) sequence.

[0112] In some embodiments of each or any of the foregoing or following embodiments, the non-antibody sequence is any one of SEQ ID NOs: 475-481, 599-655, 666-698, 727-733, 808-810, and 831-835.

[0113] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises 1 X 2 X 3 X 4 X 5 Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q).

[0114] In some embodiments of each or any of the preceding or following embodiments, X 1 X 2 X 3 X 4 X 5 The motif is The file is TIFF0007680480000013.tif72157.

[0115] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 1 X 2 X 3 X 4 X 5 Contains motifs.

[0116] In some embodiments of each or any of the preceding or following embodiments, CX 1 X 2 X 3 X 4 X 5 The motif is The file is TIFF0007680480000014.tif79159.

[0117] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4.

[0118] In some embodiments of each or any of the preceding or following embodiments, (Xa X b ) z The motif is The file is TIFF0007680480000015.tif132159.

[0119] In some embodiments of each or any of the preceding or following embodiments, (X a X b ) z The motif is YXYXYX.

[0120] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises 1 X 2 X 3 X 4 X 5 X n Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and n is 27 to 54).

[0121] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises n (X a X b ) z Motif (wherein, X a is any amino acid residue, and X bis an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4.

[0122] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises 1 X 2 X 3 X 4 X 5 X n (X a X b ) z Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and X is a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4.

[0123] In some embodiments of each or any of the preceding or following embodiments, X 1 X 2 X 3 X 4 X 5 The motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and (X a X b ) zThe motif is YXYXYX.

[0124] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 1 X 2 X 3 X 4 X 5 Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q); TIFF0007680480000016.tif186157; and (X a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4.

[0125] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 is 1 X 2 X 3 X 4 X 5 Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q); TIFF0007680480000017.tif212158; and (X a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4.

[0126] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises an additional sequence which is a linker.

[0127] In some embodiments of each or any of the foregoing or following embodiments, the linker is linked to the C-terminus, the N-terminus, or both the C-terminus and the N-terminus of the non-antibody sequence.

[0128] In some embodiments of each or any of the foregoing or following embodiments, the very long CDR3 is a ruminant CDR3.

[0129] In some embodiments of each or any of the foregoing or following embodiments, the ruminant is a bovine.

[0130] In some embodiments of each or any of the foregoing or following aspects, the humanized antibody or binding fragment thereof comprises human heavy chain variable region framework sequences.

[0131] In some embodiments of each or any of the foregoing or following aspects, the humanized antibody or binding fragment thereof comprises or is derived from a human heavy chain germline sequence.

[0132] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:735.

[0133] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:737.

[0134] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:739.

[0135] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:741.

[0136] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:743.

[0137] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:745.

[0138] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:747.

[0139] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:749.

[0140] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000018.tif105164.

[0141] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000019.tif118160.

[0142] In some embodiments of each or any of the foregoing or following aspects, the humanized antibody or binding fragment thereof comprises a light chain variable region sequence.

[0143] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises a light chain variable region comprising a sequence selected from the group of: (i) SEQ ID NO: 750, (ii) SEQ ID NO: 751, (iii) SEQ ID NO: 752, and (iv) SEQ ID NO: 753.

[0144] In some embodiments of each or any of the preceding or following embodiments, the light chain variable region sequence is a lambda light chain variable region sequence.

[0145] In some embodiments of each or any of the foregoing or following embodiments, the light chain variable region sequence is a human lambda light chain variable region sequence.

[0146] In some embodiments of each or any of the foregoing or following embodiments, the light chain variable region sequence comprises a VL1-51 germline sequence.

[0147] In some embodiments of each or any of the foregoing or following embodiments, the light chain variable region is derived from the VL1-51 germline sequence.

[0148] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises a CDR1 that comprises an Ile29Val substitution and an Asn32Gly substitution based on Kabat numbering.

[0149] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises a CDR2 comprising a DNN to GDT substitution.

[0150] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises a CDR2 that comprises a DNNKRP (SEQ ID NO: 471) to GDTSRA (SEQ ID NO: 472) substitution.

[0151] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence includes an S2A substitution, a T5N substitution, a P8S substitution, an A12G substitution, an A13S substitution, and a P14L substitution based on Kabat numbering.

[0152] In some embodiments of each or any of the foregoing or following embodiments, the VL1-51 germline sequence comprises an S2A substitution, a T5N substitution, a P8S substitution, an A12G substitution, an A13S substitution, and a P14L substitution based on Kabat numbering, and a CDR2 that comprises a DNN to GDT substitution.

[0153] In some embodiments of each or any of the above or below embodiments, a humanized antibody or binding fragment thereof comprising: (a) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742, and SEQ ID NO:743; and (b) a light chain variable region comprising SEQ ID NO:750.

[0154] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof is present in a spatially addressed format.

[0155] The present disclosure also provides a method of humanizing an antibody variable region comprising genetically combining a nucleic acid sequence encoding a very long CDR3 with a nucleic acid sequence encoding a variable region sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749.

[0156] The present disclosure also provides a method for generating a library of humanized antibodies comprising very long CDR3s, the method comprising: synthesizing a nucleic acid sequence encoding the very long CDR3 with any of the following: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO: 749 to create a nucleic acid encoding a humanized antibody with very long CDR3; and expressing the nucleic acid encoding the humanized antibody with very long CDR3 to generate a library of humanized antibodies with very long CDR3s.

[0157] The present disclosure also provides a method for generating a library of humanized antibodies or binding fragments thereof comprising very long CDR3s and comprising non-antibody sequences, the method comprising combining a nucleic acid sequence encoding the very long CDR3 and a nucleic acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO: 749 with a nucleic acid sequence encoding a non-antibody sequence to generate a nucleic acid encoding a humanized antibody or binding fragment thereof comprising an extremely long CDR3 and a non-antibody sequence; and expressing the nucleic acid encoding a humanized antibody or binding fragment thereof comprising an extremely long CDR3 and a non-antibody sequence to generate a library of humanized antibodies or binding fragments thereof comprising an extremely long CDR3 and a non-antibody sequence. In some embodiments, the extremely long CDR3 comprises a bovine sequence, a non-bovine sequence, an antibody sequence, or a non-antibody sequence.

[0158] The present disclosure also provides a library of humanized antibodies or binding fragments thereof comprising extremely long CDR3s comprising non-bovine or non-antibody sequences, including where a non-antibody sequence (e.g., a non-antibody human sequence) is inserted into the CDR3, optionally including where a portion of the CDR3 (e.g., one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g., all or substantially all of the amino acids of the CDR3) is removed.

[0159] The present disclosure also provides a method for generating a library of humanized antibodies or binding fragments thereof comprising a very long CDR3 comprising a cysteine ​​motif, the method comprising the steps of: combining a human variable region framework (FR) sequence with a nucleic acid sequence encoding a very long CDR3 and a cysteine ​​motif; introducing one or more nucleotide changes into the nucleic acid sequence encoding one or more amino acid residues located between the one or more cysteine ​​residues in the cysteine ​​motif to generate a nucleic acid encoding a humanized antibody or binding fragment thereof comprising a very long CDR3 and a cysteine ​​motif in which the one or more nucleotide changes have been introduced between the one or more cysteine ​​residues in the cysteine ​​domain from nucleotides encoding different amino acid residues; and expressing the nucleic acid encoding a humanized antibody or binding fragment thereof comprising a very long CDR3 and a cysteine ​​motif in which the one or more nucleotide changes have been introduced between the one or more cysteine ​​residues in the cysteine ​​domain to generate a library of humanized antibodies or binding fragments thereof comprising a very long CDR3 and a cysteine ​​motif in which the one or more amino acid changes have been introduced between the one or more cysteine ​​residues in the cysteine ​​domain.

[0160] The present disclosure also provides a library of humanized antibodies or binding fragments thereof comprising an extremely long CDR3 that contains a cysteine ​​motif, wherein the antibodies or binding fragments comprise one or more substitutions of amino acid residues located between the cysteine ​​residues in the cysteine ​​motif.

[0161] The present disclosure also provides a method for generating a library of humanized antibodies or binding fragments thereof comprising very long CDR3s of bovine origin, the method comprising combining a nucleic acid sequence encoding a human variable region framework (FR) sequence with a nucleic acid encoding a very long CDR3 of bovine origin, and expressing the nucleic acid encoding the human variable region framework (FR) sequence and the nucleic acid encoding the very long CDR3 of bovine origin to generate a library of humanized antibodies or binding fragments thereof comprising very long CDR3s of bovine origin.

[0162] The disclosure also provides a library of humanized antibodies or binding fragments thereof comprising: (a) an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising an extra long CDR3 of bovine origin.

[0163] The present disclosure also provides an antibody heavy chain variable region comprising a sequence of the formula V1-X-V2, where V1 is: TIFF0007680480000020.tif105164; X comprises an extremely long CDR3, which may comprise a non-human or non-antibody sequence (e.g. a non-antibody human sequence) inserted into the CDR3 sequence of the antibody, including optionally removing a portion of the CDR3 (e.g. one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g. all or substantially all of the amino acids of the CDR3); and V2 is TIFF0007680480000021.tif18164.

[0164] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000022.tif106161.

[0165] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000023.tif118160.

[0166] In some embodiments of each or any of the preceding or following embodiments, the very long CDR3 comprises: Contains the amino acid sequence of TIFF0007680480000024.tif226158.

[0167]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000025.tif25160, and TIFF0007680480000026.tif25158, and V2 is This includes cases where the amino acid sequence is selected from TIFF0007680480000027.tif5128.

[0168]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000028.tif25158, and TIFF0007680480000029.tif25160, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000030.tif5128.

[0169]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000031.tif25158, and TIFF0007680480000032.tif18158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000033.tif5128.

[0170]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000034.tif18158, and TIFF0007680480000035.tif32158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000036.tif5128.

[0171]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises One of TIFF0007680480000037.tif18158 and TIFF0007680480000038.tif18158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000039.tif5128.

[0172] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region includes cases where the very long CDR3 is 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length.

[0173] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises an extremely long CDR3, which is 35 amino acids in length or longer.

[0174] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises a very long CDR3 that comprises a cysteine ​​motif.

[0175]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises TIFF0007680480000040.tif25161, a cysteine ​​motif, and TIFF0007680480000041.tif18158, and V2 is Including the amino acid sequence of TIFF0007680480000042.tif5128, including cases.

[0176]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000043.tif25158, a cysteine ​​motif, and TIFF0007680480000044.tif25158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000045.tif5128.

[0177]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000046.tif25158, a cysteine ​​motif, and TIFF0007680480000047.tif18158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000048.tif5128.

[0178]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000049.tif18158, a cysteine ​​motif, and TIFF0007680480000050.tif32158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000051.tif5128.

[0179]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000052.tif18158, a cysteine ​​motif, and TIFF0007680480000053.tif25161, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000054.tif5128.

[0180] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region includes where VI comprises an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; the very long CDR3 comprises an amino acid sequence that is SEQ ID NO:498 and an amino acid sequence that is SEQ ID NO:499; and V2 comprises an amino acid sequence that is SEQ ID NO:500.

[0181]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises a cysteine ​​motif: TIFF0007680480000055.tif186158.

[0182]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises a cysteine ​​motif: TIFF0007680480000056.tif213159.

[0183] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes cases where the extremely long CDR3 comprises 2 to 6 disulfide bonds.

[0184] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region includes cases where the extremely long CDR3 comprises a non-antibody sequence.

[0185]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000057.tif25158, the non-antibody sequence, and TIFF0007680480000058.tif18158, and V2 is Including the amino acid sequence of TIFF0007680480000059.tif5128, including cases.

[0186]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000060.tif25158, the non-antibody sequence, and TIFF0007680480000061.tif25158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000062.tif5128.

[0187]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000063.tif25158, the non-antibody sequence, and TIFF0007680480000064.tif18158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000065.tif5128.

[0188]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000066.tif18158, the non-antibody sequence, and TIFF0007680480000067.tif32158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000068.tif5128.

[0189]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has V1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; and the very long CDR3 comprises any one of the amino acid sequences of TIFF0007680480000069.tif18158, the non-antibody sequence, and TIFF0007680480000070.tif25158, and V2 is The present invention includes cases where the amino acid sequence is selected from TIFF0007680480000071.tif5128.

[0190] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region includes where VI comprises an amino acid sequence selected from the group consisting of SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, and SEQ ID NO:749; the very long CDR3 comprises an amino acid sequence that is SEQ ID NO:498 and an amino acid sequence that is SEQ ID NO:499; and V2 comprises an amino acid sequence that is SEQ ID NO:500.

[0191] In some embodiments of each or any of the foregoing or following aspects, the antibody heavy chain variable region includes where the non-antibody sequence is a synthetic sequence.

[0192] In some embodiments of each or any of the preceding or following aspects, the antibody heavy chain variable region includes where the non-antibody sequence is a cytokine sequence, a lymphokine sequence, a chemokine sequence, a growth factor sequence, a hormone sequence, or a toxin sequence.

[0193]

[0023] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region is selected from the group consisting of an IL-8 sequence, an IL-21 sequence, an SDF-1(α) sequence, a somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence, a ziconotide sequence, an ADWX-1 sequence, an HsTx1 sequence, an OSK1 sequence, a Pi2 sequence, a hongotoxin (HgTX) sequence, a margatoxin sequence, an agitoxin-2 sequence, a Pi3 sequence, a karyotoxin sequence, an anuloctoxin sequence, a charybdotoxin sequence, a titiustoxin-K-α sequence, a maurotoxin sequence, a ceratotoxin 1 (CcoTx1) sequence, a CcoTx2 sequence, a CcoTx3 sequence, a flixotoxin 3 (PaurTx3) sequence, a hanatoxin 1 sequence, a flixoto ... sequence, Huentoxin-IV sequence, α-conotoxin ImI sequence, α-conotoxin EpI sequence, α-conotoxin PnIA sequence, α-conotoxin PnIB sequence, α-conotoxin MII sequence, α-conotoxin AuIA sequence, α-conotoxin AuIB sequence, α-conotoxin AuIC sequence, conotoxin κ-PVIIA sequence, charybdotoxin sequence, neurotoxin B-IV sequence, crotamine sequence, ω-GVIA (conotoxin) sequence, κ-heftoxin 1 sequence, Css4 sequence, Bj-xtrIT sequence, BcIV sequence, Hm-1 sequence, Hm-2 sequence, GsAF-I (β-teraphotoxin-Gr1b) sequence, protoxin I (ProTx-I sequence, β-teraphotoxin-Tp1a) sequence, protoxin II (ProTxII) sequence, Huentoxin I sequence, μ-conotoxin PIIIA sequence, Jinzaotoxin-III (β-TRTX-Cj1α) sequence, GsAF-II (κ-teraphotoxin-Gr2c) sequence, ShK (Stichodactyla toxin) sequence, HsTx1 sequence, Gangicitoxin 1E (GxTx-1E) sequence, Maurotoxin sequence, Charybdotoxin (ChTX) sequence, Iberiotoxin (IbTx) sequence, Leiurotoxin 1 (Scyllatoxin) sequence, Tamapin sequence, and Caryotoxin-1 (Calliotoxin) sequence. (KTX) sequence, purotoxin 1 (PT-1) sequence, or GpTx-1 sequence, MOKA toxin sequence, OSK1(P12, K16, D20) sequence, OSK1(K16, D20) sequence, HmK sequence, ShK(K16, Y26, K29) sequence, ShK(K16) sequence, ShK-A(K16) sequence, ShK(K16, E30) sequence, ShK(Q21) sequence, ShK(L21) sequence, ShK(F21) sequence, ShK(I21) sequence, or ShK(A21) sequence.

[0194] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes where the non-antibody sequence is any one of SEQ ID NOs: 475-481, 599-655, 666-698, 727-733, 808-810, and 831-835.

[0195] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 770-779, 784-791, 903-922, and 925-955. Thus, in some aspects, the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 941.

[0196] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region includes cases where the very long CDR3 comprises a linker sequence.

[0197] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes cases where the linker is linked to the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the non-antibody sequence.

[0198] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes cases where the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 575-598, 699-726, and 813-830, or any combination thereof.

[0199] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes cases where the linkers attached to both the N-terminus and C-terminus have the same or different amino acid sequences.

[0200] The present disclosure also provides an antibody or binding fragment thereof comprising an antibody heavy chain variable region disclosed herein.

[0201] In some embodiments of each or any of the foregoing or following embodiments, the heavy chain variable region further comprises a heavy chain constant region 1 (CH1).

[0202] In some embodiments of each or any of the aforementioned or following embodiments, the heavy chain variable region further comprises the amino acid sequence of SEQ ID NO:390.

[0203] In some embodiments of each or any of the foregoing or following aspects, the antibody or binding fragment further comprises a light chain variable region.

[0204] In some embodiments of each or any of the preceding or following embodiments, the light chain variable region further comprises a light chain constant region (CL).

[0205] The present disclosure also provides isolated polynucleotides encoding the antibody heavy chain variable regions described herein.

[0206] The present disclosure also provides vectors comprising the polynucleotides described herein.

[0207] The present disclosure also provides host cells comprising the vectors described herein.

[0208] The present disclosure also provides a nucleic acid library comprising a plurality of polynucleotides constituting a nucleic acid sequence encoding an antibody heavy chain variable region comprising a sequence of the formula V1-X-V2, wherein V1 is TIFF0007680480000072.tif106158, wherein X comprises an extremely long CDR3, which may comprise a non-human or non-antibody sequence (e.g. a non-antibody human sequence) inserted into the CDR3 sequence of the antibody, including optionally removing a portion of the CDR3 (e.g. one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g. all or substantially all of the amino acids of the CDR3); and V2 is TIFF0007680480000073.tif19158.

[0209] The present disclosure also provides a library of antibodies comprising an antibody heavy chain variable region comprising a sequence of the formula V1-X-V2, where V1 is TIFF0007680480000074.tif106158, wherein X comprises an extremely long CDR3, which may comprise a non-human or non-antibody sequence (e.g. a non-antibody human sequence) inserted into the CDR3 sequence of the antibody, including optionally removing a portion of the CDR3 (e.g. one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g. all or substantially all of the amino acids of the CDR3); and V2 comprises an amino acid sequence selected from the group consisting of: TIFF0007680480000075.tif18158.

[0210] Extremely long CDR3, X 1 X 2 X 3 X 4 X 5 Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q).

[0211]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises 1 X 2 X 3 X 4 X 5 The motif is, This includes the case where the file is TIFF0007680480000076.tif72159.

[0212]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region has a very long CDR3, a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4.

[0213]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region is a X b ) z The motif is, This includes the case where the file is TIFF0007680480000077.tif126159.

[0214]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises an extremely long CDR3, 1 X 2 X 3 X 4 X 5 X n Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and n is 27 to 54).

[0215]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises an extremely long CDR3, n (X a X b ) z Motif (wherein, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4.

[0216]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises an extremely long CDR3, 1 X 2 X 3 X 4 X 5 X n (X a X b ) z Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4.

[0217] The present disclosure also provides an antibody heavy chain variable region comprising a sequence of formula V1-X, where V1 is (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO: 749; and X comprises an extremely long CDR3, which may comprise a non-human or non-antibody sequence (e.g. a non-antibody human sequence) inserted into the CDR3 sequence of the antibody, including optionally removing a portion of the CDR3 (e.g. one or more amino acids of the CDR3) or the entire CDR3 sequence (e.g. all or substantially all of the amino acids of the CDR3).

[0218]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises an extremely long CDR3, 1 X 2 X 3 X 4 X 5 motif, where X1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); X3 is valine (V), alanine (A), threonine (T), or aspartic acid (D); X4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); and X5 is glutamine (Q).

[0219]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises the motif: This includes the case where the file is TIFF0007680480000078.tif72158.

[0220] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region includes cases where the very long CDR3 comprises a CX1X2X3X4X5 motif.

[0221]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises a CX1X2X3X4X5 motif: This includes the case where the file is TIFF0007680480000079.tif72158.

[0222] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes cases where the very long CDR3 comprises a (XaXb)z motif, where Xa is any amino acid residue, Xb is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W) and histidine (H), and z is 1 to 4.

[0223]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises a (XaXb)z motif: This includes the case where the file is TIFF0007680480000080.tif126158.

[0224] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region comprises a (XaXb)z motif where is YXYXYX.

[0225] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes cases where the very long CDR3 comprises an X1X2X3X4X5Xn motif, where X1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); X3 is valine (V), alanine (A), threonine (T), or aspartic acid (D); X4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X5 is glutamine (Q); and n is 27 to 54.

[0226] In some embodiments of each or any of the foregoing or following embodiments, the antibody heavy chain variable region includes cases where the extremely long CDR3 comprises a Xn(XaXb)z motif (wherein Xa is any amino acid residue, Xb is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W) and histidine (H), n is 27 to 54, and z is 1 to 4).

[0227]

[0023] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region comprises an extremely long CDR3, 1 X 2 X 3 X 4 X 5 X n (X a X b ) z Motif (wherein, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4. 206. The antibody heavy chain variable region of any one of claims 142-145, wherein V1 comprises the amino acid sequence of SEQ ID NO: 737, the very long CDR3 comprises the amino acid sequence of SEQ ID NO: 498 and SEQ ID NO: 499, and V2 comprises the amino acid sequence of SEQ ID NO: 500.

[0228] In some embodiments of each or any of the preceding or following embodiments, the antibody heavy chain variable region includes where V1 comprises the amino acid sequence of SEQ ID NO: 739, the very long CDR3 comprises the amino acid sequences of SEQ ID NO: 498 and SEQ ID NO: 499, and V2 comprises the amino acid sequence of SEQ ID NO: 500.

[0229] In some embodiments of each or any of the preceding or following embodiments, the humanized antibody or binding fragment thereof includes where the light chain variable region comprises the amino acid sequence of SEQ ID NO: 750, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755.

[0230] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof comprises the amino acid sequence of SEQ ID NO:756.

[0231] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof includes where the light chain variable region comprises the amino acid sequence of SEQ ID NO:751, the amino acid sequence of SEQ ID NO:754, and the amino acid sequence of SEQ ID NO:755.

[0232] In some embodiments of each or any of the aforementioned or following embodiments, the humanized antibody or binding fragment thereof further comprises the amino acid sequence of SEQ ID NO:756.

[0233] In some embodiments of each or any of the preceding or following embodiments, the humanized antibody or binding fragment thereof includes where the light chain variable region comprises the amino acid sequence of SEQ ID NO:752, the amino acid sequence of SEQ ID NO:754, and the amino acid sequence of SEQ ID NO:755.

[0234] In some embodiments of each or any of the aforementioned or following embodiments, the humanized antibody or binding fragment thereof further comprises the amino acid sequence of SEQ ID NO:756.

[0235] In some embodiments of each or any of the preceding or following embodiments, the humanized antibody or binding fragment thereof includes where the light chain variable region comprises the amino acid sequence of SEQ ID NO:753, the amino acid sequence of SEQ ID NO:754, and the amino acid sequence of SEQ ID NO:755.

[0236] In some embodiments of each or any of the aforementioned or following embodiments, the humanized antibody or binding fragment thereof further comprises the amino acid sequence of SEQ ID NO:756.

[0237] In some embodiments of each or any of the preceding or following embodiments, the humanized antibody or binding fragment thereof includes where the heavy chain variable region comprises the amino acid sequences of SEQ ID NO: 737 and SEQ ID NO: 500, and the very long CDR3 comprises the amino acid sequences of SEQ ID NO: 498 and SEQ ID NO: 499.

[0238] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 750, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755.

[0239] In some embodiments of each or any of the preceding or following embodiments, the humanized antibody or binding fragment thereof includes where the heavy chain variable region comprises the amino acid sequences of SEQ ID NO: 739 and SEQ ID NO: 500, and the very long CDR3 comprises the amino acid sequences of SEQ ID NO: 498 and SEQ ID NO: 499.

[0240] In some embodiments of each or any of the foregoing or following embodiments, the humanized antibody or binding fragment thereof further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 750, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755. [The present invention 1001] (a)(i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) an amino acid sequence selected from the group consisting of SEQ ID NO: 748 or SEQ ID NO: 749; and (b) Extremely long CDR3 A humanized antibody or binding fragment thereof comprising a heavy chain variable region comprising: [The present invention 1002] The humanized antibody or binding fragment thereof of the present invention 1001, wherein the extremely long CDR3 is 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length. [The present invention 1003] 1002. A humanized antibody or binding fragment thereof of the present invention, wherein the extremely long CDR3 is 35 amino acids in length or more. [The present invention 1004] The humanized antibody or binding fragment thereof of the present invention 1001, wherein the very long CDR3 comprises 3 or more cysteine ​​residues, 4 or more cysteine ​​residues, 5 or more cysteine ​​residues, 6 or more cysteine ​​residues, 7 or more cysteine ​​residues, 8 or more cysteine ​​residues, 9 or more cysteine ​​residues, 10 or more cysteine ​​residues, 11 or more cysteine ​​residues, or 12 or more cysteine ​​residues. [The present invention 1005] The humanized antibody or binding fragment thereof of the present invention 1004, wherein the very long CDR3 comprises three or more cysteine ​​residues. [The present invention 1006] A humanized antibody or binding fragment thereof of the present invention 1001, comprising a cysteine ​​motif. [The present invention 1007] The cysteine ​​motif is The humanized antibody or binding fragment thereof of the present invention 1006 selected from the group consisting of TIFF0007680480000081.tif192148. [The present invention 1008] The cysteine ​​motif is The humanized antibody or binding fragment thereof of the present invention 1006 selected from the group consisting of TIFF0007680480000082.tif227148. [The present invention 1009] 1001. A humanized antibody or binding fragment thereof of the present invention, wherein the extremely long CDR3 comprises 2 to 6 disulfide bonds. [The present invention 1010] 1001. A humanized antibody or binding fragment thereof of the present invention, wherein the extremely long CDR3 comprises SEQ ID NO: 40 or a derivative thereof. [The present invention 1011] 1001. A humanized antibody or binding fragment thereof of the present invention, wherein the extremely long CDR3 comprises amino acid residues 3 to 6 of any one of SEQ ID NOs: 1 to 4. [The present invention 1012] 1001. A humanized antibody or binding fragment thereof of the present invention, wherein the very long CDR3 comprises a non-human DH or a derivative thereof. [The present invention 1013] The humanized antibody or binding fragment thereof of the present invention 1012, wherein the non-human DH is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. [The present invention 1014] 1001. A humanized antibody or binding fragment thereof of the present invention, wherein the extremely long CDR3 comprises a JH sequence or a derivative thereof. [The present invention 1015] The humanized antibody or binding fragment thereof of the present invention 1014, wherein the JH sequence comprises amino acids 5 to 15 of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. [The present invention 1016] The extremely long CDR3 - a sequence derived from a non-human VH sequence or a derivative thereof; - a sequence derived from a non-human DH sequence or a derivative thereof; and / or - A sequence derived from the JH sequence or its derivatives The humanized antibody or binding fragment thereof of the present invention comprises: [The present invention 1017] 1016. The humanized antibody or binding fragment thereof of the present invention, wherein the extremely long CDR3 comprises an additional amino acid sequence comprising 2 to 6 or more amino acid residues located between the VH sequence and the DH sequence. [The present invention 1018] The humanized antibody or binding fragment thereof of the present invention 1017, wherein the additional amino acid sequence is selected from the group consisting of IR, IF, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. [The present invention 1019] The humanized antibody or binding fragment thereof of the present invention 1001, wherein the extremely long CDR3 comprises a sequence derived from or based on SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. [The present invention 1020] 1001. A humanized antibody or binding fragment thereof of the present invention, wherein the very long CDR3 comprises a non-bovine sequence or a non-antibody sequence. [The present invention 1021] The humanized antibody or binding fragment thereof of the present invention 1020, wherein the non-antibody sequence is a synthetic sequence. [The present invention 1022] The humanized antibody or binding fragment thereof of the present invention 1020, wherein the non-antibody sequence is a cytokine sequence, a lymphokine sequence, a chemokine sequence, a growth factor sequence, a hormone sequence, or a toxin sequence. [The present invention 1023] The non-antibody sequences include an IL-8 sequence, an IL-21 sequence, an SDF-1(α) sequence, a somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence, a ziconotide sequence, an ADWX-1 sequence, an HsTx1 sequence, an OSK1 sequence, a Pi2 sequence, a Hongotoxin (HgTX) sequence, a Margatoxin sequence, an Agitoxin-2 sequence, a Pi3 sequence, a Kaliotoxin sequence, an Anuroctoxin sequence, a Charybdotoxin sequence, a Tityustoxin-K-α sequence, a Maurotoxin sequence, a Ceratotoxin 1 (CcoTx1) sequence, a CcoTx2 sequence, a CcoTx3 sequence, a PaurTx3 sequence, a Hanatoxin sequence, a 1 sequence, flixotoxin 1 sequence, huentoxin-IV sequence, α-conotoxin ImI sequence, α-conotoxin EpI sequence, α-conotoxin PnIA sequence, α-conotoxin PnIB sequence, α-conotoxin MII sequence, α-conotoxin AuIA sequence, α-conotoxin AuIB sequence, α-conotoxin AuIC sequence, conotoxin κ-PVIIA sequence, charybdotoxin sequence, neuro Toxin B-IV sequence, crotamine sequence, ω-GVIA (conotoxin) sequence, κ-hefutoxin 1 sequence, Css4 sequence, Bj-xtrIT sequence, BcIV sequence, Hm-1 sequence, Hm-2 sequence, GsAF-I (β-theraphotoxin-Gr1b) sequence, protoxin I (ProTx-I sequence, β-theraphotoxin-Tp1a) sequence, protoxin II (ProTxII) sequence, Huentoxin I sequence, μ-Conotoxin PIIIA sequence, Jinzaotoxin-III (β-TRTX-Cj1α) sequence, GsAF-II (κ-Terafotoxin-Gr2c) sequence, ShK (Stichodactyla toxin) sequence, HsTx1 sequence, Guangxitoxin 1E (GxTx-1E) sequence, Maurotoxin sequence, Charybdotoxin (ChTX) sequence, Iberiotoxin (IbTx) sequence, Leiurotoxin 1 (Scyllatoxin) sequence, Tamapin (Ta mapin sequence, Karyotoxin-1 (KTX) sequence, Purotoxin 1 (PT-1) sequence, or GpTx-1 sequence, MOKA toxin sequence, OSK1(P12, K16, D20) sequence, OSK1(K16, D20) sequence, HmK sequence, ShK(K16, Y26, K29) sequence, ShK(K16) sequence, ShK-A(K16) sequence, ShK(K16, E30) sequence, ShK(Q21) sequence, ShK(L21) sequence, ShK(F21) sequence, ShK(I21) sequence, or ShK(A21) sequence. [The present invention 1024] The humanized antibody or binding fragment thereof of the present invention 1020, wherein the non-antibody sequence is any one of SEQ ID NOs: 475-481, 599-655, 666-698, 727-733, 808-810, and 831-835. [The present invention 1025] The humanized antibody or binding fragment thereof of the present invention, wherein the antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 770-779, 784-791, 903-922, and 925-955. [The present invention 1026] The humanized antibody or binding fragment thereof of the present invention 1020, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 956 or 959. [The present invention 1027] The humanized antibody or binding fragment thereof of the present invention 1020, wherein the antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 770-779, 784-791, 903-922, and 925-955, and the humanized antibody or binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 959. [The present invention 1028] The humanized antibody or binding fragment thereof of the present invention 1020, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 941, and the humanized antibody or binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 959. [The present invention 1029] The humanized antibody or binding fragment thereof of the present invention 1020, wherein the antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 770-779, 784-791, 903-922, and 925-955, and the humanized antibody or binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 956. [The present invention 1030] The humanized antibody or binding fragment thereof of the present invention 1020, wherein a non-antibody sequence replaces at least a portion of the very long CDR3. [The present invention 1031] Extremely long CDR3, X 1 X 2 X 3 X 4 X 5 Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X5 is glutamine (Q), A humanized antibody or binding fragment thereof of the present invention. [The present invention 1032] X 1 X 2 X 3 X 4 X 5 The motif is, The humanized antibody or binding fragment thereof of the present invention 1031, which is TIFF0007680480000083.tif72150. [The present invention 1033] Extremely long CDR3 is CX 1 X 2 X 3 X 4 X 5 The humanized antibody or binding fragment thereof of the present invention, comprising the motif. [The present invention 1034] CX 1 X 2 X 3 X 4 X 5 The motif is, The humanized antibody or binding fragment thereof of the present invention 1033, which is TIFF0007680480000084.tif79150. [The present invention 1035] The extremely long CDR3 (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; A humanized antibody or binding fragment thereof of the present invention. [The present invention 1036] (X a X b ) z The motif is, The humanized antibody or binding fragment thereof of the present invention 1035, which is TIFF0007680480000085.tif132150. [The present invention 1037] (X a X b ) z The humanized antibody or binding fragment thereof of the present invention 1035, wherein the motif is YXYXYX. [The present invention 1038] The extremely long CDR3 is 1 X 2 X 3 X 4 X 5 X n Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and n is 27 to 54; A humanized antibody or binding fragment thereof of the present invention. [The present invention 1039] The extremely long CDR3 is n (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4; A humanized antibody or binding fragment thereof of the present invention. [The present invention 1040] The extremely long CDR3 is 1 X 2 X3 X 4 X 5 X n (X a X b ) z Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and X is a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4; A humanized antibody or binding fragment thereof of the present invention. [The present invention 1041] X 1 X 2 X 3 X 4 X 5 The motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and (X a X b ) z The humanized antibody or binding fragment thereof of the present invention 1040, wherein the motif is YXYXYX. [The present invention 1042] The extremely long CDR3 -CX 1 X 2 X 3 X 4 X 5 A motif, wherein X 1is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q), CX 1 X 2 X 3 X 4 X 5 motif; - A cysteine ​​motif selected from the group consisting of TIFF0007680480000086.tif186149; -(X a X b ) z A motif, wherein X a is any amino acid residue, and X b (X) is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; a X b ) z motif The humanized antibody or binding fragment thereof of the present invention comprises: [The present invention 1043] The extremely long CDR3 -CX 1 X 2 X 3 X 4 X 5 A motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q), CX 1 X 2 X 3 X 4 X 5 motif; - TIFF0007680480000087.tif226150; and -(X a X b ) z A motif, wherein X a is any amino acid residue, and X b (X) is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; a X b ) z motif The humanized antibody or binding fragment thereof of the present invention comprises: [The present invention 1044] The humanized antibody or binding fragment thereof of the present invention 1020, wherein the extremely long CDR3 comprises an additional sequence which is a linker. [The present invention 1045] A humanized antibody or binding fragment thereof of the present invention 1044, wherein the linker is linked to the C-terminus, the N-terminus, or both the C-terminus and the N-terminus of the non-antibody sequence. [The present invention 1046] The humanized antibody or binding fragment thereof of the present invention 1001, wherein the very long CDR3 is a ruminant CDR3. [The present invention 1047] The humanized antibody or binding fragment thereof of the present invention 1046, wherein the ruminant is a bovine. [The present invention 1048] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 735. [The present invention 1049] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 737. [The present invention 1050] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 739. [The present invention 1051] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 741. [The present invention 1052] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 743. [The present invention 1053] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 745. [The present invention 1054] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 747. [The present invention 1055] The humanized antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 749. [The present invention 1056] The extremely long CDR3 A humanized antibody or binding fragment thereof of the present invention 1001, comprising the amino acid sequence of TIFF0007680480000088.tif126150. [The present invention 1057] The extremely long CDR3 A humanized antibody or binding fragment thereof of the present invention 1001, comprising the amino acid sequence of TIFF0007680480000089.tif139159. [The present invention 1058] The extremely long CDR3 A humanized antibody or binding fragment thereof of the present invention 1001, comprising the amino acid sequence of TIFF0007680480000090.tif233166. [The present invention 1059] The humanized antibody or binding fragment thereof of the present invention 1001, comprising a light chain variable region comprising a sequence selected from the group consisting of (i) SEQ ID NO: 750, (ii) SEQ ID NO: 751, (iii) SEQ ID NO: 752, and (iv) SEQ ID NO: 753. [The present invention 1060] The humanized antibody or binding fragment thereof of the present invention 1059, wherein the light chain variable region comprises a lambda light chain variable region sequence. [The present invention 1061] The humanized antibody or binding fragment thereof of the present invention 1059, wherein the light chain variable region comprises a human lambda light chain variable region sequence. [The present invention 1062] The humanized antibody or binding fragment thereof of the present invention, wherein the light chain variable region comprises a VL1-51 germline sequence. [The present invention 1063] The humanized antibody or binding fragment thereof of the present invention, wherein the light chain variable region is derived from the VL1-51 germline sequence. [The present invention 1064] The humanized antibody or binding fragment thereof of the present invention 1063, wherein the VL1-51 germline sequence comprises a CDR1 comprising an Ile29Val substitution and an Asn32Gly substitution according to Kabat numbering. [The present invention 1065] The humanized antibody or binding fragment thereof of the present invention 1063, wherein the VL1-51 germline sequence comprises a CDR2 comprising a DNN to GDT substitution. [The present invention 1066] The humanized antibody or binding fragment thereof of the present invention 1063, wherein the VL1-51 germline sequence comprises a CDR2 comprising a substitution of DNNKRP (SEQ ID NO: 471) to GDTSRA (SEQ ID NO: 472). [The present invention 1067] The humanized antibody or binding fragment thereof of the present invention 1063, wherein the VL1-51 germline sequence comprises an S2A substitution, a T5N substitution, a P8S substitution, an A12G substitution, an A13S substitution, and a P14L substitution based on the Kabat numbering. [The present invention 1068] The humanized antibody or binding fragment thereof of the present invention 1063, wherein the VL1-51 germline sequence comprises S2A, T5N, P8S, A12G, A13S, and P14L substitutions based on Kabat numbering, and CDR2 comprising a DNN to GDT substitution. [The present invention 1069] The humanized antibody or binding fragment thereof of the present invention, comprising: (a) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, and SEQ ID NO: 743; and (b) a light chain variable region comprising SEQ ID NO: 750. [The present invention 1070] A polynucleotide encoding a heavy chain variable region of any one of the humanized antibodies of the present invention 1001 to 1069 or a binding fragment thereof. [The present invention 1071] A polynucleotide encoding the light chain variable region of any one of the humanized antibodies 1059 to 1069 of the present invention or a binding fragment thereof. [The present invention 1072] A polynucleotide encoding a heavy chain variable region comprising a very long CDR3, the polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 494, SEQ ID NO: 495, SEQ ID NO: 496, and SEQ ID NO: 497. [The present invention 1073] A vector comprising any one of the polynucleotides of the present invention 1070 to 1072. [The present invention 1074] A host cell comprising a vector of the present invention. [The present invention 1075] A nucleic acid library comprising a plurality of polynucleotides constituting sequences encoding humanized antibodies or binding fragments thereof, the antibodies or binding fragments thereof comprising: (a)(i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) SEQ ID NO: 748 or SEQ ID NO: 749 an amino acid sequence selected from the group consisting of: (b) Extremely long CDR3 A nucleic acid library comprising a heavy chain variable region comprising: [The present invention 1076] A library of humanized antibodies or binding fragments thereof, the antibodies or binding fragments thereof comprising: (a)(i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) SEQ ID NO: 748 or SEQ ID NO: 749 an amino acid sequence selected from the group consisting of: (b) Extremely long CDR3 A library of humanized antibodies or binding fragments thereof comprising a heavy chain variable region comprising: [The present invention 1077] The library of the invention 1075 or 1076, wherein the very long CDR3 is 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length. [The present invention 1078] The library of the present invention 1077, wherein the extremely long CDR3 is 35 amino acids in length or more. [The present invention 1079] The library of the invention 1075 or 1076, wherein the very long CDR3 comprises 3 or more cysteine ​​residues, 4 or more cysteine ​​residues, 5 or more cysteine ​​residues, 6 or more cysteine ​​residues, 7 or more cysteine ​​residues, 8 or more cysteine ​​residues, 9 or more cysteine ​​residues, 10 or more cysteine ​​residues, 11 or more cysteine ​​residues, or 12 or more cysteine ​​residues. [The present invention 1080] The library of the present invention 1079, wherein the very long CDR3 comprises three or more cysteine ​​residues. [The present invention 1081] The library of the present invention 1076, wherein the antibody or binding fragment thereof comprises a cysteine ​​motif. [The present invention 1082] The cysteine ​​motif is The library of the present invention 1076 selected from the group consisting of TIFF0007680480000091.tif186148. [The present invention 1083] The cysteine ​​motif is The library of the present invention 1081 selected from the group consisting of TIFF0007680480000092.tif226148. [The present invention 1084] The library of the present invention, 1076, wherein the extremely long CDR3 contains 2 to 6 disulfide bonds. [The present invention 1085] 1076. The library of the present invention, wherein the extremely long CDR3 comprises SEQ ID NO: 40 or a derivative thereof. [The present invention 1086] The library of the present invention, 1076, wherein the very long CDR3 comprises amino acid residues 3 to 6 of any one of SEQ ID NOs: 1 to 4. [The present invention 1087] The library of the present invention 1076, wherein the extremely long CDR3 comprises a non-human DH or a derivative thereof. [The present invention 1088] The library of the present invention 1087, wherein the non-human DH is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. [The present invention 1089] The library of the present invention 1076, wherein the extremely long CDR3 comprises a JH sequence or a derivative thereof. [The present invention 1090] The library of the present invention 1089, wherein the JH sequence comprises amino acids 5 to 15 of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. [The present invention 1091] The extremely long CDR3 - a sequence derived from a non-human VH sequence or a derivative thereof; - a sequence derived from a non-human DH sequence or a derivative thereof; and / or - A sequence derived from the JH sequence or its derivatives The library of the present invention 1076 comprising: [The present invention 1092] The library of the present invention 1091, wherein the extremely long CDR3 comprises an additional amino acid sequence comprising 2 to 6 or more amino acid residues located between the VH sequence and the DH sequence. [The present invention 1093] The library of the present invention 1092, wherein the additional amino acid sequence is selected from the group consisting of IR, IF, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. [The present invention 1094] The library of the present invention 1076, wherein the extremely long CDR3 comprises a sequence derived from or based on SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. [The present invention 1095] The library of the present invention 1076, wherein the extremely long CDR3 comprises a non-bovine sequence or a non-antibody sequence. [The present invention 1096] The library of the present invention 1095, wherein the non-antibody sequence is a synthetic sequence. [The present invention 1097] The library of the present invention 1095, wherein the non-antibody sequence is a cytokine sequence, a lymphokine sequence, a chemokine sequence, a growth factor sequence, a hormone sequence, or a toxin sequence. [The present invention 1098] The non-antibody sequences include an IL-8 sequence, an IL-21 sequence, an SDF-1(α) sequence, a somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence, a ziconotide sequence, an ADWX-1 sequence, an HsTx1 sequence, an OSK1 sequence, a Pi2 sequence, a hongotoxin (HgTX) sequence, a margatoxin sequence, an agitoxin-2 sequence, a Pi3 sequence, a karyotoxin sequence, an anuloctoxin sequence, a charybdotoxin sequence, a chitiustoxin-K-α sequence, a maurotoxin sequence, a ceratotoxin 1 (CcoTx1) sequence, a CcoTx2 sequence, a CcoTx3 sequence, a flixotoxin 3 (PaurTx3) sequence, a hanatoxin 1 sequence, a flixotoxin 1 sequence, a fentoxin-IV sequence, and a-conotoxin α ... Conotoxin ImI sequence, α-conotoxin EpI sequence, α-conotoxin PnIA sequence, α-conotoxin PnIB sequence, α-conotoxin MII sequence, α-conotoxin AuIA sequence, α-conotoxin AuIB sequence, α-conotoxin AuIC sequence, conotoxin κ-PVIIA sequence, charybdotoxin sequence, neurotoxin B-IV sequence, crotamine sequence, ω-GVIA (conotoxin) sequence, κ-heftoxin 1 sequence, Css4 sequence, Bj-xtrIT sequence, BcIV sequence, Hm-1 sequence, Hm-2 sequence, GsAF-I (β-teraphotoxin-Gr1b) sequence, protoxin I (ProTx-I sequence, β-teraphotoxin-Tp1a) sequence, protoxin II (ProTxII) sequence, Huentoxin I sequence, μ-conotoxin PIIIA sequence, Jinzaotoxin-III (β-TRTX-Cj1α) sequence, GsAF-II (κ-teraphotoxin-Gr2c) sequence, ShK (Stichodactyla toxin) sequence, HsTx1 sequence, Gangicitoxin 1E (GxTx-1E) sequence, Maurotoxin sequence, Charybdotoxin (ChTX) sequence, Iberiotoxin (IbTx) sequence, Leiurotoxin 1 (Scyllatoxin) sequence, Tamapin sequence, and Karyotoxin-1 (KTX) sequence The library of the present invention 1095, wherein the library is a nucleotide sequence selected from the group consisting of a nucleotide sequence, ... [This invention 1099] The library of the present invention, wherein the non-antibody sequence is any one of SEQ ID NOs: 475-481, 599-655, 666-698, 727-733, 808-810, and 831-835. [The present invention 1100] Extremely long CDR3, X 1 X 2 X 3 X 4 X 5 Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5is glutamine (Q), The library of the present invention 1076. [The present invention 1101] X 1 X 2 X 3 X 4 X 5 The motif is, The library of the present invention 1100 is TIFF0007680480000093.tif79150. [The present invention 1102] Extremely long CDR3, CX 1 X 2 X 3 X 4 X 5 The library of the present invention 1076, comprising the motif. [The present invention 1103] CX 1 X 2 X 3 X 4 X 5 The motif is, The library of the present invention 1102 is TIFF0007680480000094.tif79150. [The present invention 1104] The extremely long CDR3 (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; The library of the present invention 1076. [The present invention 1105] (X a X b ) z The motif is, The library of the present invention 1104 is TIFF0007680480000095.tif139150. [The present invention 1106] (X a X b )z The library of the present invention 1104, wherein the motif is YXYXYX. [The present invention 1107] The extremely long CDR3 is 1 X 2 X 3 X 4 X 5 X n Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and n is 27 to 54; The library of the present invention 1076. [The present invention 1108] The extremely long CDR3 is n (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4; The library of the present invention 1076. [The present invention 1109] The extremely long CDR3 is 1 X 2 X 3 X 4 X 5 X n (X a X b ) zContains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and X is a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4; The library of the present invention 1076. [The present invention 1110] X 1 X 2 X 3 X 4 X 5 The motif is TTVHQ (SEQ ID NO: 153) or TSVHQ (SEQ ID NO: 154), and (X a X b ) z The library of the present invention 1109, wherein the motif is YXYXYX. [The present invention 1111] The extremely long CDR3 -CX 1 X 2 X 3 X 4 X 5 A motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q), CX 1 X 2 X 3 X 4 X 5 motif; - TIFF0007680480000096.tif186150; and -(X a X b ) z A motif, wherein X a is any amino acid residue, and X b (X) is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; a X b ) z motif The library of the present invention 1076 comprising: [The present invention 1112] The extremely long CDR3 -CX 1 X 2 X 3 X 4 X 5 A motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q), CX 1 X 2 X 3 X 4 X 5 motif; - TIFF0007680480000097.tif226150; and -(X a X b ) z A motif, wherein X a is any amino acid residue, and X b (X) is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; a X b ) z motif The library of the present invention 1076 comprising: [The present invention 1113] The library of the present invention 1095, wherein the extremely long CDR3 comprises an additional sequence which is a linker. [The present invention 1114] The library of the present invention 1113, wherein the linker is linked to the C-terminus, the N-terminus, or both the C-terminus and the N-terminus of the non-antibody sequence. [The present invention 1115] The library of the present invention 1076, wherein the extremely long CDR3 is a ruminant CDR3. [The present invention 1116] The library of the present invention 1115, wherein the ruminant is a cow. [The present invention 1117] The library of the present invention 1076, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 735. (113) The library of the present invention 1071, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 737. [The present invention 1118] The library of the present invention 1076, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 739. [The present invention 1119] The library of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 741. [The present invention 1120] The library of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 743. [The present invention 1121] The library of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 745. [The present invention 1122] The library of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 747. [The present invention 1123] The library of the present invention, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 749. [The present invention 1124] The extremely long CDR3 A library of the present invention 1076 comprising the amino acid sequence of TIFF0007680480000098.tif126150. [The present invention 1125] The extremely long CDR3 A library of the present invention 1076 comprising the amino acid sequence of TIFF0007680480000099.tif139157. [The present invention 1126] The library of the present invention 1076, wherein the humanized antibody or binding fragment thereof comprises a light chain variable region comprising a sequence selected from the group consisting of (i) SEQ ID NO: 750, (ii) SEQ ID NO: 751, (iii) SEQ ID NO: 752, and (iv) SEQ ID NO: 753. [The present invention 1127] The library of the present invention 1126, wherein the light chain variable region comprises a lambda light chain variable region sequence. [The present invention 1128] The library of the present invention 1126, wherein the light chain variable region sequence comprises a human λ light chain variable region sequence. [The present invention 1129] The library of the present invention 1126, wherein the light chain variable region sequence comprises a VL1-51 germline sequence. [The present invention 1130] The library of the present invention 1126, wherein the light chain variable region is derived from the VL1-51 germline sequence. [The present invention 1131] The library of the invention 1130, wherein the VL1-51 germline sequence comprises a CDR1 that contains an Ile29Val substitution and an Asn32Gly substitution based on Kabat numbering. [The present invention 1132] The library of the invention 1130, wherein the VL1-51 germline sequence comprises a CDR2 comprising a DNN to GDT substitution. [The present invention 1133] The library of the present invention 1130, wherein the VL1-51 germline sequence comprises a CDR2 comprising a substitution of DNNKRP (SEQ ID NO: 471) to GDTSRA (SEQ ID NO: 472). [The present invention 1134] The library of the invention 1130, wherein the VL1-51 germline sequence comprises an S2A substitution, a T5N substitution, a P8S substitution, an A12G substitution, an A13S substitution, and a P14L substitution based on the Kabat numbering. [This invention 1135] The library of the invention 1130, wherein the VL1-51 germline sequence comprises S2A, T5N, P8S, A12G, A13S, and P14L substitutions based on Kabat numbering, and a CDR2 comprising a DNN to GDT substitution. [The present invention 1136] The library of the present invention 1076, wherein the humanized antibody or binding fragment thereof comprises: (a) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, and SEQ ID NO: 743; and (b) a light chain variable region comprising SEQ ID NO: 750. [This invention 1137] The library of any of claims 1076 to 1136, wherein the humanized antibody or binding fragment thereof is present in a spatially addressed format. [The present invention 1138] A nucleic acid sequence encoding a very long CDR3 is (i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) SEQ ID NO: 748 or SEQ ID NO: 749 genetically combining said nucleic acid sequences with a nucleic acid sequence encoding a variable region sequence selected from the group consisting of: A method for humanizing an antibody variable region, comprising: [The present invention 1139] a) a nucleic acid sequence encoding a very long CDR3, (i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) SEQ ID NO: 748 or SEQ ID NO: 749 in combination with a nucleic acid sequence encoding a variable region sequence selected from the group consisting of: Producing a nucleic acid encoding a humanized antibody comprising a very long CDR3; and b) expressing nucleic acids encoding humanized antibodies with very long CDR3s to generate a library of humanized antibodies with very long CDR3s. A method for generating a library of humanized antibodies containing extremely long CDR3s, comprising: [The present invention 1140] a) a nucleic acid sequence encoding a very long CDR3; (i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) SEQ ID NO: 748 or SEQ ID NO: 749 A nucleic acid encoding a variable region sequence selected from the group consisting of: with a nucleic acid sequence encoding a non-antibody sequence to generate a nucleic acid encoding a humanized antibody or binding fragment thereof comprising a very long CDR3 and the non-antibody sequence; and b) expressing nucleic acids encoding humanized antibodies or binding fragments thereof comprising very long CDR3 and non-antibody sequences to generate a library of humanized antibodies or binding fragments thereof comprising very long CDR3 and non-antibody sequences. A method for generating a library of humanized antibodies or binding fragments thereof comprising an extremely long CDR3 comprising a non-antibody sequence, the method comprising: [This invention 1141] A library of humanized antibodies or binding fragments thereof comprising extremely long CDR3s that include non-antibody sequences. [This invention 1142] a) combining human variable region framework (FR) sequences with a nucleic acid sequence encoding a very long CDR3 and a cysteine ​​motif; b) introducing one or more nucleotide changes into a nucleic acid sequence encoding one or more amino acid residues located between one or more cysteine ​​residues in the cysteine ​​motif to generate a nucleic acid encoding a humanized antibody or binding fragment thereof comprising a very long CDR3 and a cysteine ​​motif in which one or more nucleotide changes have been introduced between one or more cysteine ​​residues in the cysteine ​​domain from nucleotides encoding different amino acid residues; and c) expressing nucleic acids encoding humanized antibodies or binding fragments thereof comprising a very long CDR3 and a cysteine ​​motif in which one or more nucleotide changes have been introduced between one or more cysteine ​​residues in the cysteine ​​domain to generate a library of humanized antibodies or binding fragments thereof comprising a very long CDR3 and a cysteine ​​motif in which one or more amino acid changes have been introduced between one or more cysteine ​​residues in the cysteine ​​domain. A method for generating a library of humanized antibodies or binding fragments thereof comprising an extremely long CDR3 containing a cysteine ​​motif, comprising: [This invention 1143] A library of humanized antibodies or binding fragments thereof comprising a very long CDR3 containing a cysteine ​​motif, wherein the antibodies or binding fragments comprise one or more substitutions of amino acid residues located between the cysteine ​​residues in the cysteine ​​motif. [This invention 1144] a) combining a nucleic acid sequence encoding a human variable region framework (FR) sequence with a nucleic acid encoding a very long CDR3 of bovine origin; and b) expressing nucleic acids encoding human variable region framework (FR) sequences and nucleic acids encoding very long CDR3s of bovine origin to generate a library of humanized antibodies or binding fragments thereof comprising very long CDR3s of bovine origin. A method for generating a library of humanized antibodies or binding fragments thereof comprising: [This invention 1145] (a)(i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) SEQ ID NO: 748 or SEQ ID NO: 749 an amino acid sequence selected from the group consisting of: (b) Extremely long CDR3 from bovine A library of humanized antibodies or binding fragments thereof comprising a heavy chain variable region comprising: [This invention 1146] An antibody heavy chain variable region comprising a sequence of the formula V1-X-V2, In the formula, V1 is comprising an amino acid sequence selected from the group consisting of: TIFF0007680480000100.tif113148; X comprises an extremely long CDR3; and V2 is TIFF0007680480000101.tif38128, Antibody heavy chain variable region. [This invention 1147] The extremely long CDR3 The antibody heavy chain variable region of the present invention 1146, comprising the amino acid sequence of TIFF0007680480000102.tif126148. [This invention 1148] The extremely long CDR3 The antibody heavy chain variable region of the present invention 1146, comprising the amino acid sequence of TIFF0007680480000103.tif139155. [This invention 1149] The extremely long CDR3 The antibody heavy chain variable region of the present invention 1146, comprising the amino acid sequence of TIFF0007680480000104.tif13142TIFF0007680480000105.tif248162. [The present invention 1150] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000106.tif25140, and TIFF0007680480000107.tif25151, -V2, TIFF0007680480000108.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1151] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000109.tif25138, and TIFF0007680480000110.tif25145, -V2, TIFF0007680480000111.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1152] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000112.tif25145, and TIFF0007680480000113.tif25145, -V2, TIFF0007680480000114.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1153] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000115.tif18140, and TIFF0007680480000116.tif32151, -V2, TIFF0007680480000117.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1154] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 TIFF0007680480000118.tif18135, and TIFF0007680480000119.tif18135, -V2, TIFF0007680480000120.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1155] The antibody heavy chain variable region of any of the present inventions 1146 to 1154, wherein the extremely long CDR3 is 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length. [This invention 1156] The antibody heavy chain variable region of the present invention 1155, wherein the extremely long CDR3 is 35 amino acids or more in length. [This invention 1157] The antibody heavy chain variable region of any of Nos. 1146 to 1156 of the present invention, wherein the extremely long CDR3 contains a cysteine ​​motif. [This invention 1158] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 TIFF0007680480000121.tif25141, a cysteine ​​motif, and TIFF0007680480000122.tif25141, and -V2, TIFF0007680480000123.tif5128 containing the amino acid sequence The antibody heavy chain variable region of the present invention 1146. [This invention 1159] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000124.tif25138, a cysteine ​​motif, and TIFF0007680480000125.tif25142, -V2, TIFF0007680480000126.tif5128, The antibody heavy chain variable region of the present invention 1146. [The present invention 1160] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences, cysteine ​​motifs, and TIFF0007680480000128.tif25142, -V2, TIFF0007680480000129.tif5128, The antibody heavy chain variable region of the present invention 1146. [The present invention 1161] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000130.tif19140, a cysteine ​​motif, and TIFF0007680480000131.tif32138, -V2, TIFF0007680480000132.tif5128, The antibody heavy chain variable region of the present invention 1146. [The present invention 1162] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000133.tif18135, a cysteine ​​motif, and TIFF0007680480000134.tif25140, -V2, TIFF0007680480000135.tif5128, The antibody heavy chain variable region of the present invention 1146. [The present invention 1163] -VI comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; - the very long CDR3 comprises the amino acid sequence of SEQ ID NO: 498 and the amino acid sequence of SEQ ID NO: 499; - V2 comprises the amino acid sequence of SEQ ID NO: 500; The antibody heavy chain variable region of the present invention 1146. [The present invention 1164] The cysteine ​​motif is The antibody heavy chain variable region of any one of Nos. 1157 to 1163 of the present invention, selected from the group consisting of TIFF0007680480000136.tif199152. [The present invention 1165] The cysteine ​​motif is The antibody heavy chain variable region of any one of Nos. 1157 to 1163 of the present invention, selected from the group consisting of TIFF0007680480000137.tif240151. [The present invention 1166] The antibody heavy chain variable region of any of the antibodies 1146 to 1165 of the present invention, wherein the extremely long CDR3 comprises 2 to 6 disulfide bonds. [The present invention 1167] The antibody heavy chain variable region of any of Nos. 1146 to 1165 of the present invention, wherein the extremely long CDR3 comprises a non-antibody sequence. [The present invention 1168] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000138.tif25141, the non-antibody sequence, and TIFF0007680480000139.tif25142, and -V2, TIFF0007680480000140.tif5128 containing the amino acid sequence The antibody heavy chain variable region of the present invention 1146. [The present invention 1169] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000141.tif25138, the non-antibody sequence, and TIFF0007680480000142.tif25142, -V2, TIFF0007680480000143.tif5128, The antibody heavy chain variable region of the present invention 1146. [The present invention 1170] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000144.tif25145, the non-antibody sequence, and TIFF0007680480000145.tif25142, -V2, TIFF0007680480000146.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1171] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000147.tif18140, the non-antibody sequence, and TIFF0007680480000148.tif32138, -V2, TIFF0007680480000149.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1172] - V1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; -Extremely long CDR3 any one of the amino acid sequences of TIFF0007680480000150.tif19135, the non-antibody sequence, and TIFF0007680480000151.tif18135, -V2, TIFF0007680480000152.tif5128, The antibody heavy chain variable region of the present invention 1146. [This invention 1173] -VI comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, and SEQ ID NO: 749; - the very long CDR3 comprises the amino acid sequence of SEQ ID NO: 498 and the amino acid sequence of SEQ ID NO: 499; - V2 comprises the amino acid sequence of SEQ ID NO: 500; The antibody heavy chain variable region of the present invention 1146. [This invention 1174] The antibody heavy chain variable region of the present invention 1167, wherein the non-antibody sequence is a synthetic sequence. [This invention 1175] The antibody heavy chain variable region of the present invention 1167, wherein the non-antibody sequence is a cytokine sequence, a lymphokine sequence, a chemokine sequence, a growth factor sequence, a hormone sequence, or a toxin sequence. [The present invention 1176] The non-antibody sequences include an IL-8 sequence, an IL-21 sequence, an SDF-1(α) sequence, a somatostatin sequence, a chlorotoxin sequence, a Pro-TxII sequence, a ziconotide sequence, an ADWX-1 sequence, an HsTx1 sequence, an OSK1 sequence, a Pi2 sequence, a hongotoxin (HgTX) sequence, a margatoxin sequence, an agitoxin-2 sequence, a Pi3 sequence, a karyotoxin sequence, an anuloctoxin sequence, a charybdotoxin sequence, a chitiustoxin-K-α sequence, a maurotoxin sequence, a ceratotoxin 1 (CcoTx1) sequence, a CcoTx2 sequence, a CcoTx3 sequence, a flixotoxin 3 (PaurTx3) sequence, a hanatoxin 1 sequence, a flixotoxin 1 sequence, a fentoxin-IV sequence, and a-conotoxin α ... Conotoxin ImI sequence, α-conotoxin EpI sequence, α-conotoxin PnIA sequence, α-conotoxin PnIB sequence, α-conotoxin MII sequence, α-conotoxin AuIA sequence, α-conotoxin AuIB sequence, α-conotoxin AuIC sequence, conotoxin κ-PVIIA sequence, charybdotoxin sequence, neurotoxin B-IV sequence, crotamine sequence, ω-GVIA (conotoxin) sequence, κ-heftoxin 1 sequence, Css4 sequence, Bj-xtrIT sequence, BcIV sequence, Hm-1 sequence, Hm-2 sequence, GsAF-I (β-teraphotoxin-Gr1b) sequence, protoxin I (ProTx-I sequence, β-teraphotoxin-Tp1a) sequence, protoxin II (ProTxII) sequence, Huentoxin I sequence, μ-conotoxin PIIIA sequence, Jinzaotoxin-III (β-TRTX-Cj1α) sequence, GsAF-II (κ-teraphotoxin-Gr2c) sequence, ShK (Stichodactyla toxin) sequence, HsTx1 sequence, Gangicitoxin 1E (GxTx-1E) sequence, Maurotoxin sequence, Charybdotoxin (ChTX) sequence, Iberiotoxin (IbTx) sequence, Leiurotoxin 1 (Scyllatoxin) sequence, Tamapin sequence, and Karyotoxin-1 (KTX) sequence. The antibody heavy chain variable region of the present invention is a 1167 sequence, ... [This invention 1177] The antibody heavy chain variable region of the present invention 1172, wherein the non-antibody sequence is any one of SEQ ID NOs: 475-481, 599-655, 666-698, 727-733, 808-810, and 831-835. [This invention 1178] The antibody heavy chain variable region of the present invention 1177, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 770-779, 784-791, 903-922, and 925-955. [This invention 1179] The antibody heavy chain variable region of the present invention 1167, comprising the amino acid sequence of SEQ ID NO: 941. [The present invention 1180] The heavy chain variable region of any of the antibodies according to the present invention, Nos. 1142 to 1172, wherein the extremely long CDR3 comprises a linker sequence. [This invention 1181] The antibody heavy chain variable region of the present invention 1180, wherein the linker is linked to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus of the non-antibody sequence. [The present invention 1182] The antibody heavy chain variable region of the present invention 1180 or 1181, wherein the linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 575-598, 699-726, and 813-830, or any combination thereof. [The present invention 1183] The antibody heavy chain variable region of the present invention 1181, wherein the linkers linked to both the N-terminus and C-terminus have the same or different amino acid sequences. [This invention 1184] An antibody or a binding fragment thereof comprising the heavy chain variable region of any one of antibodies 1146 to 1183 of the present invention. [This invention 1185] The antibody or binding fragment thereof of the present invention 1184, wherein the heavy chain variable region further comprises a heavy chain constant region 1 (CH1). [The present invention 1186] The antibody or binding fragment thereof of the present invention, wherein the heavy chain variable region further comprises the amino acid sequence of SEQ ID NO: 390. [This invention 1187] The antibody or binding fragment thereof of the present invention 1184, further comprising a light chain variable region. [The present invention 1188] The antibody or binding fragment thereof of the present invention 1187, wherein the light chain variable region further comprises a light chain constant region (CL). [The present invention 1189] An isolated polynucleotide encoding the heavy chain variable region of any one of antibodies 1146 to 1183 of the present invention. [The present invention 1190] A vector comprising a polynucleotide of the present invention. [The present invention 1191] A host cell comprising a vector of the present invention. [This invention 1192] A nucleic acid library comprising a plurality of polynucleotides constituting a nucleic acid sequence encoding an antibody heavy chain variable region comprising a sequence of the formula V1-X-V2, In the formula, V1 is comprising an amino acid sequence selected from the group consisting of: TIFF0007680480000153.tif125145; X comprises an extremely long CDR3; and V2 is TIFF0007680480000154.tif49128, Nucleic acid libraries. [The present invention 1193] A library of antibodies comprising an antibody heavy chain variable region comprising a sequence of the formula V1-X-V2, In the formula, V1 is TIFF0007680480000155.tif126145; X comprises an extremely long CDR3; and V2 is TIFF0007680480000156.tif49128, Antibody library. [This invention 1194] The extremely long CDR3 is 1 X 2 X 3 X 4 X 5 Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q), The antibody heavy chain variable region of the present invention 1146. [The present invention 1195] X 1 X 2 X 3 X 4 X 5The motif is TIFF0007680480000157.tif72144, the antibody heavy chain variable region of the present invention 1194. [The present invention 1196] The extremely long CDR3 (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; The antibody heavy chain variable region of the present invention 1146. [The present invention 1197] (X a X b ) z The motif is TIFF0007680480000158.tif139151, the antibody heavy chain variable region of the present invention 1196. [The present invention 1198] (X a X b ) z The antibody heavy chain variable region of the present invention 1197, wherein the motif is YXYXYX. [The present invention 1199] The extremely long CDR3 is 1 X 2 X 3 X 4 X 5 X n Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and n is 27 to 54; The antibody heavy chain variable region of the present invention 1146. [The present invention 1200] The extremely long CDR3 is n (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4; The antibody heavy chain variable region of the present invention 1146. [The present invention 1201] The extremely long CDR3 is 1 X 2 X 3 X 4 X 5 X n (X a X b ) z motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and X a is any amino acid residue, and X bis an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4; The antibody heavy chain variable region of the present invention 1146. [The present invention 1202] An antibody heavy chain variable region comprising a sequence of formula V1-X, In the formula, V1 is (i) SEQ ID NO: 734 or SEQ ID NO: 735, (ii) SEQ ID NO: 736 or SEQ ID NO: 737, (iii) SEQ ID NO: 738 or SEQ ID NO: 739, (iv) SEQ ID NO: 740 or SEQ ID NO: 741, (v) SEQ ID NO: 742 or SEQ ID NO: 743, (vi) SEQ ID NO: 744 or SEQ ID NO: 745, (vii) SEQ ID NO: 746 or SEQ ID NO: 747, and (viii) SEQ ID NO: 748 or SEQ ID NO: 749 and X comprises an extremely long CDR3, Antibody heavy chain variable region. [The present invention 1203] The extremely long CDR3 is 1 X 2 X 3 X 4 X 5 Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q), The antibody heavy chain variable region of the present invention 1202. [The present invention 1204] X 1 X 2 X 3 X 4 X 5 The motif is, TIFF0007680480000159.tif72144, the antibody heavy chain variable region of the present invention 1203. [The present invention 1205] Extremely long CDR3, CX 1 X 2 X 3 X 4 X 5 The antibody heavy chain variable region of the present invention 1202, comprising the motif. [The present invention 1206] CX 1 X 2 X 3 X 4 X 5 The motif is, TIFF0007680480000160.tif79142, the antibody heavy chain variable region of the present invention 1205. [The present invention 1207] The extremely long CDR3 (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4; The antibody heavy chain variable region of the present invention 1202. [The present invention 1208] (X a X b )z The motif is, TIFF0007680480000161.tif139151, the antibody heavy chain variable region of the present invention 1207. [The present invention 1209] (X a X b ) z The antibody heavy chain variable region of the present invention 1207, wherein the motif is YXYXYX. [The present invention 1210] Extremely long CDR3, X 1 X 2 X 3 X 4 X 5 X n Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and n is 27 to 54; The antibody heavy chain variable region of the present invention 1202. [The present invention 1211] Extremely long CDR3, X n (X a X b ) z Contains motifs, In the formula, X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), n is 27 to 54, and z is 1 to 4; The antibody heavy chain variable region of the present invention 1202. [The present invention 1212] Extremely long CDR3, X 1 X 2 X 3 X 4 X 5 X n (X a X b ) z Contains motifs, In the formula, X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and X 5 is glutamine (Q) and X is a is any amino acid residue, and X b (206) The antibody heavy chain variable region of any of the antibody heavy chain variable regions of the present inventions 1142 to 1145, wherein V1 comprises the amino acid sequence of SEQ ID NO: 737, the very long CDR3 comprises the amino acid sequences of SEQ ID NO: 498 and SEQ ID NO: 499, and V2 comprises the amino acid sequence of SEQ ID NO: 500. [The present invention 1213] The antibody heavy chain variable region of any of claims 1146 to 1149 of the present invention, wherein V1 comprises the amino acid sequence of SEQ ID NO: 739, the very long CDR3 comprises the amino acid sequences of SEQ ID NO: 498 and SEQ ID NO: 499, and V2 comprises the amino acid sequence of SEQ ID NO: 500. [The present invention 1214] The humanized antibody or binding fragment thereof of the present invention 1059, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 750, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755. [The present invention 1215] The humanized antibody or binding fragment thereof of the present invention further comprises the amino acid sequence of SEQ ID NO: 756. [The present invention 1216] The humanized antibody or binding fragment thereof of the present invention 1059, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 751, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755. [The present invention 1217] The humanized antibody or binding fragment thereof of the present invention further comprises the amino acid sequence of SEQ ID NO: 756. [The present invention 1218] The humanized antibody or binding fragment thereof of the present invention 1059, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 752, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755. [The present invention 1219] The humanized antibody or binding fragment thereof of the present invention further comprises the amino acid sequence of SEQ ID NO: 756. [The present invention 1220] The humanized antibody or binding fragment thereof of the present invention 1059, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 753, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755. [The present invention 1221] The humanized antibody or binding fragment thereof of the present invention further comprises the amino acid sequence of SEQ ID NO: 756. [The present invention 1222] The humanized antibody or binding fragment thereof of the present invention 1001, wherein the heavy chain variable region comprises the amino acid sequences of SEQ ID NO: 737 and SEQ ID NO: 500, and the very long CDR3 comprises the amino acid sequences of SEQ ID NO: 498 and SEQ ID NO: 499. [The present invention 1223] The humanized antibody or binding fragment thereof of the present invention 1222, further comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 750, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755. [The present invention 1224] The humanized antibody or binding fragment thereof of the present invention 1001, wherein the heavy chain variable region comprises the amino acid sequences of SEQ ID NO: 739 and SEQ ID NO: 500, and the very long CDR3 comprises the amino acid sequences of SEQ ID NO: 498 and SEQ ID NO: 499. [The present invention 1225] The humanized antibody or binding fragment thereof of the present invention 1224, further comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 750, the amino acid sequence of SEQ ID NO: 754, and the amino acid sequence of SEQ ID NO: 755.

[0241] The foregoing summary and the following detailed description of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the present disclosure is not limited to the precise arrangements, examples, and instrumentalities shown. [Brief description of the drawings]

[0242] [Figure 1] 1 shows sequence alignments of exemplary bovine-derived antibody variable region sequences designated BLV1H12, BLV5B8, BLV5D3, BLV8C11, BF4E9, BF1H1, or F18 that contain extremely long CDR3 sequences. [Figure 2A]Figures 2A-2C show very long CDR3 sequences. (Top) Translation from germline VHBUL, DH2, and JH. The five full-length very long CDR H3s reported in the literature contain 4-8 cysteines and are not highly homologous to each other; however, when the first cysteine ​​of these CDR H3s is "fixed" before alignment, some conservation of cysteine ​​residues with DH2 can be found. Four of the seven sequences (BLV1H12, BLV5D3, BLV8C11, and BF4E9) contain four cysteines in the same positions as DH2, but also have an additional cysteine. BLV5B8 has two cysteines in common with germline DH2. This limited homology, including some cysteine ​​conservation, suggests that these sequences may have been generated by mutation of DH2. B-L1 and B-L2 are derived from initial sequences from bovine spleen, while the remaining ones are very long CDR H3 sequences selected from deep sequencing data. The first group contains the longest CDR H3 identified and is likely clonally related. * indicates a sequence that was represented 167 times, suggesting that it was strongly selected for function. Several of the 8 cysteine ​​sequences were represented multiple times, indicated in brackets, and therefore likely selected for function. Other representative sequences of various lengths are shown in the last group. Framework cysteine ​​and tryptophan residues that define the boundaries of the CDR H3 are double underlined. The sequences BLV1H12-UL-77 (left-most column) presented in Tables 2A-C are shown divided into four segments to identify segments of amino acid residues derived from certain germline sequences and V / D / J joint sequences. From left to right, the first segment is derived from the VH germline and is represented in this disclosure as the X1X2X3X4X5 motif. The second segment represents the sequence derived from the VD junction and is represented in this disclosure as X. The third segment is a set of amino acid residues derived from the DH2 germline, and the fourth segment is a set of amino acid residues derived from the JH1 germline region. [Figure 2B] Details are as shown in Figure 2A. [Figure 2C]Details are as shown in Figure 2A. [Diagram 3] 1 shows a sequence alignment of exemplary bovine-derived very long CDR3 sequences designated BLV1H12, BLV5B8, BLV5D3, BLV8C11, BF4E9, BF1H1, or F18. [Figure 4] 1 shows an exemplary bovine germline heavy chain variable region (VH) sequence, designated VH-UL, suitable for modification by or use with a very long CDR3 sequence. [Figure 5A] Exemplary human germline heavy chain variable region sequences, designated 4-39, 4-59*03, 4-34*09, and 4-34*02, suitable for modification by or use with very long CDR3 sequences are shown. [Figure 5B] 1 shows an alignment of exemplary human germline heavy chain variable region sequences, designated 4-39, 4-59*03, 4-34*09, and 4-34*02, suitable for modification by or use with very long CDR3 sequences. [Figure 6] An exemplary bovine light chain variable region sequence, designated BLV1H12, suitable for modification by or use with very long CDR3 sequences (e.g., heavy chain variable region sequences that include very long CDR3 sequences) is shown. [Figure 7A] Exemplary light chain variable region sequences suitable for modification by or use with very long CDR3 sequences, designated Vl1-47, Vl1-40*1, Vl1-51*01, and Vl2-18*02, are shown. [Figure 7B] 1 shows an alignment of exemplary light chain variable region sequences, designated Vl1-47, Vl1-40*1, Vl1-51*01, and Vl2-18*02, suitable for modification by or use with very long CDR3 sequences. [Figure 8] 1 shows an exemplary heavy chain variable region sequence. [Figure 9]1 shows an exemplary light chain variable region sequence. [Figure 10-1] 1 shows an exemplary heavy chain variable region sequence with an IL-8 non-antibody sequence. [Figure 10-2] 1 shows an exemplary heavy chain variable region sequence with an IL-8 non-antibody sequence. [Figure 10-3] 1 shows an exemplary heavy chain variable region sequence with an IL-8 non-antibody sequence. [Figure 10-4] 1 shows an exemplary heavy chain variable region sequence with an IL-8 non-antibody sequence. [Figure 11-1] Exemplary light chain variable and constant region sequences are shown. [Figure 11-2] Exemplary light chain variable and constant region sequences are shown. [Figure 12] Exemplary amino acid sequences are shown. [Figure 13-1] 1 shows an exemplary nucleic acid linker-BsaI-linker sequence introduced into the BLV1H12 sequence. [Figure 13-2] 1 shows an exemplary nucleic acid linker-BsaI-linker sequence introduced into the BLV1H12 sequence. [Figure 13-3] 1 shows an exemplary nucleic acid linker-BsaI-linker sequence introduced into the BLV1H12 sequence. [Figure 13-4] 1 shows an exemplary nucleic acid linker-BsaI-linker sequence introduced into the BLV1H12 sequence. [Figure 14-1] 1 shows an exemplary BLV1H12 heavy chain amino acid sequence with a toxin sequence inserted into CDR3 by various linkers. [Figure 14-2] 1 shows an exemplary BLV1H12 heavy chain amino acid sequence with a toxin sequence inserted into CDR3 by various linkers. [Figure 15-1] Exemplary amino acid sequences of the heavy chain variable region and the light chain (VL-CL) are shown. [Figure 15-2] Exemplary amino acid sequences of the heavy chain variable region and the light chain (VL-CL) are shown. [Figure 15-3] Exemplary amino acid sequences of the heavy chain variable region and the light chain (VL-CL) are shown. [Figure 16-1] 1 shows the amino acid sequences of exemplary linkers. [Figure 16-2] A continuation of Figure 16-1 is shown. [Figure 16-3] A continuation of Figure 16-2 is shown. [Figure 17-1] 1 shows exemplary amino acid sequences for toxins. [Figure 17-2] 1 shows exemplary amino acid sequences for toxins. [Figure 17-3] 1 shows exemplary amino acid sequences for toxins. [Figure 17-4] 1 shows exemplary amino acid sequences for toxins. [Figure 17-5] 1 shows exemplary amino acid sequences for toxins. [Figure 17-6] 1 shows exemplary amino acid sequences for toxins. [Figure 18] 1 shows exemplary nucleic acid sequences for toxins. [Figure 19-1] Exemplary A, D, and V2 regions of a variable heavy chain from an HIV-1 neutralizing antibody are shown. [Figure 19-2] A continuation of Figure 19-1 is shown. [Figure 19-3] A continuation of Figure 19-2 is shown. [Figure 20] Exemplary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of antibody samples (VH4-34 MutE 1xG4S ShK (BID #56) and VH4-34 MutE linkerless ShK (BID #59)) purified from CHO (e.g., CHO-S) and HEK (e.g., 293F) cells. [Figure 21-1] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-2] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-3]1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-4] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-5] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-6] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-7] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-8] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-9] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-10] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-11] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-12] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-13]1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-14] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-15] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-16] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-17] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-18] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-19] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-20] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-21] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-22] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-23]1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-24] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-25] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-26] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-27] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-28] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-29] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-30] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-31] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-32] 1 shows exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-33]1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-34] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 21-35] 1A-1D provide exemplary amino acid and nucleic acid sequences for the heavy chain variable regions described and referenced herein by BID number, IgG name, and / or heavy chain name. [Figure 22-1] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-2] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-3] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-4] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-5] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-6] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-7] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-8]1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-9] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-10] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-11] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-12] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-13] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-14] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-15] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-16] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-17] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-18]1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-19] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-20] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-21] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-22] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-23] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-24] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-25] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-26] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-27] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-28]1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-29] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-30] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-31] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-32] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-33] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-34] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. [Figure 22-35] 1A-1D provide exemplary amino acid and nucleic acid sequences for the light chain variable regions described and referenced herein by BID number, IgG name, and / or light chain name. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0243] Detailed Description The disclosure provides a humanized antibody comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO 747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3 sequence, as well as materials (e.g., protein sequence, gene sequence, cells, libraries) and methods (e.g., humanization methods, library methods) for making the antibody. Such humanized antibodies may be useful for the treatment or prevention of a variety of diseases, disorders, or conditions, including inflammatory diseases, disorders, or conditions, autoimmune diseases, disorders, or conditions, metabolic diseases, disorders, or conditions, neoplastic diseases, disorders, or conditions, and cancer.

[0244] The present disclosure also relates to (a) (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749. Also provided is a humanized antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of NO:749; and (b) a very long CDR3 sequence, wherein the CDR3 sequence is 35 amino acids long or more (e.g., 40 or more, 45 or more, 50 or more, 55 or more, 60 or more), and / or the CDR3 sequence has at least three or more cysteine ​​residues (e.g., 3 or more cysteine ​​residues, 4 or more cysteine ​​residues, 5 or more cysteine ​​residues, 6 or more cysteine ​​residues, 7 or more cysteine ​​residues, 8 or more cysteine ​​residues, 9 or more cysteine ​​residues, 10 or more cysteine ​​residues, 11 or more cysteine ​​residues, or 12 or more cysteine ​​residues). Such antibodies bind (e.g., specifically or selectively bind) to a variety of targets, including, for example, protein targets such as transmembrane proteins (e.g., GPCRs, ion channels, transporters, cell surface receptors), as described herein.

[0245] The present disclosure also provides methods and materials for preparing or generating a humanized antibody comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3 sequence. Such materials include proteins, gene sequences, cells, and libraries. Such methods include humanization methods and methods of generating and screening libraries.

[0246] The disclosure provides a humanized antibody or binding fragment thereof comprising: (a) an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3. In some embodiments, the very long CDR3 may be 35 amino acids or more, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, or 55 amino acids or more, or 60 amino acids or more. In some embodiments, the very long CDR3 may contain 3 or more cysteine ​​residues, 4 or more cysteine ​​residues, 5 or more cysteine ​​residues, 6 or more cysteine ​​residues, 7 or more cysteine ​​residues, 8 or more cysteine ​​residues, 9 or more cysteine ​​residues, 10 or more cysteine ​​residues, 11 or more cysteine ​​residues, or 12 or more cysteine ​​residues. A very long CDR3 may, for example, be a CDR in which the cysteine ​​motif is: TIFF0007680480000162.tif186158. Alternatively, the very long CDR3 may be, for example, where the cysteine ​​motif is selected from the group consisting of: TIFF0007680480000163.tif52158TIFF0007680480000164.tif160158.

[0247] The present disclosure relates to a humanized antibody or binding fragment thereof comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3, wherein the very long CDR3 is X 1 X 2 X 3 X 4 X 5 motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V), and X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N), and X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); and X 5 In some embodiments, X is glutamine (Q). 1 X 2 X 3 X 4 X 5 The motif is TIFF0007680480000165.tif72158 is also fine.

[0248] The present disclosure relates to a humanized antibody or binding fragment thereof comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3, wherein the very long CDR3 is (X a X b ) z Contains the motif X a is any amino acid residue, and X b In some embodiments, the present invention provides a humanized antibody or binding fragment thereof, wherein (X a X b ) z The motif is TIFF0007680480000166.tif133158 is also fine.

[0249] The present disclosure relates to a humanized antibody or binding fragment thereof comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3, wherein the very long CDR3 is X 1 X 2 X 3 X 4 X 5 X n (X a X b ) z Contains the motif X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); and X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); and X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D); and X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q) and X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H); n is 27 to 54; and z is 1 to 4.

[0250] The present disclosure relates to a humanized antibody or binding fragment thereof comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3, wherein the very long CDR3 is selected from the group consisting of X, Y, Z, and Z; 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); and X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); and X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D); and X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 CX where Q is glutamine 1 X 2 X 3 X 4 X 5 motif, TIFF0007680480000167.tif186158, and X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4 (X a X b ) z The present invention provides a humanized antibody or binding fragment thereof comprising the motif.

[0251] The present disclosure relates to a humanized antibody or binding fragment thereof comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3, wherein the very long CDR3 is selected from the group consisting of X, Y, Z, and Z; 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); and X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); and X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D); and X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 CX where Q is glutamine 1 X 2 X 3 X 4 X 5 motif; TIFF0007680480000168.tif212158; and X a is any amino acid residue, and X b is an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4 (X a X b )z The present invention provides a humanized antibody or binding fragment thereof comprising the motif.

[0252] The disclosure also provides a method for generating a library of humanized antibodies with very long CDR3s, comprising combining a nucleic acid sequence encoding a very long CDR3 with a nucleic acid sequence encoding a human variable region framework (FR) sequence to create a nucleic acid encoding a humanized antibody with a very long CDR3; and expressing the nucleic acid encoding the humanized antibody with a very long CDR3 to generate a library of humanized antibodies with very long CDR3s.

[0253] The disclosure also provides a method for generating a library of humanized antibodies or binding fragments thereof comprising very long CDR3s comprising non-antibody sequences, comprising combining a nucleic acid sequence encoding a very long CDR3, a nucleic acid sequence encoding a human variable region framework (FR) sequence, and a nucleic acid sequence encoding a non-antibody sequence to generate a nucleic acid encoding a humanized antibody or binding fragment thereof comprising a very long CDR3 and a non-antibody sequence, and expressing the nucleic acid encoding the humanized antibody or binding fragment thereof comprising a very long CDR3 and a non-antibody sequence to generate a library of humanized antibodies or binding fragments thereof comprising a very long CDR3 and a non-antibody sequence.

[0254] The present disclosure also provides a library of humanized antibodies or binding fragments thereof comprising extremely long CDR3s that include non-antibody sequences.

[0255] The disclosure also provides a method for generating a library of humanized antibodies or binding fragments thereof comprising very long CDR3s comprising a cysteine ​​motif, comprising the steps of: combining a human variable region framework (FR) sequence and a nucleic acid sequence encoding a very long CDR3 and a cysteine ​​motif; introducing one or more nucleotide changes to the nucleic acid sequence encoding one or more amino acid residues located between the one or more cysteine ​​residues in the cysteine ​​motif such that the nucleotides encode different amino acid residues to generate a nucleic acid encoding a humanized antibody or binding fragment thereof comprising a very long CDR3 and a cysteine ​​motif having one or more nucleotide changes introduced between the one or more cysteine ​​residues in the cysteine ​​domain; and expressing the nucleic acid encoding a humanized antibody or binding fragment thereof comprising a very long CDR3 and a cysteine ​​motif having one or more nucleotide changes introduced between the one or more cysteine ​​residues in the cysteine ​​domain to generate a library of humanized antibodies or binding fragments thereof comprising a very long CDR3 and a cysteine ​​motif having one or more nucleotide changes introduced between the one or more cysteine ​​residues in the cysteine ​​domain.

[0256] The present disclosure also provides a library of humanized antibodies or binding fragments thereof comprising an extremely long CDR3 that contains a cysteine ​​motif, wherein the antibodies or binding fragments comprise one or more substitutions of amino acid residues located between the cysteine ​​residues in the cysteine ​​motif.

[0257] The disclosure also provides a method for generating a library of humanized antibodies or binding fragments thereof comprising bovine very long CDR3s, comprising combining a nucleic acid sequence encoding a human variable region framework (FR) sequence and a nucleic acid encoding a bovine very long CDR3, and expressing the nucleic acid encoding the human variable region framework (FR) sequence and the nucleic acid encoding the bovine very long CDR3 to generate a library of humanized antibodies or binding fragments thereof comprising bovine very long CDR3s.

[0258] protein The disclosure provides a humanized antibody comprising: (a) an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3 sequence.

[0259] In one embodiment, the disclosure provides a humanized antibody comprising a very long CDR3, where the CDR3 is 35 amino acids in length or more (e.g., 40 or more, 45 or more, 50 or more, 55 or more, 60 or more). Such a humanized antibody may contain at least three or more cysteine ​​residues (e.g., four or more, six or more, eight or more) in the very long CDR3.

[0260] In another embodiment, the disclosure provides a humanized antibody comprising an extremely long CDR3, the CDR3 being 35 amino acids or more in length and derived or based on a non-human sequence. The extremely long CDR3 sequence may be derived from any species that naturally produces extremely long CDR3 antibodies, including ruminants such as Bos taurus.

[0261] In another embodiment, the present disclosure provides a humanized antibody comprising an extremely long CDR3, where the CDR3 is 35 amino acids or more in length, and the CDR3 is derived from a non-antibody sequence. The non-antibody sequence may be derived from any protein family, including, but not limited to, chemokines, growth factors, peptides, cytokines, cell surface proteins, serum proteins, toxins, extracellular matrix proteins, clotting factors, secreted proteins, and the like. The non-antibody sequence may be of human or non-human origin and may include a portion of a non-antibody protein, such as a peptide or domain. The non-antibody sequence of the extremely long CDR3 may contain mutations from its native sequence, including amino acid changes (e.g., substitutions), insertions, or deletions. Additional amino acids may be engineered at the junctions between the non-antibody sequences to facilitate or enhance correct folding of the non-antibody sequence within the humanized antibody.

[0262] In another embodiment, the disclosure provides a humanized antibody comprising an extremely long CDR3, wherein the CDR3 is 35 amino acids in length or more, and wherein the CDR3 comprises at least three or more cysteine ​​residues, including, for example, four or more, six or more, and eight or more.

[0263] In another embodiment, the disclosure provides a humanized antibody comprising an extremely long CDR3, wherein the CDR3 is 35 amino acids in length or longer, wherein the CDR3 comprises at least three or more cysteine ​​residues, and wherein the extremely long CDR3 is a component of a multispecific antibody, which may be bispecific and may comprise additional valencies.

[0264] In another aspect, the disclosure provides a humanized antibody comprising a very long CDR3, wherein the CDR3 is 35 amino acids in length or longer, wherein the CDR3 comprises at least three or more cysteine ​​residues, and wherein the partially human very long CDR3 is a component of an immunoconjugate.

[0265] In another embodiment, the present disclosure provides a humanized antibody comprising an extremely long CDR3, wherein the CDR3 is 35 amino acids or more in length and the CDR3 comprises at least three or more cysteine ​​residues, and the humanized antibody comprises an extremely long CDR3 that binds to a transmembrane protein target. Such transmembrane targets may include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.

[0266] Gene sequence The present disclosure provides a genetic sequence (e.g., gene, nucleic acid, polynucleotide) encoding a humanized antibody comprising: (a) an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a heavy chain variable region comprising a very long CDR3 sequence.

[0267] The present disclosure also provides genetic sequences (eg, genes, nucleic acids, polynucleotides) that encode extremely long CDR3s.

[0268] In one embodiment, the disclosure provides a genetic sequence encoding a humanized antibody comprising a very long CDR3, where the CDR3 is 35 amino acids long or more (e.g., 40 or more, 45 or more, 50 or more, 55 or more, 60 or more). Such a humanized antibody may contain at least three or more cysteine ​​residues (e.g., four or more, six or more, eight or more) in the very long CDR3.

[0269] In another embodiment, the present disclosure provides a genetic sequence encoding a humanized antibody comprising an extremely long CDR3, the CDR3 being 35 amino acids or more in length and derived or based on a non-human sequence. The genetic sequence encoding the extremely long CDR3 may be derived from any species that naturally produces extremely long CDR3 antibodies, including ruminants such as Bos taurus.

[0270] In another embodiment, the present disclosure provides a genetic sequence encoding a humanized antibody comprising an extremely long CDR3, where the CDR3 is 35 amino acids or more in length, and the CDR3 is derived from a non-antibody protein sequence. The genetic sequence encoding the non-antibody protein sequence may be derived from any protein family, including, but not limited to, chemokines, growth factors, peptides, cytokines, cell surface proteins, serum proteins, toxins, extracellular matrix proteins, clotting factors, secreted proteins, and the like. The non-antibody protein sequence may be of human or non-human origin and may include a portion of a non-antibody protein, such as a peptide or domain. The non-antibody protein sequence of the extremely long CDR3 may contain mutations from its native sequence, including amino acid changes (e.g., substitutions), insertions, or deletions. Additional amino acids may be engineered at the junctions between the non-antibody sequences to facilitate or enhance correct folding of the non-antibody sequence within the humanized antibody.

[0271] In another aspect, the disclosure provides a genetic sequence encoding a humanized antibody comprising an extremely long CDR3, wherein the CDR3 is 35 amino acids in length or more, and wherein the CDR3 comprises at least three or more cysteine ​​residues, including, for example, four or more, six or more, eight or more.

[0272] In another embodiment, the disclosure provides a genetic sequence encoding a humanized antibody comprising a very long CDR3, where the CDR3 is 35 amino acids long or longer, where the CDR3 comprises at least three or more cysteine ​​residues, and where the very long CDR3 is a component of a multispecific antibody, which may be bispecific and may comprise additional valencies.

[0273] In another aspect, the disclosure provides a genetic sequence encoding a humanized antibody comprising a very long CDR3, wherein the CDR3 is 35 amino acids in length or longer, wherein the CDR3 comprises at least three or more cysteine ​​residues, and wherein the very long CDR3 is a component of an immunoconjugate.

[0274] In another embodiment, the present disclosure provides a genetic sequence encoding a humanized antibody comprising an extremely long CDR3, the CDR3 being 35 amino acids or more in length and comprising at least three or more cysteine ​​residues, which binds to a transmembrane protein target. Such transmembrane targets may include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.

[0275] Libraries and Arrays The present disclosure provides collections, libraries and arrays of humanized antibodies that contain extremely long CDR3 sequences.

[0276] In one embodiment, the disclosure provides a library or array of humanized antibodies comprising very long CDR3 sequences, where at least two members of the library or array differ in at least one position of cysteines in the very long CDR3 sequence. Structural diversity may be enhanced by different numbers of cysteines (e.g., at least three or more cysteine ​​residues, e.g., four or more, six or more, and eight or more) in the very long CDR3 sequence and / or by different disulfide bond formation and thus different loop structures.

[0277] In another embodiment, the present disclosure provides a library or array of humanized antibodies comprising very long CDR3 sequences, where at least two members of the library or array differ in at least one amino acid located between cysteines in the very long CDR3. In this regard, members of the library or array may contain cysteines at the same position in the CDR3, resulting in a similar overall structural fold, but with subtle differences caused by different amino acid side chains. Such libraries or arrays may be useful for affinity maturation.

[0278] In another embodiment, the present disclosure provides a library or array of humanized antibodies comprising very long CDR3 sequences, where at least two of the very long CDR3 sequences (e.g., 35 amino acids or more in length, e.g., 40 or more, 45 or more, 50 or more, 55 or more, and 60 or more) have different lengths. The amino acid and cysteine ​​content may or may not vary between members of the library or array. Due to the different lengths of the very long CDR3 sequences, the length variations may result in unique binding sites, including, for example, due to steric differences.

[0279] In another embodiment, the disclosure provides a library or array of humanized antibodies comprising very long CDR3 sequences, where at least two members of the library differ in the human framework used to construct the humanized antibody comprising the very long CDR3.

[0280] In another embodiment, the disclosure provides a library or array of humanized antibodies comprising very long CDR3 sequences, where at least two members of the library or array differ in that they have a non-antibody protein sequence that comprises a portion of the very long CDR3. Such libraries or arrays may contain multiple non-antibody protein sequences, including chemokines, growth factors, peptides, cytokines, cell surface proteins, serum proteins, toxins, extracellular matrix proteins, clotting factors, secreted proteins, viral proteins or bacterial proteins, etc. The non-antibody protein sequences may be of human or non-human origin and may consist of a portion of a non-antibody protein, such as a peptide or domain. The non-antibody protein sequence of the very long CDR3 may contain mutations from its native sequence, including amino acid changes (e.g., substitutions), insertions, or deletions. Additional amino acids may be engineered at the junctions between the non-antibody sequences within the very long CDR3 to facilitate or enhance correct folding of the non-antibody sequences within the humanized antibody.

[0281] The libraries or arrays of the present disclosure may take several formats well known in the art. The libraries or arrays may be addressable libraries or addressable arrays. The libraries or arrays may take a display format, for example, antibody sequences may be expressed on the surface of phage, ribosomes, mRNA, yeast, or mammalian cells.

[0282] cell The present disclosure provides a cell comprising: (a) an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a gene sequence encoding a humanized antibody comprising a heavy chain variable region comprising a very long CDR3 sequence.

[0283] In one embodiment, the disclosure provides a cell expressing a humanized antibody with a very long CDR3. The cell may be prokaryotic or eukaryotic, and the humanized antibody with a very long CDR3 may be expressed on the cell surface or secreted into the medium. When displayed on the cell surface, the humanized antibody optionally contains a motif for insertion into the plasmid membrane, such as a transmembrane domain or a lipid binding site at the C-terminus. In the case of bacterial cells, the humanized antibody with a very long CDR3 may be secreted into the periplasm. When the cell is eukaryotic, it may be transiently transfected with a gene sequence encoding the humanized antibody with a very long CDR3. Alternatively, a stable cell line or stable pool may be created by transfecting or introducing a gene sequence encoding the humanized antibody with a very long CDR3 by methods well known to those skilled in the art. The cells may be selected by fluorescence activated cell sorting (FACS) or by selecting for genes encoding drug resistance. Cells useful for producing humanized antibodies containing very long CDR3 sequences include prokaryotic cells such as E. coli, yeasts Saccharomyces cerevisiae and Pichia pastoris, eukaryotic cells such as Chinese hamster ovary (CHO) cells, monkey cells such as COS-1, or human cells such as HEK-293, HeLa, SP-1.

[0284] Humanization Methods The present disclosure provides a method for making a humanized antibody comprising a very long CDR3 sequence, comprising engineering a very long CDR3 sequence derived from a non-human CDR3 into a human framework. The human framework may be derived from a germline or non-germline (e.g., mutated or affinity matured) sequence. To generate a hybrid DNA sequence containing a human framework and a non-human very long CDR3, genetic engineering techniques well known to those skilled in the art, including the techniques disclosed herein, may be used. Unlike human antibodies, which may be encoded by V-region genes derived from one of seven families, bovine antibodies that produce very long CDR3 sequences appear to utilize one V-region family that may be considered most homologous to the human VH4 family. In a preferred embodiment in which a very long CDR3 sequence derived from a bovine is humanized to produce an antibody comprising a very long CDR3, a human V-region sequence derived from the VH4 family may be genetically fused with a very long CDR3 sequence derived from a bovine. Exemplary VH4 germline gene sequences in the human antibody locus are shown in FIG. 5A (eg, SEQ ID NOs:31-34; and 368-371).

[0285] The present disclosure also provides a method for humanizing an antibody variable region, comprising genetically combining a nucleic acid sequence encoding a non-human very long CDR3 (ULCDR3) with a nucleic acid sequence encoding a human variable region framework (FR) sequence. Also provided is a method for making a humanized antibody variable region sequence, comprising the steps of: selecting a human framework sequence comprising FR1, FR2, and FR3; selecting a CDR1 sequence; selecting a CDR2 sequence; selecting a very long CDR3 sequence; and combining the sequences as FR1-CDR1-FR2-CDR2-FR3-ULCDR3. Also provided is a method for making a humanized antibody variable region sequence, comprising the steps of: selecting a human antibody variable region sequence comprising a sequence encoding FR1-CDR1-FR2-CDR2-FR3; selecting a sequence encoding a non-human very long CDR3 (ULCDR3); and genetically fusing the human sequence of step (a) in frame with the non-human sequence of step (b) to generate a sequence encoding FR1-CDR1-FR2-CDR2-FR3-ULCDR3.

[0286] In one embodiment, the disclosure provides a fusion of a human VH4 framework sequence with a bovine-derived very long CDR3, which may be achieved, for example, by the following steps: First, the second cysteine ​​of the V-region gene sequence is identified together with the nucleotide sequence encoding the second cysteine. Typically, the second cysteine ​​marks the boundary between the framework and the CDR3, two residues upstream (N-terminal side) of the CDR3. Second, a second cysteine ​​is identified in the bovine-derived V-region sequence, which also marks two residues upstream (N-terminal side) of the CDR3. Third, the genetic material encoding the human V-region is combined with the genetic sequence encoding the very long CDR3. In this way, a genetic fusion may be made in which the very long CDR3 sequence is placed in frame with the human V-region sequence. Preferably, the amino acid composition of the humanized antibody with the very long CDR3 is as close to human as possible. Optionally, the J-region sequence may be mutated from the bovine-derived sequence to a human sequence. Optionally, the humanized heavy chain may also be paired with a human light chain.

[0287] In another embodiment, the disclosure provides for the pairing of a human very long CDR3 heavy chain with a non-human light chain.

[0288] In another embodiment, the present disclosure provides for pairing of a humanized heavy chain comprising a very long CDR3 with a human light chain. Preferably, the light chain is homologous to a bovine light chain that is known to pair with a very long CDR3 heavy chain of bovine. Exemplary bovine light chains are shown in Figure 7A (e.g., SEQ ID NOs: 36-39; and 373-376).

[0289] Library Method The present disclosure provides a method for generating a library comprising: (a) an amino acid sequence selected from the group consisting of (i) SEQ ID NO:734 or SEQ ID NO:735, (ii) SEQ ID NO:736 or SEQ ID NO:737, (iii) SEQ ID NO:738 or SEQ ID NO:739, (iv) SEQ ID NO:740 or SEQ ID NO:741, (v) SEQ ID NO:742 or SEQ ID NO:743, (vi) SEQ ID NO:744 or SEQ ID NO:745, (vii) SEQ ID NO:746 or SEQ ID NO:747, and (viii) SEQ ID NO:748 or SEQ ID NO:749; and (b) a humanized antibody comprising a heavy chain variable region comprising a very long CDR3 sequence. A method for generating a library of spatially addressed libraries is described in WO2010 / 054007. Methods for producing libraries in yeast, phage, E. coli, or mammalian cells are well known in the art.

[0290] The present disclosure also provides methods for screening libraries of humanized antibodies containing extremely long CDR3 sequences.

[0291] definition "Very long CDR3" or "very long CDR3 sequence" are used interchangeably herein and include CDR3 sequences or CDR3 sequences that are not derived from human antibody sequences. The very long CDR3 may be 35 amino acids or more in length, for example, 40 amino acids or more, 45 amino acids or more, 50 amino acids or more, 55 amino acids or more, or 60 amino acids or more in length. The length of the very long CDR3 may include non-antibody sequences. The very long CDR3 may include non-antibody sequences, including, for example, interleukin sequences, hormone sequences, cytokine sequences, toxin sequences, lymphokine sequences, growth factor sequences, chemokine sequences, toxin sequences, or combinations thereof. Preferably, the very long CDR3 is a heavy chain CDR3 (CDR-H3 or CDRH3). Preferably, the very long CDR3 is a sequence derived from or based on a ruminant (e.g., bovine) sequence. Preferably, the very long CDR3 comprises the amino acid sequence of SEQ ID NO:498, the amino acid sequence of SEQ ID NO:499, or both. Alternatively, or in addition, the very long CDR3 comprises Alternatively, or in addition, the very long CDR3 comprises the amino acid sequence of Contains the amino acid sequence of TIFF0007680480000170.tif112159. Alternatively, or in addition, the very long CDR3 may comprise: The CDR3 comprises the amino acid sequence of TIFF0007680480000171.tif220158. A very long CDR3 may comprise at least three or more cysteine ​​residues, e.g., four or more cysteine ​​residues, six or more cysteine ​​residues, eight or more cysteine ​​residues, ten or more cysteine ​​residues, or twelve or more cysteine ​​residues (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more). A very long CDR3 may comprise the following motifs: cysteine ​​motif, X 1 X 2 X 3 X 4X 5 Motif, CX 1 X 2 X 3 X 4 X 5 Motif, or (X a X b ) z A "cysteine ​​motif" is a very long segment of amino acid residues in the CDR3 that contains three or more cysteine ​​residues, including four or more cysteine ​​residues, five or more cysteine ​​residues, six or more cysteine ​​residues, seven or more cysteine ​​residues, eight or more cysteine ​​residues, nine or more cysteine ​​residues, ten or more cysteine ​​residues, eleven or more cysteine ​​residues, or twelve or more cysteine ​​residues. A cysteine ​​motif is TIFF0007680480000172.tif186158. Alternatively, the cysteine ​​motif may comprise an amino acid sequence selected from the group consisting of: The cysteine ​​motif may preferably comprise an amino acid sequence selected from the group consisting of X in the very long CDR3. 1 X 2 X 3 X 4 X 5 Motif and (X a X b ) z It is placed between the motifs. 1 X 2 X 3 X 4 X 5 The “X motif” is a stretch of five consecutive amino acid residues in the extremely long CDR3. 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q). 1 X 2 X 3 X 4 X 5 The motif is TIFF0007680480000174.tif72158 is also acceptable. 1 X 2 X 3 X 4 X 5 The "motif" is a very long stretch of six consecutive amino acid residues in CDR3, the first of which is a cysteine ​​and the second is an X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V); X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N); 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), and X is 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L); X 5 is glutamine (Q). In some embodiments, CX 1 X 2 X 3 X 4 X 5 The motif is TIFF0007680480000175.tif79159. a X b ) z The " motif is a repeating series of two amino acid residues in the extremely long CDR3, a is any amino acid residue, and X bis an aromatic amino acid selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and z is 1 to 4. a X b ) z The motif is In some embodiments, the (X a X b ) zThe motif is YXYXYX. The very long CDR3 may comprise an amino acid sequence derived from or based on SEQ ID NO:40 (see, e.g., amino acid residues 3-6 of SEQ ID NO:1-4; see also, e.g., the VH germline sequences in Figures 2A-C). The variable region comprising the very long CDR3 may comprise an amino acid sequence that is SEQ ID NO:1 (CTTVHQ), SEQ ID NO:2 (CTSVHQ), SEQ ID NO:3 (CSSVTQ), or SEQ ID NO:4 (CTTVHP). Such sequences may be derived from or based on bovine germline VH gene sequences (e.g., SEQ ID NO:1). Very long CDR3s may include sequences derived from or based on non-human DH gene sequences, e.g., SEQ ID NO:5 (see also, e.g., Koti, et al. (2010) Mol. Immunol. 47: 2119-2128), or alternative sequences such as SEQ ID NO:6, 7, 8, 9, 10, 11, or 12 (see also, e.g., the DH2 germline sequence in Figures 2A-C). Very long CDR3s may include sequences derived from or based on JH sequences, e.g., SEQ ID NO:13 (see also, e.g., Hosseini, et al. (2004) Int. Immunol. 16: 843-852), or alternative sequences such as SEQ ID NO:14, 15, 16, or 17 (see also, e.g., the JH1 germline sequence in Figures 2A-C).In one embodiment, the very long CDR3 may comprise a sequence derived from or based on a non-human VH sequence (e.g., SEQ ID NO:1, 2, 3, or 4; or the VH sequence of Figures 2A-C), and / or a sequence derived from or based on a non-human DH sequence (e.g., SEQ ID NO:5, 6, 7, 8, 9, 10, 11, or 12; or the DH sequence of Figures 2A-C), and / or a sequence derived from or based on a JH sequence (e.g., SEQ ID NO:13, 14, 15, 16, or 17; or the JH sequence of Figures 2A-C), optionally including an additional sequence comprising 2 to 6 or more amino acids, for example, between the VH-derived sequence and the DH-derived sequence (e.g., IR, IF, SEQ ID NO:18, 19, 20, or 21). In another embodiment, the very long CDR3 may comprise a sequence derived from or based on SEQ ID NO:22, 23, 24, 25, 26, 27, or 28 (see also, e.g., SEQ ID NO:276-359 in Figures 2A-C).

[0292] "Isolated" biological molecules disclosed herein, such as various polypeptides, polynucleotides, and antibodies, refer to biological molecules that have been identified and separated and / or recovered from at least one component of its natural environment.

[0293] "Antagonist" refers to any molecule that partially or completely blocks, inhibits, or neutralizes the activity (e.g., biological activity) of a polypeptide. "Antagonist" also encompasses molecules that completely or partially inhibit the transcription or translation of mRNA encoding a polypeptide. Suitable antagonist molecules include, for example, antagonist antibodies or antibody fragments; fragments or amino acid sequence variants of natural polypeptides; peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that encode polypeptide antagonists or antagonist antibodies. Reference to "an" antagonist encompasses a single antagonist or a combination of two or more different antagonists.

[0294] "Agonist" refers to any molecule that partially or completely mimics the biological activity of a polypeptide. "Agonist" also encompasses molecules that promote the transcription or translation of mRNA encoding a polypeptide. Suitable agonist molecules include, for example, agonist antibodies or antibody fragments; natural polypeptides; fragments or amino acid sequence variants of natural polypeptides; peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that code for polypeptide agonists or antibodies. Reference to "an" agonist encompasses a single agonist or a combination of two or more different agonists.

[0295] An "isolated" antibody refers to an antibody that has been identified and separated and / or recovered from a component of its natural environment. The contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight, preferably greater than 99% by weight, of the antibody (e.g., as determined by the Lowry method), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence (e.g., by using a spinning cup sequenator), or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions (using Coomassie™ blue or, preferably, silver stain). An isolated antibody includes the antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. Similarly, an isolated antibody includes the antibody in the medium surrounding the recombinant cell. An isolated antibody may be prepared by at least one purification step.

[0296] An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is a nucleic acid molecule other than in the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from the nucleic acid molecule in natural cells. However, an isolated nucleic acid molecule includes, for example, a nucleic acid molecule contained in a cell that expresses an antibody, in which the nucleic acid molecule is in a different chromosomal location than in natural cells.

[0297] Kabat-like variable domain residue numbering or Kabat-like amino acid position numbering and variations thereof refer to the numbering system used for the heavy or light chain variable domains of an antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). With this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion into the FRs or CDRs of the variable domain. For example, the heavy chain variable domain may contain an amino acid insertion after residue 52 of H2 (e.g., residue 52a according to Kabat), an insertion residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a particular antibody by alignment with the "standard" Kabat numbered sequence in the homologous regions of the antibody sequence.

[0298] "Substantially similar" or "substantially the same" refers to a sufficiently high degree of similarity between two numerical values ​​(typically one associated with an antibody disclosed herein and the other associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance in the context of the biological characteristic measured by said values ​​(e.g., Kd values). The difference between the two values ​​is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value of the reference / comparator antibody.

[0299] "Binding affinity" generally refers to the sum of the strengths of non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y can generally be expressed as a dissociation constant. Affinity can be measured by common methods known in the art, including the methods described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens quickly and tend to remain bound for long periods of time. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure.

[0300] "On-rate" or "rate of association" or "association rate" or "k on " can be determined using surface plasmon resonance such as with a Biacore (e.g., Biacore A100, Biacore™-2000, Biacore™-3000, Biacore, Inc., Piscataway, NJ) carboxymethylated dextran biosensor chip (CM5, Biacore Inc.) according to the supplier's instructions.

[0301] A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid." A "plasmid" refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into a host cell, and thereby are replicated along with the host genome. Additionally, certain vectors are capable of expressing genes that are operably linked to the vector. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Thus, since plasmids are a commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably.

[0302] "Gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). A polypeptide may be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional property of the full-length or fragment (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) is retained. The term also encompasses the coding region of a structural gene, as well as sequences located adjacent to the 5' and 3' ends of the coding region and extending for a distance of about 1 kb or more from both ends, such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain coding regions that are separated by non-coding sequences called "introns" or "intervening regions" or "intervening sequences." Introns are gene segments that are transcribed into nuclear RNA (hnRNA). Introns may contain regulatory elements such as enhancers. Introns are removed, or "spliced ​​out," from the nuclear or primary transcript. Introns are therefore absent from messenger RNA (mRNA) transcripts. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. In addition to containing introns, genomic forms of genes may also contain sequences located at both the 5' and 3' ends of the sequences present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present in the mRNA transcript). The 5' flanking region may contain regulatory sequences that control or affect the transcription of the gene, such as promoters and enhancers. The 3' flanking region may contain sequences that direct transcription termination, post-transcriptional cleavage, and polyadenylation.

[0303] "Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a nucleotide polymer of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or a synthetic reaction. Polynucleotides may also include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after the polymer is assembled. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after synthesis, for example, by conjugation with a label. Other types of modifications include, for example, "caps", replacement of one or more natural nucleotides with analogs, internucleotide modifications, such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications containing intercalators (e.g., acridine, psoralen, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylating agents, modifications with modified bonds (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present on the sugar may be replaced with, for example, phosphonate groups, phosphate groups, protected by standard protecting groups, activated to prepare additional bonds with additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may be derivatized to standard protecting groups.Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include phosphate as P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR. 2 ("amidate"), P(O)R, P(O)OR', CO, or CH 2 ("formacetal"). In the formula, each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or alandyl, which may contain an ether (-O-) linkage. The linkages in a polynucleotide need not all be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0304] "Oligonucleotide" refers to a short, generally single-stranded, generally synthetic polynucleotide that is generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotide applies equally and completely to oligonucleotides.

[0305] "Stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization is provided by Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). In general, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm is the temperature (at defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (when the target sequence is present in excess, at Tm 50% of the probes are occupied at equilibrium). Stringent conditions can also be obtained by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5xSSC, and 1% SDS, incubation at 42°C, or 5xSSC, 1% SDS, incubation at 65°C, and washing at 0.2xSSC, 0.1% SDS, 65°C.

[0306] "Recombinant" when used in reference to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, a recombinant cell expresses a gene not found in the native (non-recombinant) form of the cell, or expresses a native gene that is overexpressed or otherwise aberrantly expressed, e.g., a native gene expressed as a non-native fragment or splice variant. As used herein, the term "recombinant nucleic acid" generally refers to a nucleic acid that has been originally formed in vitro by genetic engineering, e.g., using polymerases and endonucleases, to create a nucleic acid in a form not normally found in nature. In this manner, different sequences are operably linked. Thus, both linear isolated nucleic acids or expression vectors formed in vitro by joining DNA molecules that are not normally linked are considered recombinant for the purposes of this disclosure. It will be understood that once a recombinant nucleic acid is made and introduced into a host cell or organism, it is replicated non-recombinantly, e.g., using the in vivo cellular machinery of the host cell, rather than by in vitro manipulation. However, such a nucleic acid, once made recombinantly, is still considered recombinant for purposes disclosed herein, even though it later replicates non-recombinantly. Similarly, a "recombinant protein" is a protein made using recombinant methods, e.g., by expressing a recombinant nucleic acid, as described above.

[0307] "Percent (%) amino acid sequence identity" in relation to a peptide or polypeptide sequence refers to the percent of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps as necessary to obtain the maximum percent sequence identity without taking into account any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MegAlign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared.

[0308] "Polypeptide," "peptide," "protein," and "protein fragment" are sometimes used interchangeably to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and to non-naturally occurring amino acid polymers.

[0309] "Amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in the same way as natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. "Amino acid analog" refers to compounds that have the same basic chemical structure as a natural amino acid, such as an α carbon bonded to a hydrogen, a carboxy group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a natural amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in the same way as a natural amino acid.

[0310] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. "Amino acid variant" refers to an amino acid sequence. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code for identical or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, essentially identical or related sequences (e.g., naturally contiguous sequences). Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for a large number of proteins. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to another of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. All nucleic acid sequences herein that code for a polypeptide also refer to silent variations of the nucleic acid. Those skilled in the art will recognize that in certain circumstances, each codon in a nucleic acid (except AUG, the normally unique codon for methionine, and TGG, the normally unique codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, silent variations of a nucleic acid encoding a polypeptide are included in the sequence described for the expression product, but not in the sequence described for the actual probe sequence. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," including cases where the change replaces an amino acid with a chemically similar amino acid. Conservative substitution tables showing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles disclosed herein.Typically, conservative substitutions include: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Protein (1984)).

[0311] "Antibodies" (Ab) and "immunoglobulins" (Ig) are glycoproteins having similar structural characteristics. While antibodies can exhibit binding specificity to a particular antigen, immunoglobulins may include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are produced, for example, at low levels by the lymphatic system and at higher levels by myelomas.

[0312] "Antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), and may include certain antibody fragments (as described in more detail herein). Antibodies may be human, humanized, and / or affinity matured. Antibodies may refer to immunoglobulins and immunoglobulin portions, whether natural, partially synthetic, or fully synthetic, including recombinantly produced immunoglobulins and immunoglobulin portions, including any portion of an immunoglobulin that contains at least a portion of the variable region of an immunoglobulin molecule sufficient to form an antigen-binding site. Thus, an antibody or portion thereof includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding site. For example, an antibody may refer to an antibody that contains two heavy chains (which may be designated H and H') and two light chains (which may be designated L and L'), where each heavy chain may be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen-binding site (e.g., heavy chains include, but are not limited to, VH, VH-CH1, and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or a portion thereof sufficient to form an antigen-binding site (e.g., light chains include, but are not limited to, VL and VL-CL chains). Each heavy chain (H and H') is paired with one light chain (L and L', respectively). Typically, an antibody contains, at a minimum, all or at least a portion of a variable heavy (VH) chain and / or a variable light (VL) chain. The antibody may also contain all or a portion of a constant region. For example, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, e.g., an antibody produced by an antibody-secreting B cell, and an antibody made synthetically having the same domains.Additionally, "antibody" refers to a protein of the immunoglobulin family or a polypeptide, including immunoglobulin fragments, capable of noncovalently, reversibly, and specifically binding to a corresponding antigen. An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains. Each pair has one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD) linked through disulfide bonds. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain regions respectively.

[0313] "Variable" refers to the fact that the sequences of certain parts of the variable domains (also called variable regions) vary considerably among antibodies and are used in the binding and specificity of each particular antibody to a particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) in both the light and heavy variable domains. The CDRs include those designated Kabat, Chothia, and IMGT as set forth herein, which are found within the variable region sequences. The highly conserved portions of the variable domains are called frameworks (FRs). Naturally occurring heavy and light chain variable domains each contain four FR regions that are primarily in a β-sheet configuration, connected by three CDRs that form loop connections and, in some cases, form part of a β-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0314] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and one "Fc" fragment. The "Fc" name reflects the ability to crystallize readily. Pepsin treatment produces an F(ab') fragment that has two antigen-binding sites and is still capable of cross-linking antigen. 2 Fragments are produced.

[0315] "Fv" refers to an antibody fragment that contains an antigen recognition site and an antigen binding site. In a two-chain Fv species, this region consists of a dimer of one non-covalently associated heavy-chain variable domain and one light-chain variable domain. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to a two-chain Fv (scFv) species. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In total, the six CDRs confer antigen binding specificity to the antibody. However, even one variable domain (or half of an Fv, containing only the three CDRs specific for an antigen) is capable of recognizing and binding an antigen, albeit with a lower affinity than the complete binding site.

[0316] Fab fragments also contain the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab') 2Antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0317] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0318] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these are divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0319] An "antibody fragment" comprises only a portion of an intact antibody, which preferably retains at least one, and preferably most or all, of the functions normally associated with that portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, Fab fragments, Fd fragments, scFv fragments, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). Other known fragments include, but are not limited to, scFab fragments (Hust et al., BMC Biotechnology (2007), 7: 14). In one embodiment, an antibody fragment comprises an antigen-binding site of an intact antibody, and thus retains the ability to bind to antigen. In another embodiment, an antibody fragment, e.g., an antibody fragment comprising an Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, e.g., FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, an antibody fragment is a monovalent antibody with an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise an antigen-binding arm linked to an Fc sequence, which may confer in vivo stability to the fragment. As another example, an antibody fragment or portion refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody (e.g., one or more CDRs) sufficient to form an antigen-binding site, and thus retains the binding specificity and / or activity of the full-length antibody. Antibody fragments include antibody derivatives produced by enzymatic processing of the full-length antibody, as well as synthetic, e.g., recombinantly produced derivatives.

[0320] "dsFv" refers to an Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.

[0321] "Fd fragment" refers to an antibody fragment containing the variable domain (VH) and one constant region domain (CH1) of the antibody heavy chain.

[0322] "Fab fragment" refers to an antibody fragment containing a portion of a full-length antibody produced by papain treatment of a full-length immunoglobulin, or a synthetic, e.g., recombinantly produced, fragment having the same structure. A Fab fragment contains a light chain (containing the VL and CL portions) and another chain containing the variable domain of the heavy chain (VH) and one constant region domain portion of the heavy chain (CH1). A "Fab fragment" may be recombinantly produced.

[0323] "F(ab')2 fragment" refers to an antibody fragment generated by pepsin digestion of an immunoglobulin at pH 4.0-4.5, or a synthetic, e.g., recombinantly produced, antibody having the same structure. An F(ab')2 fragment contains two Fab fragments, but each heavy chain portion contains a few additional amino acids, including a cysteine ​​residue that forms a disulfide bond connecting the two fragments. An "F(ab')2 fragment" may be recombinantly produced.

[0324] "Fab' fragment" refers to a fragment containing half of an F(ab')2 fragment (one heavy chain and one light chain).

[0325] "Fd' fragment" refers to an antibody fragment containing one heavy chain portion of the F(ab')2 fragment.

[0326] "Fv' fragment" refers to a fragment containing only the VH and VL domains of an antibody molecule.

[0327] "scFv fragment" refers to an antibody fragment containing a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker. The linker is of a length such that it bridges the two variable domains without substantial interference. An exemplary linker is (Gly-Ser)n residues, with some Glu or Lys residues dispersed throughout to enhance solubility.

[0328] Diabodies are dimeric scFvs. Diabodies typically have shorter peptide linkers than scFvs and dimerize selectively.

[0329] "HsFv" refers to an antibody fragment in which the constant domains normally present in the Fab fragment are replaced by a heterodimeric coiled-coil domain (see, e.g., Arndt et al. (2001) J Mol Biol. 7:312:221-228).

[0330] "Hypervariable region", "HVR", or "HV", as well as "complementary determining region" or "CDR" may refer to antibody variable domain regions that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable or CDR regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Many hypervariable region or CDR diagrams are in use and are encompassed herein. The Kabat complementarity determining regions (Kabat CDRs) are based on sequence variability and are the most commonly used (Kabat et al., 2002). Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Instead, Chothia refers to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions, which nicely satisfy both the Kabat CDRs and the Chothia structural loops (Chothia "CDRs"), are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residues in each of these hypervariable regions are shown below. TIFF0007680480000177.tif97156IMGT is a library of the International Immunogenetics Information System (IISS) as described in Lefrace et al., Nucl. Acids, Res. 37; D1006-D1012 (2009), which includes, for example, the CDRs designated by IMGT for antibodies. I m M Uno G ene T ics Information System).

[0331] The hypervariable regions may include "extended hypervariable regions" such as 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0332] "Framework" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein. A "framework region" (FR) is a domain located within an antibody variable region domain that contains framework residues located in the β-sheets. FR regions are relatively more conserved in amino acid sequence than hypervariable regions.

[0333] A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, for example, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variations that may occur during the production of the monoclonal antibody. Such variations are generally present in minor amounts. Such monoclonal antibodies typically include antibodies that include a target-binding polypeptide sequence obtained by a process that includes selecting one target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should also be understood that the selected target-binding sequence can be further modified, for example, to improve affinity for the target, to humanize the target-binding sequence, to improve production of the target-binding sequence in cell culture, to reduce immunogenicity of the target-binding sequence in vivo, to create multispecific antibodies, etc., and that antibodies that include modified target-binding sequences are also monoclonal antibodies of the present disclosure. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (e.g., epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically free of contaminating other immunoglobulins. The modifier "monoclonal" indicates the characterization of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be produced using, for example, hybridoma methodology (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1-2):119-132 (2004). (2004)), as well as a variety of techniques, including techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci and genes encoding human immunoglobulin sequences (e.g., WO1998 / 24893; WO1996 / 34096; WO1996 / 33735; WO1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Patent Nos. 5,545,806; 5,569,825; 5,591,669; 5,545,807; WO1997 / 17852; U.S. Patent No. 5,545,807; U.S. Patent No. 5,545,806; U.S. Patent No. 5,569,825; U.S. Patent No. 5,625,126; U.S. Patent No. 5,633,425; (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

[0334] "Humanized" or "engineered human" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain amino acids represented by human immunoglobulin sequences, including, for example, chimeric antibodies whose minimal sequence is derived from a non-human immunoglobulin. For example, a humanized antibody may be a human antibody in which some hypervariable region residues, and possibly some FR residues, are replaced by residues from analogous sites in a non-human (e.g., rodent) antibody. Alternatively, a humanized or engineered human antibody may be a non-human (e.g., rodent) antibody in which some residues are replaced by residues from analogous sites in a human antibody (see, for example, U.S. Pat. No. 5,766,886). Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable region have been replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that has the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin have been replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient antibody or the donor antibody, including non-antibody sequences such as, for example, chemokines, growth factors, peptides, cytokines, cell surface proteins, serum proteins, toxins, extracellular matrix proteins, clotting factors, or secreted protein sequences. Such modifications may be made to further refine antibody performance. Humanized antibodies include engineered human antibodies, for example, as described in U.S. Pat. No. 5,766,886, which includes methods for preparing modified antibody variable domains. A humanized antibody may contain substantially all of at least one variable domain, typically two variable domains, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0335] A "hybrid antibody" refers to an immunoglobulin molecule in which a pair of heavy and light chains derived from antibodies with different antigenic determinant regions are assembled together such that the resulting tetramer can recognize and bind two different epitopes or two different antigens.

[0336] A "chimeric" antibody (immunoglobulin) has a portion of the heavy and / or light chain that is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as in fragments of such antibodies, so long as the desired biological activity is exhibited (see, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Humanized antibodies refer to a subset of chimeric antibodies.

[0337] A "single-chain Fv" or "scFv" antibody fragment may comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0338] "Antigen" refers to a defined antigen to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other natural or synthetic compound. Preferably, the target antigen is a polypeptide.

[0339] "Epitope" or "antigenic determinant", used interchangeably herein, refers to a portion of an antigen that a particular antibody can recognize and specifically bind to. When the antigen is a polypeptide, epitopes can be formed from both contiguous amino acids and non-contiguous amino acids adjacent by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies may bind to the same epitope on an antigen or to different epitopes on an antigen. Antibodies may be characterized by different epitope bins. Whether an antibody binds to the same epitope or a different epitope as another antibody (e.g., a reference antibody or a benchmark antibody) can be determined by competition between the antibodies in an assay (e.g., a competitive binding assay).

[0340] Competition between antibodies can be determined by an assay in which the immunoglobulin being tested inhibits specific binding to a reference antibody and a common antigen. There are numerous types of competitive binding assays, such as: direct or indirect solid-phase radioimmunoassays (RIA), direct or indirect solid-phase enzyme immunoassays, or enzyme-linked immunosorbent assays (EIA or ELISA), sandwich competitive assays including ELISA assays (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); direct solid-phase biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619 (1986)); direct solid-phase labeled assays, direct solid-phase labeled sandwich assays (see Harlow and Lane, "Antibodies, A Laboratory Manual", Cold Spring Harbor Press (1988)); direct solid-phase labeled RIA using I-125 label (Morel et al., Molec. Immunol. 25(1):7-15 (1988); direct solid-phase biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol., 32:77-82 (1990)). Competitive binding assays can be performed, for example, using surface plasmon resonance (SPR) with a Biacore® instrument for kinetic analysis of binding interactions. In such assays, humanized antibodies containing very long CDR3s with unknown epitope specificity can be evaluated for their ability to compete for binding against a comparison antibody (e.g., the BA1 or BA2 antibodies described herein). The assay can involve the use of purified antigen bound to a solid surface, or cells bearing either of these, unlabeled test immunoglobulins, and labeled reference immunoglobulins. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess.The assay (competing antibody) may include an antibody that binds to the same epitope as the reference antibody and an antibody that binds to an adjacent epitope that is close enough to the epitope bound by the reference antibody that steric hindrance occurs. Typically, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to a common antigen by at least 50%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or about 100%.

[0341] An antibody "selectively binds" or "specifically binds" means that the antibody reacts or associates with an antigen or epitope more frequently, rapidly, for a longer period of time, with greater affinity, or a combination thereof, than with other substances, including unrelated proteins. "Selectively binds" or "specifically binds" means, for example, that the antibody reacts or associates with an antigen or epitope with a K of at least about 0.1 mM. D or a K of at least about 1 μM D or at least about 0.1 μM or better D or at least about 0.01 μM or better D Since there is sequence identity between homologous proteins in different species, specific binding can also include an antibody that recognizes a particular antigen in multiple species.

[0342] "Non-specific binding" and "background binding," when used in reference to the interaction of an antibody and a protein or peptide, refer to interactions that are not dependent on the presence of a particular structure (e.g., an antibody generally binds to a protein and not to a particular structure, such as an epitope).

[0343] "Diabody" refers to a small antibody fragment that contains a heavy chain variable domain (VH) and a light chain variable domain (VL) connected in the same polypeptide chain (VH-VL) with two antigen binding sites. By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with complementary domains on another chain, creating two antigen binding sites. Diabodies are described in further detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et. al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0344] A "human antibody" refers to one that contains an amino acid sequence that corresponds to that of an antibody produced by a human and / or that is made using any of the techniques disclosed herein for making human antibodies. Notably, this definition of a human antibody excludes humanized antibodies that contain non-human antigen-binding residues.

[0345] "Affinity matured" antibody refers to an antibody that has one or more changes in one or more CDRs of the antibody, which results in improved affinity of the antibody to antigen compared to a parent antibody that does not have these changes.Preferred affinity matured antibodies have nanomolar or even picomolar affinity to the target antigen.Affinity matured antibodies are produced by techniques known in the art.Marks et al., Bio / Technology 10:779-783(1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0346] Antibody "effector function" refers to the biological activity attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and varies depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0347] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a type of cytotoxicity in which secreted Ig binds to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) to enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are absolutely necessary for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA 95:652-656 (1998).

[0348] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells may be isolated from a natural source, e.g., blood.

[0349] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds an IgG antibody (gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in the cytoplasmic domain (see review in M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein includes other FcRs, including those that will be identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulates immunoglobulin homeostasis. For example, antibody variants with improved or diminished binding to FcRs have been described (see, e.g., Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0350] Methods for measuring binding to FcRn are known (see, e.g., Ghetie 1997, Hinton 2004). Binding to human FcRn in vivo and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the Fc variant polypeptides are administered.

[0351] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0352] Polypeptide variants with altered Fc region amino acid sequences that have increased or decreased Clq binding ability have been described (see also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000)).

[0353] "Polypeptide comprising an Fc region" refers to a polypeptide, such as an antibody or immunoadhesin (see definition below), that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during polypeptide purification or by recombinantly manipulating the nucleic acid encoding the polypeptide.

[0354] A "blocking" or "antagonist" antibody refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0355] An "agonist" antibody refers to an antibody that mimics (eg, partially or fully) at least one of the functional activities of a polypeptide of interest.

[0356] "Acceptor human framework" refers to a framework that comprises the amino acid sequence of a VL framework or a VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain pre-existing amino acid sequence changes. If there are pre-existing amino acid sequence changes, there are preferably no more than 5, preferably no more than 4, or no more than 3 pre-existing amino acid changes.

[0357] "Human consensus framework" refers to a framework that corresponds to the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al., supra.

[0358] "Disorder" or "disease" refers to any condition that would benefit from treatment with a substance / molecule disclosed herein (e.g., a humanized antibody comprising an extra-long CDR3 as disclosed herein) or a method disclosed herein. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0359] "Treatment" refers to clinical intervention that attempts to change the natural course of the individual or cell being treated, and may be performed for prophylaxis or during clinical pathology. The desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, remission or palliation of disease state, and improving remission or prognosis. In some embodiments, the antibodies disclosed herein are used to delay the onset of disease or disorder.

[0360] An "individual" (e.g., "subject") refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (e.g., cows), sport animals, pets (e.g., cats, dogs, and horses), primates, mice, and rats.

[0361] A "mammal" for treatment refers to any animal classified as a mammal, including humans, rodents (e.g., mice and rats), and monkeys; domestic and farm animals; and zoo, sport, laboratory, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal is selected from a human, a rodent, or a monkey.

[0362] "Pharmaceutically acceptable" means approved or approvable by a federal or state regulatory agency for use in animals, including humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0363] "Pharmaceutically acceptable salt" refers to a salt of a compound that is pharma-ceutically acceptable and that possesses the desired pharmacological activity of the parent compound.

[0364] "Pharmaceutically acceptable excipient, carrier, or adjuvant" refers to an excipient, carrier, or adjuvant that can be administered to a subject together with at least one antibody of the present disclosure and that does not destroy its pharmacological activity and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the compound.

[0365] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the disclosure is administered.

[0366] "Providing a prognosis," "prognostic information," or "predictive information" refers to providing information, including, for example, the presence of cancer cells in a subject's tumor, regarding the impact of the presence of cancer (e.g., as determined by the diagnostic methods of the present disclosure) on the subject's future health (e.g., expected morbidity or mortality, likelihood of developing cancer, and risk of metastasis).

[0367] The terms "treat" or "treatment" or "attempting to treat" or "alleviate" or "attempting to alleviate" and the like refer to 1) therapeutic measures that cure, slow, relieve the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and 2) prophylactic or preventative measures that prevent and / or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder; those susceptible to developing the disorder; and those in whom the disorder is to be prevented.

[0368] "Providing a diagnosis" or "diagnostic information" refers to any information, including, for example, the presence of cancer cells, that is useful in confirming whether a patient has a disease or condition, and / or classifying a disease or condition into a phenotypic category or any category that has meaning regarding the prognosis of the disease or condition or the likely response to treatment of the disease or condition (either treatment in general or any specific treatment). Similarly, diagnosis refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have a condition (e.g., a tumor), whether a subject's tumor contains cancer stem cells, what the tumor is like or a classification of the tumor, e.g., high-risk or low-risk tumor, information regarding prognosis, and / or information useful in selecting an appropriate treatment. Selecting a treatment may include selecting a particular chemotherapeutic agent or other treatment modality, such as surgery or radiation, or selecting whether to withhold or deliver therapy.

[0369] "Human consensus framework" refers to a framework that is the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al., supra.

[0370] For purposes herein, "acceptor human framework" refers to a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0371] "Antigen binding site" refers to the interface formed by one or more complementarity determining regions. An antibody molecule has two antigen binding sites, each of which contains a portion of a heavy chain variable region and a portion of a light chain variable region. An antigen binding site may contain other portions of the variable region domain in addition to the CDRs.

[0372] "Antibody light chain" or "antibody heavy chain" refers to a polypeptide that constitutes a VL or a polypeptide that constitutes a VH, respectively. The VL is encoded by minigenes V (variable) and J (joining), and the VH is encoded by minigenes V, D (diversity), and J. Each VL or VH comprises CDRs and framework regions. In this application, the antibody light chain and / or antibody heavy chain are sometimes collectively referred to as "antibody chains". These terms encompass antibody chains that contain mutations that do not destroy the basic structure of the VL or VH, as those skilled in the art will readily appreciate.

[0373] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the light chain variable domain, followed by a constant light (CL) domain. Antibody light chains may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0374] "Combinatorial library" refers to a collection of compounds formed by reacting different combinations of interchangeable chemical "building blocks" to generate a collection of compounds based on the exchange of building blocks. In the case of an antibody combinatorial library, the building blocks are the component V, D, and J regions (or modified versions thereof) from which antibodies are formed. For purposes herein, the terms "library" or "collection" are used interchangeably.

[0375] A "combinatorial antibody library" refers to a collection of antibodies (or portions thereof, e.g., Fabs), in which the antibodies are encoded by nucleic acid molecules generated by the combination of V, D, and J gene segments, particularly human V, D, and J germline segments. A combinatorial library herein typically contains at least 50 different antibody (or antibody portion or fragment) members, and typically contains at least 50, 100, 500, 10, 1x10, 2x10, 3x10, 4x10, 5x10, 6x10, 7x10, 8x10, 9x ... or more distinct members, or about 50, about 100, about 500, about 103, about 1x103, about 2x103, about 3x103, about 4x103, about 5x103, about 6x103, about 7x103, about 8x103, about 9x103, about 1x104, about 2x104, about 3x104, about 4x104, about 5x104, about 6 x10, about 7x10, about 8x10, about 9x10, about 1x10, about 2x10, about 3x10, about 4x10, about 5x10, about 6x10, about 7x10, about 8x10, about 9x10, about 10, about 10, about 10, about 10, about 10, about 10, or more different members. The resulting library or collection of antibodies or portions thereof can be screened for binding to a target protein or for modulating a functional activity.

[0376] A "human combinatorial antibody library" refers to a collection of antibodies or portions thereof, each member of which contains a VL chain and a VH chain, or a sufficient portion thereof to form an antigen-binding site, encoded by nucleic acid containing human germline segments generated as described herein.

[0377] "Variable germline segment" refers to the V, D, and J groups, subgroups thereof, genes, or alleles. Gene segment sequences can be obtained from public databases (e.g., the National Center for Biotechnology Information (NCBI), the International Immunogenetics Information System® (IMGT), the Kabat database, and Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). Tables 3-5 list exemplary human variable germline segments. Exemplary VH, DH, JH, Vκ, Jκ, Vλ, and / or Jλ, germline segment sequences are set forth in SEQ ID NOs: 10-451 and 868. For purposes herein, germline segments include modified sequences thereof that have been modified according to the sequence editing rules provided herein to enable the methods to be performed. For example, germline gene segments can be selected from the group consisting of SEQ ID NOs: 10-451, ... This includes germline gene segments that contain a single amino acid deletion or insertion at the 5' or 3' end compared to any of the nucleotide sequences set forth in NOs:10-451,868.

[0378] "Editing," "editing," "combining," "combining," "rearrangement," "rearrangement," or other similar terms or grammatical variations thereof, refer to the process by which germline segments are ordered or assembled into a nucleic acid sequence corresponding to a gene. For example, variable heavy chain germline segments are assembled such that the VH segment is 5' to the DH segment, which is 5' to the JH segment, thereby resulting in a nucleic acid sequence encoding a VH chain. Variable light chain germline segments are assembled such that the VL segment is 5' to the JL segment, thereby resulting in a nucleic acid sequence encoding a VL chain. Constant gene segments can also be assembled at the 3' end of the nucleic acid encoding a VH or VL chain.

[0379] "Linked" or "linked" or other grammatical variations with respect to germline segments refer to germline segments being joined together. Linkage can be direct or indirect. Germline segments can be directly linked with no additional nucleotides between the segments, additional nucleotides can be added such that the entire segment is in-frame, or nucleotides can be deleted such that the resulting segment is in-frame. It is understood that the linker nucleotides are selected such that the resulting nucleic acid molecule is in-frame and encodes a functional and productive antibody.

[0380] "In-frame" or "linked in frame" with respect to the linking of human germline segments means that nucleotides are inserted and / or deleted at the spliced ​​junctions of the germline segments such that the resulting nucleic acid molecule is in-frame with the 5' start codon (ATG), thereby producing a "productive" or functional full-length polypeptide. The selection of nucleotides to be inserted or deleted from the germline segments, particularly the junctions where the various VD, DJ, and VJ segments are joined, follows the rules provided in the methods herein for making V(D)J junctions. For example, germline segments are assembled such that the VH segment is 5' to the DH segment, and the DH segment is 5' to the JH segment. Nucleotides can be inserted or deleted from individual VH, DH, or JH segments at the junctions where the VH and DH segments are joined and at the junctions where the DH and JH segments are joined, such that the resulting nucleic acid molecule containing the spliced ​​VDJ segments is in-frame with the 5' start codon (ATG).

[0381] A portion of an antibody includes sufficient amino acids to form an antigen-binding site.

[0382] "Reading frame" refers to a contiguous, non-overlapping set of three nucleotide codons in DNA or RNA. Since three codons code for one amino acid, there are three reading frames for a particular nucleotide sequence: reading frame 1, 2, or 3. For example, the sequence ACTGGTCA is ACT GGT CA for reading frame 1, A CTG GTC A for reading frame 2, and AC TGG TCA for reading frame 3. Generally, to practice the methods described herein, nucleic acid sequences are combined such that the V sequence is in reading frame 1.

[0383] "Stop codon" refers to a three nucleotide sequence that signals the termination of protein synthesis during translation, including the amber stop codon (UAG or TAG), the ochre stop codon (UAA or TAA), and the opal stop codon (UGA or TGA), or any sequence that codes for that sequence (e.g., a DNA sequence that codes for an RNA stop codon sequence). A stop codon does not necessarily signal translation termination in every cell or every organism. For example, in suppressor strain host cells, such as amber suppressor strains and partial amber suppressor strains, translation proceeds at least some of the way through one or more stop codons (e.g., the amber stop codon in the case of an amber suppressor strain).

[0384] A "variable heavy" (VH) chain or a "variable light" (VL) chain (also referred to as a VH domain or a VL domain) refers to a polypeptide chain that constitutes the variable domain of an antibody. For purposes herein, heavy chain germline segments are designated VH, DH, and JH, which, when edited, result in a nucleic acid encoding a VH chain. Light chain germline segments are designated VL or JL, and include kappa and lambda light chains (Vκ and Jκ; Vλ and Jλ), which, when edited, result in a nucleic acid encoding a VL chain. It is understood that the light chain is either a kappa or lambda light chain, and does not include kappa / lambda combinations due to editing of Vκ and Jλ.

[0385] A "degenerate codon" refers to a three nucleotide codon that specifies the same amino acid as a codon in a parent nucleotide sequence. Those of skill in the art are familiar with the degeneracy of the genetic code and are able to identify degenerate codons.

[0386] "Diversity" with respect to members in a collection refers to the number of unique members in the collection. Thus, diversity refers to the number of different amino acid sequences or different nucleic acid sequences among the similar polypeptide members of the collection. For example, a collection of polynucleotides with a diversity of 104 contains 104 different nucleic acid sequences among the similar polynucleotide members. In one example, the diversity of the provided collection of polynucleotides and / or polypeptides is at least or about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, or more.

[0387] "Sequence diversity" refers to a representation of nucleic acid sequence similarity and is determined using sequence alignment, diversity score, and / or sequence clustering. Any two sequences can be aligned by placing the sequences side by side and analyzing the nucleotide differences position by position along the length of these sequences. Sequence alignment can be evaluated in silico using BLAST (Basic Local Alignment Search Tool), an NCBI tool for comparing nucleic acid and / or protein sequences. The use of BLAST for sequence alignment is well known to those skilled in the art. The Blast search algorithm compares two sequences and calculates the statistical significance of each match (Blast score). Sequences that are most similar to each other have a high Blast score, whereas sequences that are most different have a low Blast score.

[0388] A "polypeptide domain" refers to a polypeptide portion (a sequence of three or more, typically five or seven or more amino acids) that can be structurally and / or functionally distinct or definable. Exemplary polypeptide domains are those that can form independently folded structures within a polypeptide, that are composed of one or more structural motifs (e.g., combinations of alpha helices and / or beta strands connected by loop regions), and / or that are recognized by a certain functional activity, such as enzymatic activity or antigen binding. A polypeptide may have one, and typically several, distinct domains. For example, a polypeptide may have one or more structural domains and one or more functional domains. A polypeptide domain can be distinguished based on structure and function. A domain may encompass a contiguous linear amino acid sequence. Alternatively, a domain may encompass several non-contiguous amino acid portions that are not contiguous along the linear amino acid sequence of the polypeptide. Typically, a polypeptide contains several domains. For example, each heavy chain and each light chain of an antibody molecule contains several immunoglobulin (Ig) domains, and each immunoglobulin (Ig) domain is about 110 amino acids long.

[0389] An "Ig domain" is recognized as such by those skilled in the art and refers to a domain that is distinguished by a structure called the immunoglobulin (Ig) fold, which contains two beta pleated sheets, each containing an antiparallel beta strand of amino acids connected by a loop. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and conserved intrachain disulfide bonds. Furthermore, individual immunoglobulin domains within an antibody chain can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), whereas a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each of the VL, CL, VH, and CH domains is an example of an immunoglobulin domain.

[0390] "Variable domain" in reference to an antibody refers to a specific Ig domain of an antibody heavy or light chain that contains an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain (VL and VH). The variable domain confers antigen specificity and is therefore responsible for antigen recognition. Each variable region contains CDRs and framework regions (FRs) that are part of the antigen binding site domain.

[0391] "Constant region domain" refers to a domain of an antibody heavy or light chain that contains an amino acid sequence that is relatively more conserved among antibodies than the variable region domain. Each light chain has one light chain constant region (CL) domain, and each heavy chain has one or more heavy chain constant region (CH) domains, including CH1, CH2, CH3, and CH4. Full-length IgA, IgD, and IgG isotypes contain CH1, CH2, CH3, and a hinge region, whereas IgE and IgM contain CH1, CH2, CH3, and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigens and the rotation of the antibody arm. The antibody constant region can perform effector functions, such as, but not limited to, the elimination of antigens, pathogens, and toxins that the antibody specifically binds, by interacting with various cells, biomolecules, and tissues.

[0392] "An antibody or a portion of an antibody sufficient to form an antigen-binding site" means that the antibody or portion thereof contains at least one or two, typically three, four, five, or all six CDRs of VH and VL sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody containing all six CDRs. Generally, a sufficient antigen-binding site requires at least the CDR3 of the heavy chain (CDRH3). Typically, a sufficient antigen-binding site also requires the CDR3 of the light chain (CDRL3). As described herein, the skilled artisan knows and can identify CDRs based on Kabat numbering or Chothia numbering (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). For example, based on Kabat numbering, CDR-LI corresponds to residues L24 to L34, CDR-L2 corresponds to residues L50 to L56, CDR-L3 corresponds to residues L89 to L97, CDR-H1 corresponds to residues H31 to H35, or 35a or 35b depending on the length, CDR-H2 corresponds to residues H50 to H65, and CDR-H3 corresponds to residues H95 to H102.

[0393] "Peptide mimetics" refers to peptides that mimic the activity of a polypeptide. For example, erythropoietin (EPO) peptide mimetics are peptides that mimic the activity of Epo, such as binding to and activating the EPO receptor.

[0394] "Address" refers to a unique identifier for each location in a collection that allows identification of an addressed member (e.g., an antibody). An addressed moiety is one that can be identified by its location or location. Addressing can be by location on a surface, such as a microplate well. For example, a protein address of F9 in a microwell plate means that the protein is in row F, column 9 of the microwell plate. Addressing can also be done with other identifiers, such as tags coded with bar codes or other symbologies, chemical tags, electronic tags, such RF tags, color-coded tags, or other such identifiers.

[0395] An "array" refers to a collection of elements, such as antibodies, containing three or more members.

[0396] "Spatial array" refers to an array in which members are separated or occupy distinct spaces in the array. Thus, a spatial array is a type of addressable array. Examples of spatial arrays include microtiter plates, where each well of the plate is an address in the array. Spatial arrays include any arrangement in which a plurality of different molecules, e.g., polypeptides, are held, presented, placed, placed, or supported. Arrays may include microtiter plates, e.g., 48-well, 96-well, 144-well, 192-well, 240-well, 288-well, 336-well, 384-well, 432-well, 480-well, 576-well, 672-well, 768-well, 864-well, 960-well, 1056-well, 1152-well, 1248-well, 1344-well, 1440-well, or 1536-well plates, tubes, slides, chips, flasks, or any other suitable laboratory equipment. Additionally, arrays may also include a plurality of subarrays. Multiple subarrays include arrays in which multiple configurations are used to arrange the polypeptides, for example, multiple 96-well plates can constitute multiple subarrays and an array.

[0397] An "addressable library" or "spatially addressed library" refers to a collection of molecules, such as nucleic acid molecules or protein drugs such as antibodies. Each member of the collection is identifiable by its address.

[0398] "Addressable array" refers to an array in which the members of the array are identifiable by address, by location within a spatial array such as a microtiter plate well or by location on a solid support, or by an identifiable or detectable label, such as color, fluorescence, electronic signal (i.e., RF, microwave, or other frequencies that do not substantially alter the interaction of the molecules of interest), barcode or other symbology, chemical label, or other such label. Thus, in general, the members of the array are placed at identifiable locations on a solid surface, or directly or indirectly linked or otherwise bound to an identifiable label, for example, attached to a microsphere or other particulate support (herein referred to as beads), suspended in solution, or spread on a surface.

[0399] An "addressable combinatorial antibody library" refers to an antibody collection in which member antibodies are identifiable and antibodies with the same identifier, e.g., location in a spatial array or on a solid support, or chemical or RF tag, all bind to the same antigen and are generally substantially identical in amino acid sequence. For purposes herein, reference to an "addressable arr...

Claims

1. (a) a heavy chain variable domain; (b) a light chain variable domain; A humanized antibody comprising: The heavy chain variable domain comprises: (i) amino acids 1 to 95 of the amino acid sequence of SEQ ID NO:33, wherein SEQ ID NO:33 contains at least one amino acid substitution selected from Q5R, Q6E, G31D, Y32K, and E50S, and up to five additional amino acids may be substituted, except at positions 5, 6, 31, 32, and 50 of SEQ ID NO:33; (ii) an extremely long CDR3 that is at least 35 amino acids in length; and (iii) Framework region 4 (FR4) in order, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:805, except for one or more amino acid substitutions selected from the group consisting of S2A, T5N, P8S, A12G, A13S, P14L, K46R, L47T, D51G, N52D, and N53T, and wherein up to ten additional amino acids may be substituted, except at positions 2, 5, 8, 12, 13, 14, 46, 47, 51, 52, and 53 of SEQ ID NO:805; Humanized antibodies.

2. The humanized antibody of claim 1, wherein SEQ ID NO:33 contains at least two amino acid substitutions selected from Q5R, Q6E, G31D, Y32K, and E50S.

3. 2. The humanized antibody of claim 1, wherein SEQ ID NO:33 comprises three, four, or five amino acid substitutions selected from Q5R, Q6E, G31D, Y32K, and E50S.

4. The humanized antibody of claim 1, wherein the light chain variable domain is a VL1-51 light chain variable domain or a variant thereof.

5. The humanized antibody of claim 1, wherein the light chain variable domain contains at least two amino acid substitutions.

6. FR4, (i) WGHGTAVTVSS (SEQ ID NO: 570), (ii) WGKGTTVTVSS (SEQ ID NO: 571); (iii) WGRGTLVTVSS (SEQ ID NO: 573), and (iv) WGQGLLVTVSS (SEQ ID NO: 500) The humanized antibody of claim 1, comprising an amino acid sequence selected from the group consisting of:

7. The humanized antibody of claim 1 which is a single-chain variable fragment.

8. The humanized antibody of claim 1, wherein the heavy chain variable domain and the light chain variable domain are on different polypeptides.

Images