MULTISPECIFIC ANTIBODIES, COMPOSITIONS COMPRISING THE SAME, AND VECTORS AND USES THEREOF
The present disclosure provides multispecific antibodies having increased in vivo sustainability, the multispecific antibodies comprising one or more bioactive effector moieties linked to either or both of an N-terminal and a C-terminal of an antigen binding fragment Fab that binds to human serum albumin.
1 . A multispecific antibody comprising a structural formula of:
wherein the antigen binding fragment (Fab) is a serum albumin Fab;
wherein R 1 and R 2 are bioactive effector moieties linked to an N-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
wherein R 3 and R 4 are bioactive effector moieties linked to a C-terminus of the Fab, each linked to a heavy chain variable domain or a light chain variable domain of the Fab;
wherein m is 0 or an integer of 1 or greater; and
wherein n is 0 or an integer of 1 or greater.
2 . The antibody of claim 1 , wherein R 1 and R 2 are same or different single-chain variable fragments (scFv).
3 . The antibody of claim 1 or 2 , wherein R 3 and R 4 are same or different Fv fragments or disulfide-stabilized Fv (dsFv) fragments.
4 . The antibody of any of claims 1 - 3 , wherein each of R 1 , R 2 , R 3 , and R 4 is linked to the Fab by one or more linkers.
5 . The antibody of any of claims 1 - 4 , wherein the Fab comprises a heavy chain variable domain comprising
(a) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61),
a heavy chain CDR2 comprising the amino acid sequence of WINTYSGGTKYAQKFQG (SEQ ID NO:62), and
a heavy chain CDR3 comprising the amino acid sequence of
(SEQ ID NO: 63)
LGHCQRGICSDALDT
(b) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61),
a heavy chain CDR2 comprising the amino acid sequence of RINTYNGNTGYAQRLQG (SEQ ID NO:64), and
a heavy chain CDR3 comprising the amino acid sequence of
(SEQ ID NO: 63)
LGHCQRGICSDALDT
(c) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of NYGIH (SEQ ID NO:65),
a heavy chain CDR2 comprising the amino acid sequence of SISYDGSNKYYADSVKG (SEQ ID NO:66), and
a heavy chain CDR3 comprising the amino acid sequence of DVHYYGSGSYYNAFDI (SEQ ID NO:67;
(SEQ ID NO: 67)
DVHYYGSGSYYNAFDI
(d) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:68),
a heavy chain CDR2 comprising the amino acid sequence of VISHDGGFQYYADSVKG (SEQ ID NO:69), and
a heavy chain CDR3 comprising the amino acid sequence of
(SEQ ID NO: 70)
AGWLRQYGMDV
(e) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYWIA (SEQ ID NO:71),
a heavy chain CDR2 comprising the amino acid sequence of MIWPPDADARYSPSFQG (SEQ ID NO:72), and
a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:73); or
(f) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74),
a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and
a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76).
6 . The antibody of any of claims 1 - 5 , wherein the Fab comprises a light chain variable domain comprising
(g) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSISRYLN (SEQ ID NO:77),
a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:78), and
a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:79);
(h) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO:80),
a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:81), and
a light chain CDR3 comprising the amino acid sequence of QQSYSTPPYT (SEQ ID NO:82);
(i) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSIFNYVA (SEQ ID NO:83),
a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:84), and
a light chain CDR3 comprising the amino acid sequence of QQRSKWPPTWT (SEQ ID NO:85);
(j) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASETVSSRQLA (SEQ ID NO:86),
a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and
a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:88);
(k) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVSSSSLA (SEQ ID NO:89),
a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and
a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:90); or
(l) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91),
a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and
a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
7 . The antibody of any of claims 1 - 6 , wherein the Fab comprises
a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76), and
a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
8 . The antibody of any of claims 1 - 7 , wherein the Fab comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99.
9 . The antibody of any of claims 1 - 8 , wherein the Fab comprises a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105.
10 . The antibody of any of claims 1 - 9 , wherein the Fab comprises a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99, and a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105, respectively.
11 . The antibody of any of claims 1 - 10 , wherein the Fab comprises a heavy chain domain comprising an amino acid sequence of SEQ ID NO:45 (V H −CH1 domain) and a light chain domain comprising an amino acid sequence of SEQ ID NO:46 (V L −C L domain).
12 . The antibody of any of claims 1 - 11 , wherein each linker comprises 1 to 20 amino acids.
13 . The antibody of any of claims 1 - 12 , wherein each linker comprises an amino acid sequence having at least 90% identity to SEQ ID NO:3 or SEQ ID NO:4.
14 . The antibody of any of claims 1 - 13 , wherein each linker comprises an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
15 . The antibody of any of claims 1 - 14 , wherein each of the R 1 and R 2 is an anti-CD40L hu5c8 scFv.
16 . The antibody of any of claims 1 - 15 , wherein each of the R 1 and R 2 is an anti-CD40L hu5c8 scFv comprising an amino acid sequence having at least 80% identity to SEQ ID NO:47 or SEQ ID NO:48.
17 . The antibody of any of claims 1 - 16 , wherein each of the R 1 and R 2 is an anti-CD40L hu5c8 scFv comprising an amino acid sequence of SEQ ID NO:47 or SEQ ID NO:48.
18 . The antibody of any of claims 1 - 17 , wherein each of R 3 and R 4 is one or more bioactive effector moieties selected from the group consisting of anti-TNF-α Fv, anti-TNF-α disulfide-stabilized Fv (dsFv), anti-TL-23 Fv, anti-TL-23 dsFv, anti-IFNAR1, and anti-IFNAR1 dsFv.
19 . The antibody of any of claims 1 - 18 , wherein each of R 3 and R 4 is one or more bioactive effector moieties comprising an anti-TNF-α Fv comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:49 and a light chain amino acid sequence having 80% identity to SEQ ID NO:50, anti-TNF-α disulfide-stabilized Fv (dsFv) comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:51 and a light chain amino acid sequence having 80% identity to SEQ ID NO:52, anti-TL-23 Fv comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:53 and a light chain amino acid sequence having 80% identity to SEQ ID NO:54, anti-TL-23 dsFv comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:55 and a light chain amino acid sequence having 80% identity to SEQ ID NO:56, anti-IFNAR1 comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:57 and a light chain amino acid sequence having 80% identity to SEQ ID NO:58, and/or anti-IFNAR1 dsFv comprising a heavy chain amino acid sequence having 80% identity to SEQ ID NO:59 and a light chain amino acid sequence having 80% identity to SEQ ID NO:60.
20 . The antibody of any of claims 1 - 19 , wherein each of R 3 and R 4 is one or more bioactive effector moieties comprising an anti-TNF-α Fv comprising a heavy chain of SEQ ID NO:49 and a light chain of SEQ ID NO:50, anti-TNF-α disulfide-stabilized Fv (dsFv) comprising a heavy chain of SEQ ID NO:51 and a light chain of SEQ ID NO:52, anti-IL-23 Fv comprising a heavy chain of SEQ ID NO:53 and a light chain of SEQ ID NO:54, anti-IL-23 dsFv comprising a heavy chain of SEQ ID NO:55 and a light chain of SEQ ID NO:56, anti-IFNAR1 comprising a heavy chain of SEQ ID NO:57 and a light chain of SEQ ID NO:58, and/or anti-IFNAR1 dsFv comprising a heavy chain of SEQ ID NO:59 and a light chain of SEQ ID NO:60.
21 . A composition comprising the multispecific antibody of any of claims 1 - 20 and an excipient.
22 . A pharmaceutical composition comprising the multispecific antibody of any of claims 1 - 20 and a pharmaceutically accepted excipient.
23 . A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 22 to the subject.
24 . An expression vector comprising:
(a) a promoter,
(b) a first nucleic acid molecule encoding an antigen binding fragment (Fab) that binds to serum albumin, and
(c) a second nucleic acid molecule encoding a bioactive effector moiety and a linker,
wherein the promoter, the first nucleic acid sequence, and the second nucleic acid molecules are operably linked.
25 . The vector of claim 24 , wherein the second nucleic acid molecule encodes 2 or more bioactive effector moieties and linkers.
26 . The vector of claim 24 or 25 , wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising
(a) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61),
a heavy chain CDR2 comprising the amino acid sequence of WINTYSGGTKYAQKFQG (SEQ ID NO:62), and
a heavy chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO:63;
(SEQ ID NO: 63)
LGHCQRGICSDALDT
(b) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:61),
a heavy chain CDR2 comprising the amino acid sequence of RINTYNGNTGYAQRLQG (SEQ ID NO:64), and
a heavy chain CDR3 comprising the amino acid sequence of LGHCQRGICSDALDT (SEQ ID NO:63;
(SEQ ID NO: 63)
LGHCQRGICSDALDT
(c) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of NYGIH (SEQ ID NO:65),
a heavy chain CDR2 comprising the amino acid sequence of SISYDGSNKYYADSVKG (SEQ ID NO:66), and
a heavy chain CDR3 comprising the amino acid sequence of DVHYYGSGSYYNAFDI (SEQ ID NO:67;
(SEQ ID NO: 67)
DVHYYGSGSYYNAFDI
(d) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of SYAMS (SEQ ID NO:68),
a heavy chain CDR2 comprising the amino acid sequence of VISHDGGFQYYADSVKG (SEQ ID NO:69), and
a heavy chain CDR3 comprising the amino acid sequence of
(SEQ ID NO: 70)
AGWLRQYGMDV
(e) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYWIA (SEQ ID NO:71),
a heavy chain CDR2 comprising the amino acid sequence of MIWPPDADARYSPSFQG (SEQ ID NO:72), and
a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:73); or
(f) a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74),
a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and
a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76).
27 . The vector of any of claims 24 - 26 , wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising
(g) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSISRYLN (SEQ ID NO:77),
a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:78), and
a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:79);
(h) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO:80),
a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:81), and
a light chain CDR3 comprising the amino acid sequence of QQSYSTPPYT (SEQ ID NO:82);
(i) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSIFNYVA (SEQ ID NO:83),
a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:84), and
a light chain CDR3 comprising the amino acid sequence of QQRSKWPPTWT (SEQ ID NO:85);
(j) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASETVSSRQLA (SEQ ID NO:86),
a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and
a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:88);
(k) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVSSSSLA (SEQ ID NO:89),
a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:87), and
a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:90); or
(l) a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91),
a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and
a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
28 . The vector of any of claims 24 - 27 , wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising
a heavy chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of AYSMN (SEQ ID NO:74), a heavy chain CDR2 comprising the amino acid sequence of SISSSGRYIHYADSVKG (SEQ ID NO:75), and a heavy chain CDR3 comprising the amino acid sequence of ETVMAGKALDY (SEQ ID NO:76), and
a light chain complementarity determining domain 1 (CDR1) comprising the amino acid sequence of RASQSVGSNLA (SEQ ID NO:91), a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:92), and a light chain CDR3 comprising the amino acid sequence of QQYYSFLAKT (SEQ ID NO:93).
29 . The vector of any of claims 24 - 28 , wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99.
30 . The vector of any of claims 24 - 29 , wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105.
31 . The vector of any of claims 24 - 30 , wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:94, 95, 96, 97, 98, or 99, and a light chain variable domain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:100, 101, 102, 103, 104, or 105, respectively.
32 . The vector of any of claims 24 - 31 , wherein the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab comprising a heavy chain domain comprising an amino acid sequence of SEQ ID NO:45 (V H −C H1 domain) and a light chain domain comprising an amino acid sequence of SEQ ID NO:46 (V L −C L domain).
33 . The vector of any of claims 24 - 32 , wherein the bioactive effector moieties are anti-TNF-α Fv, anti-TNF-α dsFv, anti-IL-23 Fv, anti-IL-23 dsFv, anti-IFNAR1 Fv, and/or anti-IFNAR1 dsFv.
34 . The vector of any of claims 23 - 33 , wherein the second nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence of one or more of SEQ ID NOs: 49-60.