Immunoglobulin variable region cassette exchange
Inventors
Bebbington, Christopher Robert • Luehrsen, Kenneth R. • Yarranton, Geoffrey T.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10919983-B2
Publication Date
2021-02-16
Expiration Date
Abstract
The invention provides methods for generating human antibodies with the specificity of a reference antibody by replacement of portions of the VH and VL sequences of the reference antibody with sequences from human antibody repertoires. The invention also provides novel compositions comprising hybrid immunoglobulin variable domains containing a combination of frameworks (FRs) and CDRs from different antibody clones.
Core Innovation
The invention relates to engineering an antibody that retains the binding specificity of a reference antibody for a target antigen while reducing potential for immunogenicity in humans. It addresses modifying a reference antibody in a way that preserves binding specificity and affinity to the target antigen and results in human exchanged antibody variants with reduced immunogenicity potential.
The disclosed approach uses variable region engineering based on replacement of an exchange cassette derived from a V gene segment framework and CDR structure, including FR1-CDR1-FR2, FR2-CDR2-FR3, FR3-CDR3-FR4, and CDR3-FR4. Exchange cassettes include at least one intact CDR adjoined to at least one intact FR that are together naturally occurring, and hybrid V-regions are generated by replacing the reference antibody exchange cassette with corresponding human exchange cassettes from human V-gene segments that are germline or from a human antibody gene.
Hybrid V-regions are paired with a complementary V-region and antibodies comprising hybrid V-regions are selected for binding specificity and reduced potential for immunogenicity in humans. The framework further includes repeating or iterative cassette exchange so that alternative exchange cassettes are serially replaced, and combining at least two human exchanged cassettes to obtain an engineered antibody with the binding specificity of the reference antibody and a higher binding affinity for the target antigen than the reference antibody.
Claims Coverage
The provided claim set includes three independent claims (clm-00001, clm-00015, clm-00017). Across these claims, the core inventive features involve generating hybrid V-regions by replacing specified exchange-cassette or FR-CDR segments with human exchange-cassette or FR(-CDR)-FR segments, pairing with a complementary V-region, selecting for binding specificity or detectable affinity, and producing engineered antibodies with reduced potential for immunogenicity in humans, with at least some embodiments requiring higher binding affinity than the reference antibody.
Hybrid V-region via human exchange cassette replacement under less than three framework regions constraint
Replacing at least one exchange cassette obtained from the V gene segment, FR1-CDR1-FR2-CDR2-FR3, of the variable region of the reference antibody with a library of corresponding human exchange cassettes from human V-gene segments comprised of immunoglobulin sequences that are germline or a V-gene segment having a human exchange cassette from one human antibody gene, generating a library of hybrid V-regions with the replaced exchange cassette encoded by different genes, with the proviso that the exchange cassette has less than three framework regions.
Intact naturally occurring CDR and FR within the exchanged cassette
Wherein the at least one exchange cassette obtained from the V-segment of the variable region of the reference antibody includes at least one intact CDR adjoined to at least one intact FR of the V-segment that are together naturally occurring.
Pairing hybrid V-region library with complementary V-region and selecting reduced-immunogenicity specificity-retaining antibodies
Pairing the library of hybrid V regions with a complementary V-region; and selecting an antibody comprising a hybrid V region having at least one human exchanged cassette that has the binding specificity of the reference antibody and has a reduced potential for immunogenicity in humans.
Iterative serial cassette replacement and multi-cassette combining for higher affinity with retained specificity
Repeating such that alternative exchange cassettes from the antibody comprising a hybrid V region are replaced with human V region cassette sequences; and combining the at least one human exchanged cassette of the engineered antibody with a second human exchange cassette of the antibody to obtain an antibody with the binding specificity of the reference antibody and a higher binding affinity for the target antigen than the reference antibody and having a reduced potential for immunogenicity in humans, wherein the antibody has a hybrid V-region that comprises at least two human exchanged cassettes.
Segmented human germline FR1-CDR1-FR2 replacement and selection with detectable affinity
Replacing the FR1-CDR1-FR2 of the variable region of the reference antibody with a library of human FR1-CDR1-FR2 regions from human V-gene segments comprised of immunoglobulin sequences that are germline to create a library of hybrid variable regions; pairing the hybrid variable regions with a complementary variable region; and selecting an antibody having a detectable affinity for the target antigen.
Segmented human germline FR2-CDR2-FR3 replacement and selection with detectable affinity
Replacing the FR2-CDR2-FR3 of the variable region of the reference antibody with a library of human FR2-CDR2-FR3 regions from human V-gene segments comprised of immunoglobulin sequences that are germline to create a library of hybrid variable regions; pairing the hybrid variable regions with a complementary variable region; and selecting an antibody having a detectable affinity for the target antigen.
Combining selected human FR1-CDR1-FR2 and human FR2-CDR2-FR3 to yield higher-affinity reduced-immunogenicity antibody
Combining the human FR1-CDR1-FR2 of the hybrid variable region of the antibody selected in (b) with the human FR2-CDR2-FR3 of the hybrid variable region of the antibody selected in (c) to obtain an antibody with the human FR1-CDR1-FR2-CDR2-FR3 variable region V gene segment, which antibody has the binding specificity of the reference antibody and has a higher binding affinity for the target antigen than the reference antibody and has a reduced potential for immunogenicity in humans.
Human germline FR1-CDR1-FR2 and CDR2-FR3 replacement with detectable affinity selection
Replacing the FR1-CDR1-FR2 of the variable region of the reference antibody with a library of human FR1-CDR1-FR2 regions comprised of immunoglobulin sequences that are germline to create a library of hybrid variable regions, pairing the hybrid variable regions with a complementary variable region, and selecting an antibody having a detectable affinity for the target antigen; replacing the CDR2-FR3 of the variable region of the reference antibody with a library of human CDR2-FR3 regions comprised of immunoglobulin sequences that are germline to create a library of hybrid variable regions, pairing the hybrid variable regions with a complementary variable region, and selecting an antibody having a detectable affinity for the target antigen.
Combining selected FR1-CDR1-FR2 and CDR2-FR3 to yield higher-affinity reduced-immunogenicity antibody
Combining the FR1-CDR1-FR2 of the hybrid variable region of the antibody selected in (b) with the CDR2-FR3 of the hybrid variable region of the antibody selected in (c) to obtain an antibody with a human variable region V segment, which antibody has the binding specificity of the reference antibody and a higher binding affinity for the target antigen than the reference antibody and has a reduced potential for immunogenicity in humans.
Across the independent claims, the inventive coverage focuses on constructing hybrid V-regions by replacing specified FR-CDR segments or exchange cassettes of a reference antibody with human germline or human-antibody-gene-derived corresponding cassette or segment libraries, maintaining intact naturally occurring CDR/FR structure within the exchanged cassette, pairing with a complementary V-region, and selecting variants that retain binding specificity or detectable affinity while reducing potential for immunogenicity; in several independent-claim formulations, the resulting antibody is also required to have higher binding affinity than the reference antibody.
Stated Advantages
Retains the binding specificity of the reference antibody for the target antigen.
Reduced potential for immunogenicity in humans.
Higher binding affinity for the target antigen than the reference antibody.
Preserves binding specificity while engineering using human exchanged cassettes or segments.
Documented Applications
Engineering antibodies for a target antigen, including selecting antigen-binding hybrids with retained binding specificity and reduced potential for immunogenicity in humans.
Cytokine target antigen example reported as a target in the described experimental examples.
Interested in licensing this patent?