US2003190598A1PendingUtilityA1
Single-domain antigen-binding antibody fragments derived from llama antibodies
Priority: May 26, 2000Filed: May 25, 2001Published: Oct 9, 2003
Est. expiryMay 26, 2020(expired)· nominal 20-yr term from priority
C40B 40/02C12N 15/1037C07K 16/00C07K 2317/22C07K 2317/569
42
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Claims
Abstract
A phage display library of variable heavy domain (V H H or VH) fragments (sdAb fragments) derived from the antibody repertoire of a non-immunized llama is disclosed. The sdAb fragments of the library are characterized by the absence of cysteine residues in complementarity determining regions (CDRs) and a very low presence of residues of glutamic acid, arginine and glycine at positions 44, 45 and 47 respectively, of the VL interface of the variable heavy domain V H H. The large size of the library (in the order of 10 9 ) makes it a source of antigen-binding fragments having high affinity to almost any antigen of interest. The library is preferably generated using a modified fd-tet phage growing in plaques in the absence of a tetracycline.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A phage display library of antigen-binding fragments derived from llama antibodies, each antigen-binding fragment comprising at least a part of the variable heavy domain (V H H or VH) of a llama antibody.
2 . A phage display library according to claim 1 , wherein the antigen-binding fragment comprises a complete variable heavy domain (V H H or VH).
3 . A phage display library according to claim 2 , wherein the antigen-binding fragment consist essentially of a variable heavy domain (V H H or VH) of a llama antibody.
4 . A phage display library according to claim 3 , wherein the library is derived from the antibody repertoire of a non-immunized llama.
5 . A phage display library according to claim 4 , wherein the library is of a size of at least 10 9 .
6 . A phage display library according to claim 5 , wherein the library is of a size of at least 10 8 .
7 . A phage display library according to claim 4 , wherein the phage vector is a modified fd-tet phage.
8 . A phage display library according to claim 7 , wherein the library is generated in the absence of a tetracycline.
9 . A phage display library according to claim 8 , wherein the library is generated as plaques.
10 . An antigen-binding fragment derived from a llama antibody, said fragment comprising at least a part of the variable heavy domain (V H H or VH) of the antibody.
11 . An antigen-binding fragment according to claim 10 , wherein said fragment comprises a complete variable heavy domain (V H H or VH) of the antibody.
12 . An antigen-binding fragment according to claim 11 , wherein said fragment consists essentially of the variable heavy domain (V H H or VH) of a llama antibody.
13 . An antigen-binding fragment according to claim 12 , wherein the antibody is selected from the antibody repertoire of a non-immunized lama.
14 . An antigen-binding fragment according to claim 13 , wherein the complementarity determining regions CDR1/H1, CDR2 and CDR3 of the variable heavy domain (V H H or VH) are essentially free of cysteine residues.
15 . An antigen-binding fragment according to claim 14 , wherein the CDR1/H1 region of the variable heavy domain (V H H or VH) is selected from the group consisting of:
GFTFSSYAMS
(SEQ ID NO: 85)
GFTFSSYYMS
(SEQ ID NO: 86)
GFTFDEHAIG
(SEQ ID NO: 87)
GFTVSSNHMT
(SEQ ID NO: 88)
GFTFSSYHMA
(SEQ ID NO: 89)
GFTFSRHQMS
(SEQ ID NO: 91)
GFTFRTYYMN
(SEQ ID NO: 92)
GFIFSSYAMS
(SEQ ID NO: 93)
GFTFSTYAMT
(SEQ ID NO: 95)
GFTFSGYAMS
(SEQ ID NO: 99)
GFAFSNYRMT
(SEQ ID NO: 100)
GFTFSRYAMS
(SEQ ID NO: 101)
16 . An antigen-binding fragment according to claim 14 , wherein the CDR2 region of the variable heavy domain (V H H or VH) is selected from the group consisting of:
GIEGGGGITRYADSVKG
(SEQ ID NO: 102)
TIKPGGGSTYYADSVKG
(SEQ ID NO: 103)
TIDIGGGRTYADSVKG
(SEQ ID NO: 104)
RISSDGRNTYYADSVKG
(SEQ ID NO: 105)
TINPGDGSTYYADSVKG
(SEQ ID NO: 106)
HIDTGGSTWYAASVKG
(SEQ ID NO: 107)
TINIDGSSTYYADSVRG
(SEQ ID NO: 109)
GINSFGGSKYYADSVKG
(SEQ ID NO: 110)
TINTSGRGTYYADSVKG
(SEQ ID NO: 112)
AINSGGGSTSYADSVKG
(SEQ ID NO: 113)
HIDTGGGSTWYAASVKG
(SEQ ID NO: 114)
DINSGGDSTRNADSVKG
(SEQ ID NO: 115)
SINSGGGSTYYADSVKG
(SEQ ID NO: 116)
RINSIGDRISYADSVKG
(SEQ ID NO: 117)
17 . An antigen-binding fragment according to claim 14 , wherein the CDR3 region of the variable heavy domain (V H H or VH) is selected from the group consisting of:
AHGGYGAFGS
(SEQ ID NO: 119)
YSGGALDA
(SEQ ID NO: 122)
LSQGAMDY
(SEQ ID NO: 124)
IDRERAFTS
(SEQ ID NO: 127)
IDWERAFTS
(SEQ ID NO: 128)
QGYAGSYDY
(SEQ ID NO: 129)
LGVPGTFDY
(SEQ ID NO: 130)
TNRGIFDY
(SEQ ID NO: 131)
TPGSSGVYEY
(SEQ ID NO: 132)
TQTGSHDY
(SEQ ID NO: 133)
QVGTAYDY
(SEQ ID NO: 134)
RRGSSGVYEY
(SEQ ID NO: 135)
18 . An antigen-binding fragment according to claim 14 , wherein said fragment has at position 45 a residue of an amino acid other than cysteine.
19 . An antigen-binding fragment according to claim 18 , wherein amino acid residues of the VL interface of the variable heavy domain (V H H or VH) are Gly at position 44, Leu, Phe, Pro, or Arg at position 45, and Trp, Tyr, or Phe at position 47.
20 . An antigen-binding fragment according to claim 19 , wherein amino acid residues at positions 44, 45 and 47 are Gly, Leu and Trp, respectively.
21 . An antigen-binding fragment according to claim 19 , wherein amino acid residues at positions 44, 45 and 47 are Gly, Pro and Trp, respectively.
22 . An antigen-binding fragment according to claim 18 , wherein amino acid residues of the VL interface of the variable heavy domain (V H H or VH) are Glu at position 44, Arg at position 45, and Phe, Ile, Val, or Gly at position 47.
23 . An antigen-binding fragment according to claim 18 , wherein amino acid residues of the VL interface of the variable heavy domain (V H H or VH) are Gln, Gly, Lys, Ala, or Asp at position 44, Arg at position 45, and Leu, Phe, or Trp at position 47.
24 . An antigen-binding fragment according to claim 18 , wherein amino acid residues at positions 6, 23, 74, 82a, 83, 84, 93 and 108 are Ala, Ala, Ala, Asn, Lys, Pro, Ala and Gln, respectively.
25 . A cDNA library comprising nucleotide sequences coding for antigen-binding fragments of llama antibodies, said library obtained by performing the steps of:
(a) isolating lymphocytes from a biological sample obtained from a non-immunized llama; (b) isolating total RNA from the lymphocytes; (c) reverse-transcribing and amplifying RNA sequences coding for the antigen-binding fragments; (d) cloning the amplified cDNA in a vector, and (e) recovering the obtained clones.
26 . A cDNA library according to claim 25 , wherein each antigen-binding fragment comprises at least a part of the variable heavy domain (V H H or VH) of the antibody.
27 . A cDNA library according to claim 26 , wherein the antigen-binding fragment comprises a complete variable heavy domain (V H H or VH) of the antibody.
28 . A cDNA library according to claim 27 , wherein the antigen-binding fragment consists essentially of the variable heavy domain (V H H or VH) of a llama heavy chain antibody.
29 . A cDNA library according to claim 28 , wherein the vector is a filamentous bacteriophage.
30 . A cDNA library according to claim 29 , wherein the filamentous bacteriophage is fd-tet phage.
31 . A process for the preparation of an antigen-binding fragment of a llama antibody, said fragment binding to a predetermined antigen, said process comprising the steps of:
(a) isolating lymphocytes from a biological sample obtained from a non-immunized llama; (b) isolating total RNA from the lymphocytes; (c) reverse-transcribing and amplifying RNA sequences coding for antigen-binding fragments; (d) cloning the cDNA sequences so obtained into a cloning vector, said first vector capable of a surface display of the corresponding antigen-binding fragments; (e) subjecting the clones to antigen affinity selection and recovering clones having the desired affinity; (f) for the recovered clones, amplifying DNA sequences coding for antigen-binding fragments; (g) cloning the amplified DNA sequences into an expression vector; (h) transforming host cells with the expression vector under conditions allowing expression of DNA coding for antigen binding fragments; and (i) recovering the antibody fragments having the desired specificity.
32 . A process according to claim 31 , wherein the antigen-binding fragment comprises at least a part of the variable heavy domain (V H H or VH) of the llama antibody.
33 . A process according to claim 32 , wherein the antigen-binding fragment comprises a complete variable heavy domain (V H H or VH) of the llama antibody.
34 . A process according to claim 33 , wherein the antigen-binding fragment consists essentially of the variable heavy domain (V H H or VH) of a llama antibody.
35 . A process according to claim 34 , wherein the cloning vector is selected from the group consisting of bacteriophages, bacteria, and yeasts.
36 . A process according to claim 35 , wherein the cloning vector is a filamentous bacteriophage.
37 . A process according to claim 36 , wherein the filamentous bacteriophage is fd-tet phage.
38 . A process according to claim 31 , wherein the expression vector is a plasmid, a phage, a virus, a YAC, or a cosmid.
39 . A process according to claim 31 , wherein the host cells are prokaryotic cells or eukaryotic cells.
40 . A process according to claim 39 , wherein the eukaryotic cells are selected from the group consisting of yeast cells, mammalian cells, plant cells and protozoan cells.Join the waitlist — get patent alerts
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