US2017022291A1PendingUtilityA1
Multispecific antibody analogs comprising a common light chain, and methods of their preparation and use
Est. expiryApr 1, 2034(~7.7 yrs left)· nominal 20-yr term from priority
C07K 2317/21C07K 2317/64C07K 16/32C07K 16/2863C07K 2317/522C07K 2317/31C07K 2317/56C07K 2317/35C07K 2317/565C07K 2317/526C07K 2317/515C07K 2317/24C07K 2317/55C07K 2317/92C07K 2317/524C07K 16/468C07K 2317/53C07K 2317/567C07K 16/065C07K 16/005
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Claims
Abstract
Multispecific antibody analogs that co-engage at least two different antigens or epitopes (also referred to targets, used interchangeably throughout), said analogs comprising a common light chain, are provided, as well as methods for their production and use.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method of making a multispecific antibody analog comprising at least two first antigen binding regions and at least two second antigen binding regions, said first and second antigen binding regions having a common light chain, wherein first antigen binding regions have a different antigen specificity than the second antigen binding regions, the method comprising;
i) obtaining at least one light chain from a first antigen binding region having specificity for the first antigen, wherein the first antigen binding region comprises said at least one light chain and a heavy chain; ii) obtaining heavy chains from the output of a selection performed from a naïve library against a second antigen; iii) preparing a restricted library comprising heavy chains obtained in step ii) and the at least one light chain obtained in step i); iv) performing a second selection against the second antigen from the library prepared in step iii); v) obtaining an multispecific antibody comprising the second antigen binding region from the selection performed in step iv), wherein the second antigen binding region comprises the at least one light chain obtained in step i); vi) incorporating the first antigen binding region and the second antigen binding region into a multispecific antibody format, wherein the format comprises: an IgG moiety comprising either: a) the first antigen binding region; or b) the second antigen binding region; and two Fab moieties, wherein each Fab moiety comprises either: a) the second antigen binding region; or b) the first antigen binding region; wherein the N-terminus of the heavy chain of one Fab moiety is linked to the C-terminus of the Fc region of one heavy chain of the IgG moiety via a linker moiety, and the N-terminus of the heavy chain of the other Fab moiety is linked to the C-terminus of the Fc region of the other heavy chain of the IgG moiety via a linker moiety; thereby generating the multispecific antibody analog.
2 . The method according to claim 1 , wherein each linker moiety independently comprises a peptide from 1 to 75 amino acids in length, inclusive.
3 . The method according to any one of claims 1 and 2 , wherein one or more of the linker moieties independently comprises at least one of the 20 naturally occurring amino acids.
4 . The method according to any one of claims 1 through 3 , wherein the one or more of the linker moieties independently comprises at least one non-natural amino acid incorporated by chemical synthesis, post-translational chemical modification or by in vivo incorporation by recombinant expression in a host cell.
5 . The method according to any one claims of 1 through 4 , wherein the one or more of the linker moieties independently comprises one or more amino acids selected from the group consisting of serine, glycine, alanine, proline, asparagine, glutamine, glutamate, aspartate, and lysine.
6 . The method according to any one of claims 1 through 5 , wherein the one or more of the linker moieties independently comprises a majority of amino acids that are sterically unhindered.
7 . The method according to any one of claims 1 through 6 , wherein the one or more of the linker moieties independently comprises one or more of the following: an acidic linker, a basic linker, and a structural motif.
8 . The method according to any one of claims 1 through 7 , wherein one or more of the linker moieties independently comprises: polyglycine, polyalanine, poly(Gly-Ala), or poly(Gly-Ser).
9 . The method according to any one of claims 1 through 8 , wherein one or more of the linker moieties independently comprises: a polyglycine selected from the group consisting of: (Gly)3 (SEQ ID NO: 1), (Gly)4 (SEQ ID NO: 2), and (Gly)5 (SEQ ID NO: 3).
10 . The method according to any one of claims 1 through 9 wherein one or more of the linker moieties independently comprises (Gly) 3 Lys(Gly) 4 (SEQ ID NO: 4); (Gly) 3 AsnGlySer(Gly) 2 (SEQ ID NO: 5); (Gly) 3 Cys(Gly) 4 (SEQ ID NO: 6); and GlyProAsnGlyGly (SEQ ID NO: 7).
11 . The method according to any one of claims 1 through 10 , wherein one or more of the linker moieties independently comprises a combination of Gly and Ala.
12 . The method according to any one of claims 1 through 11 , wherein one or more of the linker moieties independently comprises a combination of Gly and Ser.
13 . The method according to any one of claims 1 through 12 , wherein one or more of the linker moieties independently comprises a combination of:
Gly and Glu; or
Gly and Asp.
14 . The method according to any one of claims 1 through 13 , wherein one or more of the linker moieties independently comprises a combination of Gly and Lys.
15 . The method according to any one of claims 1 through 14 , wherein one or more of the linker moieties independently comprises a sequence selected from group consisting of: [Gly-Ser] n (SEQ ID NO: 8); [Gly-Gly-Ser] n (SEQ ID NO: 9); [Gly-Gly-Gly-Ser] n (SEQ ID NO: 10); [Gly-Gly-Gly-Gly-Ser] n (SEQ ID NO: 11); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 12); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 13); [Gly-Gly-Gly-Gly-Ser Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 14); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 15); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 16); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 17); and combinations thereof; where n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.
16 . The method according to any one of claims 1 through 15 , wherein one or more of the linker moieties independently comprises a sequence selected from the group consisting of: [Gly-Glu] n (SEQ ID NO: 18); [Gly-Gly-Glu] n (SEQ ID NO: 19); [Gly-Gly-Gly-Glu] n (SEQ ID NO: 20); [Gly-Gly-Gly-Gly-Glu] n (SEQ ID NO: 21); [Gly-Asp]n (SEQ ID NO: 22); [Gly-Gly-Asp] n (SEQ ID NO: 23); [Gly-Gly-Gly-Asp] n (SEQ ID NO: 24); [Gly-Gly-Gly-Gly-Asp] n (SEQ ID NO: 25); and combinations thereof; where n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.
17 . The method according to any one of claims 1 through 16 , wherein at least one of the first and second antigen binding regions comprises at least one humanized variable heavy domain or at least one humanized variable light domain.
18 . The method according to any one of claims 1 through 17 , wherein at least one of the first and second antigen binding regions comprises at least one complimentary determining region CDR that is derived from a non-human multispecific antibody or multispecific antibody fragment.
19 . The method according to any one of claims 1 through 18 , wherein at least one of the first and second antigen binding regions binds an epitope from a tumor associated antigen, a hormone receptor, a cytokine receptor, chemokine receptor, a growth factor receptor, an immune activating receptor, a hormone, a cytokine, a chemokine, a growth factor, a G protein-coupled receptor, or a transmembrane receptor.
20 . The method according to any one of claims 1 through 19 , wherein at least one of the first and second antigen binding regions binds a target associated with an autoimmune disorder, an inflammatory disorder, an oncological disorder, neuromuscular disorder, a neurodegenerative disorder, a metabolic disorder, or an infectious disease.
21 . The method according to any one of claims 1 through 20 , wherein the multispecific antibody analog binds at least two different targets.
22 . The method according to any one of claims 1 through 21 , wherein the multispecific analog binds at least three different targets.
23 . The method according to any one of claims 1 through 22 , wherein the multispecific antibody analog binds at least four different targets.
24 . The method according to any one of claims 1 through 23 , wherein the multispecific antibody analog binds at least one target monovalently.
25 . The method according to any one of claims 1 through 24 , wherein the multispecific antibody analog binds at least two targets monovalently.
26 . The multivalent multispecific antibody analog according to any one of claims 1 through 72 , wherein the multispecific antibody analog binds at least three targets monovalently.
27 . The method according to any one of claims 1 through 26 , wherein the multispecific antibody analog binds at least four targets monovalently.
28 . The method according to any one of claims 1 through 27 , wherein at least one of the antigen binding regions comprises or is derived from a non-human species.
29 . The method according to any one of claims 1 through 28 , wherein at least one of the antigen binding sites comprises a humanized variable domain or a humanized CDR.
30 . The method according to any one of claims 1 through 29 , wherein at least one VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 each independently selected from the following:
a VH CDR1 amino acid sequence selected from the group consisting of:
GSVSSGSYYWS;
(SEQ ID NO: 26)
GSISSGGYYWS;
(SEQ ID NO: 27)
GSINSSSYYWQ;
(SEQ ID NO: 28)
FTLSGDWIH;
(SEQ ID NO: 29)
FNIKDTYIH;
(SEQ ID NO: 30)
FSLTNYGVH;
(SEQ ID NO: 31)
GSISSGGDYWQ;
(SEQ ID NO: 32)
a VH CDR2 amino acid sequence selected from the group consisting of:
YIYYSGSTNYNPSLKS;
(SEQ ID NO: 33)
IIYYSGWTNYNPSLKS;
(SEQ ID NO: 34)
EIAYSGSTYYNPSLKS;
(SEQ ID NO: 35)
EISAAGGYTDYADSVKG;
(SEQ ID NO: 36)
RIYPTNGYTRYADSVKG;
(SEQ ID NO: 37)
VIWSGGNTDYNTPFTSR;
(SEQ ID NO: 38)
and
a VH CDR3 selected from the group consisting of:
ARTNLYSTPFDI;
(SEQ ID NO: 39)
ARGVGPDFWSGYSYSSYFDL;
(SEQ ID NO: 40)
ARGQQWAAFDI;
(SEQ ID NO: 41)
ARESRVSFEAAMDY;
(SEQ ID NO: 42)
SRWGGDGFYAMDY;
(SEQ ID NO: 43)
RALTYYDYEFAYW.
(SEQ ID NO: 44)
31 . The method according to any one of claims 1 through 30 , wherein at least one VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 each independently selected from the following:
a VL CDR1 amino acid sequence selected from the group consisting of:
RASQDISSWLA;
(SEQ ID NO: 45)
RASQAISSWLA;
(SEQ ID NO: 46)
RASQNIATDVA;
(SEQ ID NO: 47)
RASQDVNTAVA;
(SEQ ID NO: 48)
RASQSIGTNIH;
(SEQ ID NO: 49)
a VL CDR2 amino acid sequence selected from the group consisting of:
AASSLQS;
(SEQ ID NO: 50)
DASSLES;
(SEQ ID NO: 51)
AASSLQS;
(SEQ ID NO: 52)
SASFLYS;
(SEQ ID NO: 53)
YASESIS;
(SEQ ID NO: 54)
and
a VL CDR3 amino acid sequence selected from the group consisting of:
QQEHDFPWT;
(SEQ ID NO: 55)
HQYQSYSWT;
(SEQ ID NO: 56)
QQEHDFPWT;
(SEQ ID NO: 57)
QQSEPEPYT;
(SEQ ID NO: 58)
QQHYTTPPT;
(SEQ ID NO: 59)
QQNNNWPTT.
(SEQ ID NO: 60)
32 . The method according to any one of claims 1 through 31 , wherein the multispecific antibody analog comprises at least one heavy chain framework region that corresponds to or is derived from VH1-46, VH3-23, VH4-39, or VH4-61, and wherein at least one light chain framework region that corresponds to or is derived from VK1-05, VK1-12, or VK3-11.
33 . The method according to any one of claims 1 through 32 , wherein the multispecific antibody analog comprises a VH region that comprises an amino acid sequence selected from the group consisting of:
(SEQ ID NO: 99)
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWI
GYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCART
NLYSTPFDIWGQGTMVTVSS;
(SEQ ID NO: 62)
QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWI
GIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARG
VGPDFWSGYSYSSYFDLWGRGTLVTVSS;
(SEQ ID NO: 63)
QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWI
GEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
QQWAAFDIWGQGTMVTVSS;
(SEQ ID NO: 64)
EVQLVESGGGLVQPGGSLRLSCAASGFTLSGDWIHWVRQAPGKGLEWVGE
ISAAGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARES
RVSFEAAMDYWGQGTLVTVSS;
(SEQ ID NO: 65)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSS;
(SEQ ID NO: 66)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSDDTAIYYCARALT
YYDYEFAYWGQGTLVTVSS;
and
(SEQ ID NO: 67)
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGGDYWQWIRQPPGKGLEWI
GEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG
QQWAAFDIWGQGTMVTVSS.
34 . The method according to any one of claims 1 through 33 , wherein the multispecific antibody analog comprises a VL region that comprises an amino acid sequence selected from the group consisting of:
(SEQ ID NO: 68)
DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGG
GTKVEIK;
(SEQ ID NO: 69)
DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYD
ASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGG
GTKVEIK;
(SEQ ID NO: 70)
DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGG
GTKVEIK;
(SEQ ID NO: 71)
DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTFGQ
GTKVEIK;
(SEQ ID NO: 72)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEIK;
and
(SEQ ID NO: 73)
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPRLLIKY
ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA
GTKLELK.
35 . The method according to any one of claims 1 through 34 , wherein the multispecific antibody analog comprises a polypeptide comprising, from N-terminus to C-terminus, a first VH region, a CH1, a hinge region, a CH2 region, a CH3 region, a second VH region, and a CH1 region, the amino acid sequence of which comprises an amino acid sequence selected from the group consisting of:
(SEQ ID NO: 74)
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGK
GLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVY
YCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGKGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSIN
SSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQF
SLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC;
(SEQ ID NO: 75)
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGK
GLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVY
YCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSIS
SGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQF
SLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
C;
(SEQ ID NO: 76)
QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGK
GLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVY
YCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTC
TVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTIS
VDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
C;
(SEQ ID NO: 77)
QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGK
GLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVY
YCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSS
GSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFS
LKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC.
36 . The method according to any one of claims 1 through 35 , wherein the multispecific antibody analog comprises four copies of a polypeptide comprising, from N-terminus to C-terminus, a VL region, and a CK region, and wherein said polypeptide heterodimerizes with compatible VH regions of the multispecific antibody analog, the amino acid sequence of which comprises an amino acid sequence selected from the group consisting of:
(SEQ ID NO: 78)
DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPK
LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFP
WTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC;
(SEQ ID NO: 79)
DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPK
LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYS
WTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC;
(SEQ ID NO: 80)
DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPK
LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEP
YTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC;
(SEQ ID NO: 81)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK
LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP
PTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC;
and
(SEQ ID NO: 82)
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPR
LLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWP
TTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC.
37 . The method according to any one of claims 1 through 36 wherein the multispecific antibody analog has binding specificity for an oncology target.
38 . The method according to any one of claims 1 through 37 , wherein the multispecific antibody analog has binding specificity for one or more targets selected from the group consisting of: EGFR, HER2, and HER3.
39 . The method according to any one of claims 1 through 38 , wherein the multispecific antibody analog has binding specificity for EGFR and HER2.
40 . The method according to any one of claims 1 through 39 , wherein the polypeptide multispecific antibody analog has binding specificity for EGFR and HER3.
41 . The method according to any one of claims 1 through 40 , wherein the multispecific antibody analog has binding specificity for EGFR, HER2, and HER3.
42 . The method according to any one of claims 1 through 41 , wherein the multispecific antibody analog is selected from the group consisting of the multispecific antibody analogs described in the Examples.
43 . The method according to any one of claims 1 through 42 , wherein the multispecific antibody analog is expressed by a prokaryotic host cell or a eukaryotic host cell.
44 . The method according to any one of claims 1 through 43 , wherein the multispecific antibody analog is expressed by a eukaryotic host cell.
45 . The method according to any one of claims 1 through 44 , wherein the multispecific antibody analog is expressed by a eukaryotic host cell selected from the group consisting of: yeast cells; Saccharomyces cerevisiae cells; Pichia cells; mammalian cells; Chinese hamster ovary (CHO) cells; human embryonic kidney (HEK) cells; insect cells; Sf9 cells; and Sf21 cells.
46 . A multispecific antibody analog prepared by performing a method according to any one of claims 1 through 44 .
47 . A multispecific antibody analog comprising at least two first antigen binding regions and at least two second antigen binding regions, said first and second antigen binding regions having a common light chain, wherein first antigen binding regions have a different antigen specificity than the second antigen binding regions.
48 . A multispecific antibody analog comprising at least two first antigen binding regions and at least two second antigen binding regions, said first and second antigen binding regions having a common light chain, wherein first antigen binding regions have a different antigen specificity than the second antigen binding regions, wherein the analog prepared is by a method comprising:
i) obtaining at least one light chain from a first antigen binding region having specificity for the first antigen, wherein the first antigen binding region comprises said at least one light chain and a heavy chain; ii) obtaining heavy chains from the output of a selection performed from a naïve library against a second antigen; iii) preparing an multispecific antibody library comprising heavy chains obtained in step ii) and the at least one light chain obtained in step i); iv) performing a second selection against the second antigen from the library prepared in step iii); v) obtaining an multispecific antibody comprising the second antigen binding region from the selection performed in step iv); vi) incorporating the first antigen binding region and the second antigen binding region into a multispecific antibody format, wherein the format comprises: an IgG moiety comprising either: a) the first antigen binding region; or b) the second antigen binding region; and two Fab moieties, wherein each Fab moiety comprises either: a) the second antigen binding region; or b) the first antigen binding region; wherein the N-terminus of the heavy chain of one Fab moiety is linked to the C-terminus of the Fc region of one heavy chain of the IgG moiety via a linker moiety, and the N-terminus of the heavy chain of the other Fab moiety is linked to the C-terminus of the Fc region of the other heavy chain of the IgG moiety via a linker moiety; thereby generating the multispecific antibody analog.
49 . The multispecific antibody analog to claim 48 , wherein each linker moiety independently comprises a peptide from 1 to 75 amino acids in length, inclusive.
50 . The multispecific antibody analog according to any one of claims 48 through 49 , wherein one or more of the linker moieties independently comprises at least one of the 20 naturally occurring amino acids.
51 . The multispecific antibody analog according to any one of claims 48 through 50 , wherein the one or more of the linker moieties independently comprises at least one non-natural amino acid incorporated by chemical synthesis, post-translational chemical modification or by in vivo incorporation by recombinant expression in a host cell.
52 . The multispecific antibody analog according to any one claims of 48 through 51 , wherein the one or more of the linker moieties independently comprises one or more amino acids selected from the group consisting of serine, glycine, alanine, proline, asparagine, glutamine, glutamate, aspartate, and lysine.
53 . The multispecific antibody analog according to any one of claims 48 through 52 , wherein the one or more of the linker moieties independently comprises a majority of amino acids that are sterically unhindered.
54 . The multispecific antibody analog according to any one of claims 48 through 53 , wherein the one or more of the linker moieties independently comprises one or more of the following: an acidic linker, a basic linker, and a structural motif.
55 . The multispecific antibody analog according to any one of claims 48 through 54 , wherein one or more of the linker moieties independently comprises: polyglycine, polyalanine, poly(Gly-Ala), or poly(Gly-Ser).
56 . The multispecific antibody analog according to any one of claims 48 through 55 , wherein one or more of the linker moieties independently comprises: a polyglycine selected from the group consisting of: (Gly)3 (SEQ ID NO: 1), (Gly)4 (SEQ ID NO: 2), and (Gly)5 (SEQ ID NO: 3).
57 . The multispecific antibody analog according to any one of claims 48 through 56 wherein one or more of the linker moieties independently comprises (Gly) 3 Lys(Gly) 4 (SEQ ID NO: 4); (Gly) 3 AsnGlySer(Gly) 2 (SEQ ID NO: 5); (Gly) 3 Cys(Gly) 4 (SEQ ID NO: 6); and GlyProAsnGlyGly (SEQ ID NO: 7).
58 . The multispecific antibody analog according to any one of claims 48 through 57 , wherein one or more of the linker moieties independently comprises a combination of Gly and Ala.
59 . The multispecific antibody analog according to any one of claims 48 through 58 , wherein one or more of the linker moieties independently comprises a combination of Gly and Ser.
60 . The multispecific antibody analog according to any one of claims 48 through 59 , wherein one or more of the linker moieties independently comprises a combination of:
Gly and Glu; or
Gly and Asp.
61 . The multispecific antibody analog according to any one of 48 through 60 , wherein one or more of the linker moieties independently comprises a combination of Gly and Lys.
62 . The multispecific antibody analog according to any one of claims 48 through 61 , wherein one or more of the linker moieties independently comprises a sequence selected from group consisting of: [Gly-Ser] n (SEQ ID NO: 8); [Gly-Gly-Ser] n (SEQ ID NO: 9); [Gly-Gly-Gly-Ser] n (SEQ ID NO: 10); [Gly-Gly-Gly-Gly-Ser] n (SEQ ID NO: 11); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 12); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 13); [Gly-Gly-Gly-Gly-Ser Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 14); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 15); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 16); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly] n (SEQ ID NO: 17); and combinations thereof; where n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.
63 . The multispecific antibody analog according to any one of claims 48 through 62 , wherein one or more of the linker moieties independently comprises a sequence selected from the group consisting of: [Gly-Glu] n (SEQ ID NO: 18); [Gly-Gly-Glu] n (SEQ ID NO: 19); [Gly-Gly-Gly-Glu] n (SEQ ID NO: 20); [Gly-Gly-Gly-Gly-Glu] n (SEQ ID NO: 21); [Gly-Asp]n (SEQ ID NO: 22); [Gly-Gly-Asp] n (SEQ ID NO: 23); [Gly-Gly-Gly-Asp] n (SEQ ID NO: 24); [Gly-Gly-Gly-Gly-Asp] n (SEQ ID NO: 25); and combinations thereof; where n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.
64 . The multispecific antibody analog according to any one of claims 48 through 63 , wherein at least one of the first and second antigen binding regions comprises at least one humanized variable heavy domain or at least one humanized variable light domain.
65 . The multispecific antibody analog according to any one of claims 48 through 64 , wherein at least one of the first and second antigen binding regions comprises at least one complimentary determining region CDR that is derived from a non-human multispecific antibody or multispecific antibody fragment.
66 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 65 , wherein at least one of the first and second antigen binding regions binds an epitope from a tumor associated antigen, a hormone receptor, a cytokine receptor, chemokine receptor, a growth factor receptor, an immune activating receptor, a hormone, a cytokine, a chemokine, a growth factor, a G protein-coupled receptor, or a transmembrane receptor.
67 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 66 , wherein at least one of the first and second antigen binding regions binds a target associated with an autoimmune disorder, an inflammatory disorder, an oncological disorder, neuromuscular disorder, a neurodegenerative disorder, a metabolic disorder, or an infectious disease.
68 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 67 , wherein the multispecific antibody analog binds at least two different targets.
69 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 68 , wherein the multispecific analog binds at least three different targets.
70 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 69 , wherein the multispecific antibody analog binds at least four different targets.
71 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 70 , wherein the multispecific antibody analog binds at least one target monovalently.
72 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 71 , wherein the multispecific antibody analog binds at least two targets monovalently.
73 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 72 , wherein the multispecific antibody analog binds at least three targets monovalently.
74 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 73 , wherein the multispecific antibody analog binds at least four targets monovalently.
75 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 74 , wherein at least one of the antigen binding regions comprises or is derived from a non-human species.
76 . The method according to any one of claims 1 through 45 , or the multispecific antibody analog according to any one of claims 46 through 75 , wherein at least one of the antigen binding sites comprises a humanized variable domain or a humanized CDR.
77 . A method if obtaining or identifying one or more common light chains for use in preparing a multispecific antibody or multispecific antibody analog, the method comprising:
i) performing a first selection against a first antigen from a first library and obtaining one or more light chains from the output that has specificity for the first antigen; ii) performing a second selection against a second antigen from a second library and obtaining heavy chains from the output that has specificity for the second antigen; iii) generating a restricted library comprising the one or more light chains obtained in step i) and the heavy chains obtained in step ii); iv) performing a third selection against the first antigen from the restricted library generated in step iii) and obtaining one or more antibodies form the output of the third selection, wherein the one or more antibodies comprise one or more light chains that each have specificity for the first antigen and the second antigen; thereby obtaining or identifying the one or more common light chains.
78 . The method according to claim 77 , wherein:
a) the first library comprises a naïve library; b) the second library comprises a naïve library; or c) the first library comprises a naïve library and the second library comprises a naïve library.
79 . The method according to claim 77 or claim 78 , wherein the method further comprises:
a) performing a subsequent selection against the first antigen from a maturation library;
b) performing a subsequent selection against the second antigen from a maturation library; or
c) performing a subsequent selection against the first antigen from a maturation library and performing a subsequent selection against the second antigen from a maturation library;
after performing:
a) step i;
b) step ii
c) step iii; and/or
d) step iv.
80 . A method of making a multispecific antibody analog comprising
contacting the one or more common light chains obtained or identified according to any one of claims 77 through 79 with: i) a first polypeptide comprising a heavy chain that has specificity for the first antigen; and ii) A second polypeptide comprising a heavy chain that has specificity for the second antigen.
81 . The method according to claim 81 , wherein the one or more common light chains, the first polypeptide, and the second polypeptide are expressed by host cells.
82 . The method according to claim 81 or claim 82 , wherein the one or more common light chains, the first polypeptide, and the second polypeptide are expressed by the same host cell.
83 . A multispecific antibody analog comprising a common light chain obtained or identified by performing a method according to any one of claims 77 through 81 .Cited by (0)
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