NOVEL RABBIT ANTIBODY HUMANIZATION METHODS AND HUMANIZED RABBIT ANTIBODIES
First Claim
1. A humanized antibody or antibody fragment containing at least one heavy and light chain polypeptide wherein the light chain polypeptide is a humanized light chain polypeptide which contains at least the following (i) the amino acid residues spanning the first residue of FR1 through the terminus of FR3 including the CDR1 and CDR2 regions of a human light chain germline sequence that is selected from a library of human germline sequences based on its greater homology (percent sequence identity) of the selected amino acid residues spanning FR1 through FR3 (relative to other human germline sequences in the library) to the corresponding amino acid residues of the light chain of a parent rabbit antibody having specificity to a desired antigen that is to be humanized and (ii) further wherein the CDR residues in CDR1 and CDR2 corresponding to “
- selectivity determining residues”
in the light chain of the same parent rabbit antibody are replaced with the corresponding rabbit selectivity determining residues;
(iii) the amino acid residues encompassing the entire CDR3 region of the same parent rabbit antibody;
(iv) the amino acid residues encompassing the entire FR4 region of an antibody light chain derived from a library of human germline sequences based on its greater homology (sequence identity) to the corresponding FR4 region contained in the light chain of the same parent rabbit antibody; and
(v) wherein few or none of the FR residues of the human FR1, FR2, FR3 and FR4 regions in the selected homologous human FR regions are substituted with the corresponding rabbit FR residues.
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Abstract
The present invention is directed to novel and improved methods for humanizing rabbit heavy and light variable regions. The resulting humanized rabbit heavy and light chains and antibodies and antibody fragments containing are well suited for use in immunotherapy and immunodiagnosis as they retain the antigen binding affinity of the parent antibody and based on their very high level of sequence identity to human antibody sequences should be essentially non-immunogenic in humans. The invention exemplifies the protocol for the manufacture of therapeutic humanized anti-human TNF-alpha and anti-human IL-6 antibodies.
158 Citations
91 Claims
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1. A humanized antibody or antibody fragment containing at least one heavy and light chain polypeptide wherein the light chain polypeptide is a humanized light chain polypeptide which contains at least the following (i) the amino acid residues spanning the first residue of FR1 through the terminus of FR3 including the CDR1 and CDR2 regions of a human light chain germline sequence that is selected from a library of human germline sequences based on its greater homology (percent sequence identity) of the selected amino acid residues spanning FR1 through FR3 (relative to other human germline sequences in the library) to the corresponding amino acid residues of the light chain of a parent rabbit antibody having specificity to a desired antigen that is to be humanized and (ii) further wherein the CDR residues in CDR1 and CDR2 corresponding to “
- selectivity determining residues”
in the light chain of the same parent rabbit antibody are replaced with the corresponding rabbit selectivity determining residues;
(iii) the amino acid residues encompassing the entire CDR3 region of the same parent rabbit antibody;
(iv) the amino acid residues encompassing the entire FR4 region of an antibody light chain derived from a library of human germline sequences based on its greater homology (sequence identity) to the corresponding FR4 region contained in the light chain of the same parent rabbit antibody; and
(v) wherein few or none of the FR residues of the human FR1, FR2, FR3 and FR4 regions in the selected homologous human FR regions are substituted with the corresponding rabbit FR residues. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 22, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91)
- selectivity determining residues”
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11. A humanized antibody or antibody fragment containing at least one heavy chain and light chain polypeptide wherein the heavy chain is a humanized heavy chain polypeptide which contains at least the following (i) the amino acid residues spanning the first residue of FR1 through the terminus of FR3 including the CDR1 and CDR2 regions encoded by a human germline sequence that is selected from a library of human germline sequences based on its greater homology (percent sequence identity) of the selected amino acid residues spanning FR1 through FR3 (relative to other human germline sequences in the library) to the corresponding amino acid residues of the heavy chain of a parent rabbit antibody having specificity to a desired antigen that is to be humanized and (ii) further wherein the CDR residues in the CDR1 and CDR2 regions of the human heavy chain corresponding to “
- selectivity determining residues”
in the CDR1 and CDR2 regions of the heavy chain of the same parent rabbit antibody are replaced with the corresponding heavy chain selectivity determining residues contained in the CDR1 and CDR2 regions of the rabbit heavy chain;
(iii) the amino acid residues encompassing the entire CDR3 region of the same parent rabbit antibody;
(iv) the FR4 region derived from a library of human germline sequences based on its greater homology (sequence identity) to the corresponding FR4 region contained in the heavy chain of the same parent rabbit antibody; and
(v) wherein the final 1-3 amino acids of the human heavy FR1 region are optionally replaced with the terminal 1-3 amino acids of the corresponding rabbit heavy chain FR1 residues; and
/or the terminal amino acid of the human heavy chain framework 2 region is optionally replaced with the corresponding terminal amino acid residue of the rabbit heavy chain framework 2; and
/or the fourth amino acid from the terminus of the rabbit heavy chain CDR2 (typically a tryptophan) is optionally replaced with the corresponding human CDR2 residue (typically a serine); and
(vi) wherein few or none of the remaining FR residues of the selected homologous human FR regions are substituted with the corresponding rabbit FR residues. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
- selectivity determining residues”
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23. A humanization strategy for producing a humanized light chain antibody sequence comprising the following steps:
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(i) obtaining a DNA encoding rabbit light chain antibody sequence from a rabbit antibody that specifically binds to a desired antigen and identifying the amino residues spanning the beginning of Framework 1 (FR1) to the end of Framework 3 (FR3) inclusive; (ii) conducting a homology search using said rabbit light antibody amino acid sequence spanning the beginning of FR1 to the end of FR3 sequence against a library containing human light chain antibody sequences and identifying a human light chain antibody sequence that exhibits substantial sequence homology thereto relative to other human germline antibody light chain sequences; (iii) identifying in both the rabbit and human light chain sequences the arrangement and the specific residues thereof that correspond to FR1. FR2, FR3, CDR1, CDR2 regions and aligning these discrete regions in the rabbit and selected human antibody light chain; (iv) constructing a DNA or amino acid sequence wherein the CDR1 and CDR2 regions of the selected homologous human light chain sequence are substituted by the corresponding selectivity determining residues contained in the CDR1 and CDR2 regions of the rabbit light chain sequence; (v) further attaching to the DNA or amino acid sequence obtained by step (iv) a DNA sequence encoding or polypeptide containing the corresponding amino acid residues of the rabbit CDR3 light chain antibody sequence; (vi) further selecting a human light chain framework 4 region (FR4) that is homologous to the FR4 contained in the rabbit light chain and which preferably differs therefrom by at most 2-4 amino acid residues and attaching a DNA sequence encoding said human FR4 or the corresponding amino residues of said human FR4 onto the DNA or amino acid sequence obtained after step (v); and (vii) synthesizing a DNA or amino acid sequence encoding or containing the humanized rabbit light chain sequence that results from steps (i) through (vi). - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 68, 69, 70, 71, 82, 83, 84, 85, 86, 87, 88)
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43. A humanization strategy for producing a humanized heavy chain antibody sequence from a rabbit heavy chain antibody sequence comprising the following steps:
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(i) obtaining a rabbit heavy chain antibody sequence from an rabbit antibody that specifically binds to a desired antigen and identifying the amino residues spanning the beginning of Framework 1 (FR1) to the end of Framework 3 (FR3) inclusive; (ii) conducting a homology search (e.g., by BLAST searching of human germline antibody sequence containing libraries) using said rabbit heavy antibody amino acid sequence spanning the beginning of FR1 to the end of FR3 sequence and identifying a human heavy chain antibody sequence that is homologous thereto, i.e. which preferably possesses at least 80%-90% identical thereto at the amino acid level; (iii) identifying in both the rabbit and human heavy chain sequences the arrangement of and the specific residues thereof that correspond to FR1, FR2, FR3, CDR1, CDR2 regions and aligning these discrete regions of the rabbit against the corresponding regions of the selected homologous human antibody heavy chain; (iv) constructing a DNA or amino acid sequence wherein the residues in the CDR1 and CDR2 regions of the selected homologous human heavy chain sequence are substituted by the selectivity determining residues contained in the corresponding CDR1 and CDR2 regions of the rabbit heavy chain sequence and optionally replacing the terminal 1-3 amino acids of the human heavy FR1 region with the corresponding terminal 1-3 amino acids of the rabbit heavy chain FR1; and
/or optionally replacing the terminal amino acid of the human heavy chain framework 2 region with the corresponding terminal amino acid residue of the rabbit heavy chain framework 2 and/or optionally replacing the fourth amino acid from the terminus of the rabbit heavy chain CDR2 (typically a tryptophan) with the corresponding human CDR2 residue (typically a serine);(v) further attaching to the DNA or amino acid sequence obtained by step (iv) a DNA sequence encoding or having the corresponding amino acid residues of the rabbit heavy chain CDR3 which is contained in the same rabbit heavy chain antibody sequence; (vi) further selecting a human heavy chain framework 4 region (FR4) that is homologous thereto (preferably differs from the FR4 contained in the humanized rabbit antibody heavy chain sequence by at most 4 amino acid residues) and attaching a DNA sequence encoding said selected homologous human FR4 or the corresponding amino residues of said human FR4 onto the DNA or amino acid sequence obtained after step (v)); and (vii) synthesizing a DNA or amino acid sequence encoding or containing the humanized rabbit heavy chain sequence that results from steps (i) through (vi). - View Dependent Claims (44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67)
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Specification