Nucleic acids coding for humanized antibodies for binding sphingosine-1-phosphate
View Patent ↗The present invention relates to anti-S1P agents, particularly humanized monoclonal antibodies (and antigen binding fragments thereof) specifically reactive with S1P, compositions containing such antibodies (or fragments), and the use of such antibodies (or fragments), for example, to treat diseases and conditions associated with aberrant levels of S1P.
1. An isolated, non-naturally occurring nucleic acid molecule that encodes an immunoglobulin heavy chain or an immunoglobulin light chain of an antibody or antigen binding fragment thereof that binds sphingosine-1-phosphate (S1P), wherein:
A. when the nucleic acid molecule encodes an immunoglobulin heavy chain, wherein the immunoglobulin heavy chain is selected from the group consisting of:
(i) an immunoglobulin heavy chain that comprises a first sequence of amino acid residues of sequence DHTIH (SEQ ID NO: 13), a second sequence of amino acid residues AISPRHDITKYNEMFRG (SEQ ID NO: 31), and a third sequence of amino acid residues of sequence GGFYGSTIWFDF (SEQ ID NO: 15);
(ii) an immunoglobulin heavy chain that comprises a heavy chain variable domain comprising a sequence of amino acid residues having an amino acid sequence:
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLE
WMGAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDT
AMYFCARGGFYGSTIWFDFWGQGTMVTVSS (SEQ ID NO: 32,
residues 20-140, inclusive); and
(iii) an immunoglobulin heavy chain that comprises a sequence of amino acid residues having an amino acid sequence:
(SEQ ID NO: 38)
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLEWMGA
ISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMYFCARGG
GFYGSTIWFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK,
residues 20-455, inclusive); and
and
B. when the nucleic acid molecule encodes an immunoglobulin light chain, the immunoglobulin light chain is selected from the group consisting of:
(i) an immunoglobulin light chain that comprises a first sequence of amino acid residues of sequence ITTTDIDDDMN (SEQ ID NO: 10), a second sequence of amino acid residues of sequence EGNILRP (SEQ ID NO: 11), a third sequence of amino acid residues of sequence LQSDNLPFT (SEQ ID NO: 12);
(ii) an immunoglobulin light chain that comprises a light chain variable domain comprising a sequence of amino acid residues having an amino acid sequence:
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLL
ISEGNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNL
PFTFGQGTKLEIK (SEQ ID NO: 33, residues 21-127,
inclusive); and
(iii) an immunoglobulin light chain that comprises a sequence of amino acid residues having an amino acid sequence:
(SEQ ID NO: 37)
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLLISE
GNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNLPFTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC,
residues 21-234, inclusive).
2. An expression cassette that comprises a promoter and an isolated, non-naturally occurring nucleic acid molecule according to claim 1 .
3. An engineered plasmid comprising an expression cassette according to claim 2 .
4. An engineered host cell transfected with an isolated, non-naturally occurring nucleic acid molecule according to claim 1 , optionally wherein the host cell is a non-human mammalian cell, optionally a CHO cell.
5. An engineered host cell transfected with a plasmid according to claim 3 , optionally wherein the host cell is a non-human mammalian cell, optionally a CHO cell.
6. An engineered plasmid comprising a first expression cassette and a second expression cassette, wherein each of the first and second expression cassettes comprises a promoter and an isolated, non-naturally occurring nucleic acid molecule according to claim 1 , wherein the first expression cassette directs the expression of an immunoglobulin heavy chain and the second expression cassette directs the expression of an immunoglobulin light chain.
7. An engineered host cell transfected with a plasmid according to claim 6 , optionally wherein the host cell is a non-human mammalian cell, optionally a CHO cell.
8. An engineered host cell transfected with a first plasmid and a second plasmid, wherein each of the first and second plasmids is a plasmid according to claim 2 , wherein the first plasmid comprises a first expression cassette that comprises a promoter and an isolated, non-naturally occurring nucleic acid molecule that encodes an immunoglobulin heavy chain and the second plasmid comprises a second expression cassette that comprises a promoter and an isolated, non-naturally occurring nucleic acid molecule that encodes an immunoglobulin light chain.
9. An isolated, non-naturally occurring nucleic acid molecule according to claim 1 that comprises a first nucleic acid molecule that encodes an immunoglobulin heavy chain and a second nucleic acid molecule that encodes an immunoglobulin light chain, wherein:
A. the immunoglobulin heavy chain encoded by the first nucleic acid molecule is selected from the group consisting of:
(i) an immunoglobulin heavy chain that comprises a first sequence of amino acid residues of sequence DHTIH (SEQ ID NO: 13), a second sequence of amino acid residues AISPRHDITKYNEMFRG (SEQ ID NO: 31), and a third sequence of amino acid residues of sequence GGFYGSTIWFDF (SEQ ID NO: 15);
(ii) an immunoglobulin heavy chain that comprises a heavy chain variable domain comprising a sequence of amino acid residues having an amino acid sequence: EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLE WMGAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDT AMYFCARGGFYGSTIWFDFWGQGTMVTVSS (SEQ ID NO: 32, residues 20-140, inclusive); and
(iii) an immunoglobulin heavy chain that comprises a sequence of amino acid residues having an amino acid sequence:
(SEQ ID NO: 38)
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLEWMGA
ISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMYFCARGG
GFYGSTIWFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK,
residues 20-455, inclusive); and
B. the immunoglobulin light chain encoded by the second nucleic acid molecule is selected from the group consisting of:
(i) an immunoglobulin light chain that comprises a first sequence of amino acid residues of sequence ITTTDIDDDMN (SEQ ID NO: 10), a second sequence of amino acid residues of sequence EGNILRP (SEQ ID NO: 11), a third sequence of amino acid residues of sequence LQSDNLPFT (SEQ ID NO: 12);
(ii) an immunoglobulin light chain that comprises a light chain variable domain comprising a sequence of amino acid residues having an amino acid sequence:
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLL
ISEGNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNL
PFTFGQGTKLEIK (SEQ ID NO: 33, residues 21-127,
inclusive); and
(iii) an immunoglobulin light chain that comprises a sequence of amino acid residues havina an amino acid sequence:
(SEQ ID NO: 37)
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLLISE
GNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNLPFTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC,
residues 21-234, inclusive).
10. An isolated, non-naturally occurring nucleic acid molecule according to claim 9 wherein (i) the first nucleic acid molecule is disposed in a first expression cassette that further comprises a promoter to direct expression of the immunoglobulin heavy chain encoded by the first nucleic acid molecule and (ii) the second nucleic acid molecule is disposed in a first expression cassette that further comprises a promoter to direct expression of the immunoglobulin heavy chain encoded by the first nucleic acid molecule.
11. An engineered plasmid comprising an isolated, non-naturally occurring nucleic acid molecule according to claim 10 .
12. An engineered host cell transfected with a plasmid according to claim 11 , optionally wherein the host cell is a non-human mammalian cell, optionally a CHO cell.
13. An engineered host cell that comprises an isolated, non-naturally occurring nucleic acid molecule according to claim 1 , wherein the engineered host cell is selected from the group consisting of an engineered host cell deposited under accession number ATCC PTA-8421 and an engineered host cell deposited under accession number ATCC PTA-8422.
14. An engineered plasmid that encodes an antibody that binds sphingosine-1-phosphate (S1P), wherein the plasmid comprises a first protein coding sequence that encodes an immunoglobulin light chain and a second protein coding sequence that encodes an immunoglobulin heavy chain, each of which immunoglobulin heavy and light chains can be expressed in a recombinant host cell, wherein:
A. the immunoglobulin heavy chain is selected from the group consisting of:
(i) an immunoglobulin heavy chain that comprises a first sequence of amino acid residues of sequence DHTIH (SEQ ID NO: 13), a second sequence of amino acid residues AISPRHDITKYNEMFRG (SEQ ID NO: 31), and a third sequence of amino acid residues of sequence GGFYGSTIWFDF (SEQ ID NO: 15);
(ii) an immunoglobulin heavy chain that comprises a heavy chain variable domain comprising a sequence of amino acid residues having an amino acid sequence:
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLE
WMGAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDT
AMYFCARGGFYGSTIWFDFWGQGTMVTVSS (SEQ ID NO: 32,
residues 20-140, inclusive); and
(iii) an immunoglobulin heavy chain that comprises a sequence of amino acid residues having an amino acid sequence:
(SEQ ID NO: 38)
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLEWMGA
ISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMYFCARGG
GFYGSTIWFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK,
residues 20-455, inclusive); and
B. the immunoglobulin light chain is selected from the group consisting of:
(i) an immunoglobulin light chain that comprises a first sequence of amino acid residues of sequence ITTTDIDDDMN (SEQ ID NO: 10), a second sequence of amino acid residues of sequence EGNILRP (SEQ ID NO: 11), a third sequence of amino acid residues of sequence LQSDNLPFT (SEQ ID NO: 12);
(ii) an immunoglobulin light chain that comprises a light chain variable domain comprising a sequence of amino acid residues having an amino acid sequence:
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLL
ISEGNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNL
PFTFGQGTKLEIK (SEQ ID NO: 33, residues 21-127,
inclusive); and
(iii) an immunoglobulin light chain that comprises a sequence of amino acid residues having an amino acid sequence:
(SEQ ID NO: 37)
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLLISE
GNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNLPFTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC,
residues 21-234, inclusive).
15. An engineered plasmid according to claim 14 wherein the first protein coding sequence further comprises a promoter to direct expression of the immunoglobulin light chain from the first protein coding sequence and the second protein coding sequence further comprises a promoter to direct expression of the immunoglobulin heavy chain from the second protein coding sequence.
16. An engineered host cell transfected with a plasmid according to claim 15 , optionally wherein the host cell is a non-human mammalian cell, optionally a CHO cell.
17. A recombinant host cell capable of expressing an antibody that binds sphingosine-1-phosphate (SIP), wherein:
A. the host cell includes a first nucleic acid molecule that encodes an immunoglobulin heavy chain of the antibody that binds S1P, wherein the immunoglobulin heavy chain is selected from the group consisting of:
(i) an immunoglobulin heavy chain that comprises a first sequence of amino acid residues of sequence DHTIH (SEQ ID NO: 13), a second sequence of amino acid residues AISPRHDITKYNEMFRG (SEQ ID NO: 31), and a third sequence of amino acid residues of sequence GGFYGSTIWFDF (SEQ ID NO: 15);
(ii) an immunoglobulin heavy chain that comprises a heavy chain variable domain comprising a sequence of amino acid residues having an amino acid sequence:
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLE
WMGAISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDT
AMYFCARGGFYGSTIWFDFWGQGTMVTVSS (SEQ ID NO: 32,
residues 20-140, inclusive); and
(iii) an immunoglobulin heavy chain that comprises a sequence of amino acid residues having an amino acid sequence:
(SEQ ID NO: 38)
EVQLVQSGAEVKKPGESLKISCQSFGYIFIDHTIHWMRQMPGQGLEWMGA
ISPRHDITKYNEMFRGQVTISADKSSSTAYLQWSSLKASDTAMYFCARGG
GFYGSTIWFDFWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK,
residues 20-455, inclusive); and
B. the host cell includes a second nucleic acid molecule that encodes an immunoglobulin light chain of the antibody that binds S1P, wherein the immunoglobulin light chain is selected from the group consisting of:
(i) an immunoglobulin light chain that comprises a first sequence of amino acid residues of sequence ITTTDIDDDMN (SEQ ID NO: 10), a second sequence of amino acid residues of sequence EGNILRP (SEQ ID NO: 11), a third sequence of amino acid residues of sequence LQSDNLPFT (SEQ ID NO: 12);
(ii) an immunoglobulin light chain that comprises a light chain variable domain comprising a sequence of amino acid residues having an amino acid sequence:
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLL
ISEGNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNL
PFTFGQGTKLEIK (SEQ ID NO: 33, residues 21-127,
inclusive); and
(iii) an immunoglobulin light chain that comprises a sequence of amino acid residues having an amino acid sequence:
(SEQ ID NO: 37)
ETTVTQSPSFLSASVGDRVTITCITTTDIDDDMNWFQQEPGKAPKLLISE
GNILRPGVPSRFSSSGYGTDFTLTISKLQPEDFATYYCLQSDNLPFTFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC,
residues 21-234, inclusive).