CN117751142A - Treatment of cardiovascular diseases with TREM-1 antigen binding proteins - Google Patents

Treatment of cardiovascular diseases with TREM-1 antigen binding proteins Download PDF

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CN117751142A
CN117751142A CN202280053372.3A CN202280053372A CN117751142A CN 117751142 A CN117751142 A CN 117751142A CN 202280053372 A CN202280053372 A CN 202280053372A CN 117751142 A CN117751142 A CN 117751142A
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seq
amino acid
hcdr
lcdr
heavy chain
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S·谢特利
S·杰克逊
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Amgen Inc
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Amgen Inc
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Priority claimed from PCT/US2022/034834 external-priority patent/WO2022272018A1/en
Publication of CN117751142A publication Critical patent/CN117751142A/en
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Abstract

The present disclosure relates generally to methods of treating cardiovascular diseases (e.g., atherosclerosis or myocardial infarction) using antigen binding proteins that bind to TREM-1 and compositions thereof.

Description

Treatment of cardiovascular diseases with TREM-1 antigen binding proteins
Cross Reference to Related Applications
The present application claims the benefit of priority from U.S. provisional patent application number 63/215,260 filed on 25 th 6 of 2021 and U.S. provisional patent application number 63/353,223 filed on 17 th 6 of 2022, which are hereby incorporated by reference in their entireties.
Technical Field
The present disclosure relates to antigen binding portions specific for myeloid cell trigger receptor 1 (TREM-1) and compositions thereof for use in the treatment of cardiovascular disorders, such as atherosclerosis or Myocardial Infarction (MI).
Incorporation of electronically submitted materials by reference
A sequence listing as part of the present disclosure is presented concurrently with the present specification in the form of a text file. The text file containing the sequence listing is named "56967p2_seqlising. Txt", created at 2022, 6, 17 and is 1368364 bytes in size. The subject matter of the sequence listing is incorporated herein by reference in its entirety.
Background
Myeloid cell trigger receptor 1 (TREM 1, TREM-1, cd 354) is a member of the Ig family expressed in neutrophils, monocytes and macrophages. TREM-1 is a mediator of the myeloid cellular immune response, inducing inflammatory cytokines including IL-8, MCP/CCL2, and tumor necrosis factor alpha (TNF alpha) by TREM-1 activation. PGLYRP1 (peptidoglycan recognition protein 1) has recently been reported to be a ligand for TREM-1 (Read,J.Immunol [ journal of immunology ]]194:1417-1421,2015) and potentially other ligands remain to be determined. TREM-1 is associated with up-regulation of inflammatory responses in sepsis (Ford et al Curr Op Immunol. [ current point of immunology]2009; 21:38-46), and blocking TREM-1 activity by TREM-1-Fc or an antagonistic peptide (e.g., LP 17) reduced endotoxin effects in sepsis animals (Ford, supra, qian et al, J.int J Clin Exp Med. [ J.International journal of clinical and experimental medicine]2014 7 (7):1650-8). TREM-1 knockout mice are viable and protected against DSS colitis and T cell transfer colitis (Weber,PLoS Pathog [ public science library-pathogen ]],10(1):1003900,2014)。
Treatment with TREM-1-Fc fusion increased survival and decreased TNFα induction following LPS challenge in mice (Bouchon, nature, 410:1103-7,2001). anti-TREM-1 antibodies have been reported to reduce the secretion of inflammatory cytokines by lamina propria cells isolated from IBD patients under stimulation of the TREM-1 agonist PGLYRP-1/peptidoglycan (Brynjolfsson et al, inflamm Bowel Dis [ inflammatory bowel disease ]22 (8): 1803-11, 2016). In addition, nanganibotide is a chemically synthesized 12L-amino acid peptide derived from the protein TREM-like transcript-1 (TLT-1) and has been studied for the treatment of septic shock (Cuvier et al, br J Clin Pharmacol [ J. Clin. Clinical pharmacology ]2018, month 10; 84 (10): 2270-2279). Although TREM-1 is expressed primarily on monocytes/macrophages and neutrophils, it is also found on bronchial, corneal, gastric epithelial and hepatic endothelial cells during inflammation.
Disclosure of Invention
The present disclosure provides antigen binding proteins that are specific for human myeloid cell trigger receptor 1 (TREM-1) and useful for the treatment of cardiovascular diseases.
Provided herein are methods of treating a cardiovascular disease (e.g., atherosclerosis or myocardial infarction) comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein that binds to myeloid cell triggering receptor 1 (TREM-1), the antigen binding protein comprising:
a. a light chain variable domain comprising:
i. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, and 544;
light chain CDR2 comprising an amino acid sequence selected from SEQ ID NOs 11, 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271 and 545;
light chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272 and 546; and
b. a heavy chain variable domain comprising:
i. a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NOs 16, 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276 and 550;
Heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277 and 551; and
heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278 and 552.
In various embodiments, the antigen binding protein comprises:
the light chain CDR1 sequences set forth in SEQ ID NO. 10, 30, 50, 90, 130, 150, or 270;
the light chain CDR2 sequences set forth in SEQ ID NO. 11, 31, 51, 91, 131, 151, or 271;
the light chain CDR3 sequences set forth in SEQ ID NO. 12, 32, 52, 92, 132, 152, or 272;
the heavy chain CDR1 sequences set forth in SEQ ID NO. 16, 36, 56, 96, 136, 156, or 276;
the heavy chain CDR2 sequences set forth in SEQ ID NO 17, 37, 57, 97, 137, 157, or 277; and
the heavy chain CDR3 sequences set forth in SEQ ID NO. 18, 38, 58, 98, 138, 158, or 278.
In various embodiments, the antigen binding protein comprises:
the light chain CDR1 sequence set forth in SEQ ID NO. 30 or 90;
the light chain CDR2 sequences set forth in SEQ ID NO. 31 or 91;
the light chain CDR3 sequences set forth in SEQ ID NO. 32 or 92;
The heavy chain CDR1 sequences set forth in SEQ ID NO. 36 or 096;
e.the heavy chain CDR2 sequences set forth in SEQ ID NO 37 or 97; and
the heavy chain CDR3 sequences set forth in SEQ ID NO. 38 or 98.
Consensus sequences of the TREM-1 antibody/antigen binding protein heavy and light chain CDRs and/or variable region sequences disclosed herein are also contemplated. For example, in various embodiments, a TREM-1 antibody comprises an antigen binding domain comprising a sequence having a light chain variable region comprising an LCDR1 amino acid sequence selected from the group consisting of:
X 1 ASQSX 2 X 3 X 4 NLA (SEQ ID NO: 553), wherein X 1 Is R or Q, wherein X 2 Is V or I, wherein X 3 Is N or S, and wherein X 4 S, H, I, V or a;
QASX 1 DIX 2 X 3 X 4 LN (SEQ ID NO: 558), wherein X 1 Is R or Q, wherein X 2 R, S, N or F, where X 3 Is K or N, and wherein X 4 H, Y or D;
RASQSVNSNLA(SEQ ID NO:566)；
QASQDIRKHLN(SEQ ID NO:567)；
RASQDISSNLN(SEQ ID NO:568)；
QASQDIHLN(SEQ ID NO:569)；
RASQGIRKWLA(SEQ ID NO:570)；
RASQSVNSNLA (SEQ ID NO: 571) and
SGDKLGERVS(SEQ ID NO:572)。
in various embodiments, the TREM-1 antigen binding protein comprises an antigen binding domain comprising a sequence having a light chain variable region comprising an LCDR2 amino acid sequence selected from the group consisting of:
GAX 1 X 2 RAT (SEQ ID NO: 554), wherein X 1 Is S or Y, and wherein X 2 Is T or I;
X 1 X 2 X 3 X 4 LET (SEQ ID NO: 560), wherein X 1 D, G or H, wherein X 2 A, V or T, wherein X 3 S, A or Y, and wherein X 4 Is T or N;
GASTRAT(SEQ ID NO:573)；
DANLET (SEQ ID NO: 574); and
AASRLQS(SEQ ID NO:575)。
in various embodiments, the TREM-1 antigen binding protein comprises an antigen binding domain comprising a sequence having a light chain variable region comprising an LCDR3 amino acid sequence selected from the group consisting of:
QX 1 X 2 X 3 X 4 X 5 X 6 PX 7 t (SEQ ID NO: 555); wherein X is 1 Q, H or E, wherein X 2 Is F or Y, wherein X 3 K, Y or I, wherein X 4 N, T, L, I, or M; wherein X is 5 W, F, H or Y, wherein X 6 Absence or P; wherein X is 7 W, N, Y, H or L;
QX 1 YX 3 X 4 X 5 PX 6 t (SEQ ID NO: 561), wherein X 1 Is Q or H, wherein X 2 D, A or G, wherein X 3 Is N or K; wherein X is 4 Is L or I, and wherein X 5 Is I or L;
QQFKNWPPT(SEQ ID NO:576)；
QHYDNLPIT(SEQ ID NO:577)；
LQAHGFPWT(SEQ ID NO:578)；
QQYDNLPLT (SEQ ID NO: 579) and
QFWPPWT(SEQ ID NO:580)。
in various embodiments, the TREM-1 antigen binding protein comprises an antigen binding domain comprising a sequence having a heavy chain variable region comprising an HCDR1 amino acid sequence selected from the group consisting of:
X 1 X 2 X 3 MX 4 (SEQ ID NO: 556), wherein X 1 A, R, T or S, wherein X 2 Is Y or N, wherein X 3 Is A or W, and wherein X 4 Is S or N;
X 1 YDIN (SEQ ID NO: 563) wherein X 1 Is R or S; GYYX 1 H, where X 1 Is M or I;
AYAMS(SEQ ID NO:581)；
RYDIN (SEQ ID NO: 582); and
SYWMS(SEQ ID NO:583)。
in various embodiments, the TREM-1 antigen binding protein comprises an antigen binding domain comprising a sequence having a heavy chain variable region comprising an HCDR2 amino acid sequence selected from the group consisting of:
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YYX 10 X 11 X 12 VKG (SEQ ID NO: 559), wherein X 1 T, E, or S, wherein X 2 Absence or M, V, or I, wherein X 3 S, R or K, wherein X 4 Is G or Q, wherein X 5 S, D or H, wherein X 6 G, S, L, or A, wherein X 7 Is S, G, or R, wherein X 8 T, S, P or E, wherein X 9 Is T or I, wherein X 10 Is A or V, wherein X 11 Is D or E, and wherein X 12 Is S or A;
X 1 X 2 NPX 3 X 4 GX 5 X 6 GX 7 X 8 X 9 X 10 FX 11 X 12 (SEQ ID NO: 564), wherein X 1 Is W or R, wherein X 2 Is M or L, wherein X 3 N, Q, or K, wherein X 4 Is S, A, or R, wherein X 5 Is N, or Q, wherein X 6 Is S, A, or T, wherein X 7 S, Q, or Y, wherein X 8 Is V or T, wherein X 9 Q or K, wherein X 10 Is K or N, wherein X 11 Is R or Q, and wherein X 12 Is G or D;
TSGSGSTTYYADSVKG(SEQ ID NO:584)；
WMNPNSGNSSVQKFRG(SEQ ID NO:585)；
NIKQDGSEEYYVDSVKG (SEQ ID NO: 586); and
TSGSGTYYADSVKG(SEQ ID NO:841)。
in various embodiments, the TREM-1 antigen binding protein comprises an antigen binding domain comprising a sequence having a heavy chain variable region comprising an HCDR3 amino acid sequence selected from the group consisting of:
X 1 X 2 X 3 X 4 X 5 X 6 X 7 FX 8 YYX 9 (SEQ ID NO: 557) wherein X 1 V, E, A or G, wherein X 2 A, F, Y or G, wherein X 3 G, S, Y or W, wherein X 4 Is S or R, wherein X 5 Absent or N, where X 6 F, S, Y, or is absent, where X 7 Is L or F or is absent, wherein X 8 Is D or E, and wherein X 9 Y, H or S;
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 FX 13 X 14 (SEQ ID NO: 565); wherein X is 1 G, L or R, wherein X 2 Is G, I, or R, wherein X 3 Y, R, I, G, or A, wherein X 4 T, S, Y, or V, where X 5 Is S or Y, wherein X 6 Is S, A, I, or R, wherein X 7 W, A, or S, wherein X 8 Absence or S, wherein X 9 Absence or F, W, or Y, wherein X 10 R, S, H, K, or E, wherein X 11 W, H, Y, or F, where X 12 Y, V, A, or S, wherein X 13 Is D or Q, and wherein X 14 L, Y, I, or H;
VAGSNFLFDY(SEQ ID NO:842)；
GGYTSSWRWYFDL(SEQ ID NO:843)；
GGYTSSWSRWYFDL (SEQ ID NO: 844); and
DYGDSFDY(SEQ ID NO:845)。
in various embodiments, provided herein are isolated antigen binding proteins, wherein the antigen binding proteins:
a. is an antibody or antibody fragment;
b. binds to human TREM-1 having the amino acid sequence set forth in SEQ ID NO. 2;
c. comprising a light chain variable domain comprising:
i. comprising the amino acid sequence X 1 ASQSX 2 X 3 X 4 Light chain CDR1 of NLA (SEQ ID NO: 553), wherein X 1 Is R or Q, wherein X 2 Is V or I, wherein X 3 Is N or S, and wherein X 4 S, H, I, V or a;
comprising the amino acid sequence GAX 1 X 2 Light chain CDR2 of RAT (SEQ ID NO: 554), wherein X 1 Is S or Y, and wherein X 2 Is T or I; and
comprising the amino acid sequence QX 1 X 2 X 3 X 4 X 5 X 6 PX 7 T (SEQ ID NO: 555) light chain CDR3; wherein X is 1 Q, H or E, wherein X 2 Is F or Y, wherein X 3 K, Y or I, wherein X 4 N, T, L, I, or M; wherein X is 5 W, F, H or Y, wherein X 6 Absence or P; wherein X is 7 W, N, Y, H or L; and
d. comprising a heavy chain variable domain comprising:
i. comprising the amino acid sequence X 1 X 2 X 3 MX 4 (SEQ ID NO: 556) heavy chain CDR1, wherein X 1 A, R, T or S, wherein X 2 Is Y or N, wherein X 3 Is A or W, and wherein X 4 Is S or N;
comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YYX 10 X 11 X 12 VKG (SEQ ID NO: 559), wherein X is a heavy chain CDR2 1 T, E, or S, wherein X 2 Absence or M, V, or I, wherein X 3 S, R or K, wherein X 4 Is G or Q, wherein X 5 S, D or H, wherein X 6 G, S, L, or A, wherein X 7 Is S, G, or R, wherein X 8 T, S, P or E, wherein X 9 Is T or I, wherein X 10 Is A or V, wherein X 11 Is D or E, and wherein X 12 Is S or A; and
comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 FX 8 YYX 9 (SEQ ID NO: 557) heavy chain CDR3, wherein X 1 V, E, A or G, wherein X 2 A, F, Y or G, wherein X 3 G, S, Y or W, wherein X 4 Is S or R, wherein X 5 Absent or N, where X 6 F, S, Y, or is absent, where X 7 Is L or F or absentIn which X is 8 Is D or E, and wherein X 9 Y, H or S.
In various embodiments, the antigen binding protein comprises:
a. a light chain variable domain comprising:
i. a light chain CDR1 comprising amino acid sequence RASQSVNSNLA (SEQ ID NO: 556);
light chain CDR2 comprising the amino acid sequence GASTRAT (SEQ ID NO: 573);
light chain CDR3 comprising amino acid sequence QQFKNWPPT (SEQ ID NO: 576); and
b. a heavy chain variable domain comprising:
i. a heavy chain CDR1 comprising the amino acid sequence AYAMS (SEQ ID NO: 581);
heavy chain CDR2 comprising amino acid sequence TSGSGSTTYYADSVKG (SEQ ID NO: 584); and
heavy chain CDR3 comprising amino acid sequence VAGSNFLFDY (SEQ ID NO: 842).
In various embodiments, the disclosure provides an isolated antigen binding protein, wherein the antigen binding protein:
a. is an antibody or antibody fragment;
b. binds to human TREM-1 having the amino acid sequence set forth in SEQ ID NO. 2;
c. Comprising a light chain variable domain comprising:
i. comprising the amino acid sequence QASX 1 DIX 2 X 3 X 4 LN (SEQ ID NO: 558) light chain CDR1, wherein X 1 Is R or Q, wherein X 2 R, S, N or F, where X 3 Is K or N, and wherein X 4 H, Y or D;
comprising the amino acid sequence X 1 X 2 X 3 X 4 Light chain CDR2 of LET (SEQ ID NO: 560), wherein X 1 D, G or H, wherein X 2 A, V or T, wherein X 3 S, A or Y, and wherein X 4 Is T or N;
comprising the amino acid sequence QX 1 YX 3 X 4 X 5 PX 6 T (SEQ ID NO: 561) light chain CDR3, wherein X 1 Is Q or H, wherein X 2 D, A or G, wherein X 3 Is N or K; wherein X is 4 Is L or I, and wherein X 5 Is I or L; and
d. comprising a heavy chain variable domain comprising:
i. comprising the amino acid sequence X 1 Heavy chain CDR1 of YDIN (SEQ ID NO: 563) wherein X 1 Is R or S;
comprising the amino acid sequence X 1 X 2 NPX 3 X 4 GX 5 X 6 GX 7 X 8 X 9 X 10 FX 11 X 12 Heavy chain CDR2 of (SEQ ID NO: 564), wherein X 1 Is W or R, wherein X 2 Is M or L, wherein X 3 N, Q, or K, wherein X 4 Is S, A, or R, wherein X 5 Is N, or Q, wherein X 6 Is S, A, or T, wherein X 7 S, Q, or Y, wherein X 8 Is V or T, wherein X 9 Q or K, wherein X 10 Is K or N, wherein X 11 Is R or Q, and wherein X 12 Is G or D; and
comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 FX 13 X 14 (SEQ ID NO: 565) a heavy chain CDR3; wherein X is 1 G, L or R, wherein X 2 Is G, I, or R, wherein X 3 Y, R, I, G, or A, wherein X 4 T, S, Y, or V, where X 5 Is S or Y, wherein X 6 Is S, A, I, or R, wherein X 7 W, A, or S, wherein X 8 Absence or S, wherein X 9 Absence or F, W, or Y, and wherein X 10 R, S, H, K, or E, wherein X 11 W, H, Y, or F, where X 12 Y, V, A, or S, wherein X 13 Is D or Q, and wherein X 14 L, Y, I, or H.
In various embodiments, the antigen binding protein comprises:
a. a light chain variable domain comprising:
i. a light chain CDR1 comprising amino acid sequence QASQDIRKHLN (SEQ ID NO: 567);
light chain CDR2 comprising the amino acid sequence DASLET (SEQ ID NO: 574); and
light chain CDR3 comprising amino acid sequence QHYDNLPIT (SEQ ID NO: 577); and
b. a heavy chain variable domain comprising:
i. heavy chain CDR1 comprising the amino acid sequence RYDIN (SEQ ID NO: 582);
heavy chain CDR2 comprising amino acid sequence WMNPNSGNSSVQKFRG (SEQ ID NO: 585); and
heavy chain CDR3 comprising amino acid sequence GGYTSSWRWYFDL (SEQ ID NO: 843) or GGYTSSWSRWYFDL (SEQ ID NO: 844).
In various embodiments, the disclosure provides methods for treating a cardiovascular disease (e.g., atherosclerosis or myocardial infarction) comprising administering an antigen binding protein, wherein the antigen binding protein:
a. is an antibody or antibody fragment;
b. binds to human myeloid cell trigger receptor 1 (TREM-1) having the amino acid sequence set forth in SEQ ID NO. 2;
c. comprising a set of CDR sequences selected from the group consisting of:
i) SEQ ID NO. 10 (LCDR 1), SEQ ID NO. 11 (LCDR 2), SEQ ID NO. 12 (LCDR 3), SEQ ID NO. 16 (HCDR 1), SEQ ID NO. 17 (HCDR 2) and SEQ ID NO. 18 (HCDR 3);
ii) SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3);
iii) SEQ ID NO:50 (LCDR 1), SEQ ID NO:51 (LCDR 2), SEQ ID NO:52 (LCDR 3), SEQ ID NO:56 (HCDR 1), SEQ ID NO:57 (HCDR 2) and SEQ ID NO:58 (HCDR 3);
iv) SEQ ID NO:70 (LCDR 1), SEQ ID NO:71 (LCDR 2), SEQ ID NO:72 (LCDR 3), SEQ ID NO:76 (HCDR 1), SEQ ID NO:77 (HCDR 2) and SEQ ID NO:78 (HCDR 3);
v) SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3);
vi) SEQ ID NO:110 (LCDR 1), SEQ ID NO:111 (LCDR 2), SEQ ID NO:112 (LCDR 3), SEQ ID NO:116 (HCDR 1), SEQ ID NO:117 (HCDR 2) and SEQ ID NO:118 (HCDR 3);
vii) SEQ ID NO:130 (LCDR 1), SEQ ID NO:131 (LCDR 2), SEQ ID NO:132 (LCDR 3), SEQ ID NO:136 (HCDR 1), SEQ ID NO:137 (HCDR 2) and SEQ ID NO:138 (HCDR 3);
viii) SEQ ID NO:150 (LCDR 1), SEQ ID NO:151 (LCDR 2), SEQ ID NO:152 (LCDR 3), SEQ ID NO:156 (HCDR 1), SEQ ID NO:157 (HCDR 2) and SEQ ID NO:158 (HCDR 3);
ix) SEQ ID NO:170 (LCDR 1), SEQ ID NO:171 (LCDR 2), SEQ ID NO:172 (LCDR 3), SEQ ID NO:176 (HCDR 1), SEQ ID NO:177 (HCDR 2) and SEQ ID NO:178 (HCDR 3);
x) SEQ ID NO. 190 (LCDR 1), SEQ ID NO. 191 (LCDR 2), SEQ ID NO. 192 (LCDR 3), SEQ ID NO. 196 (HCDR 1), SEQ ID NO. 197 (HCDR 2) and SEQ ID NO. 198 (HCDR 3);
xi) SEQ ID NO:210 (LCDR 1), SEQ ID NO:211 (LCDR 2), SEQ ID NO:212 (LCDR 3), SEQ ID NO:216 (HCDR 1), SEQ ID NO:217 (HCDR 2) and SEQ ID NO:218 (HCDR 3);
xii) SEQ ID NO:230 (LCDR 1), SEQ ID NO:231 (LCDR 2), SEQ ID NO:232 (LCDR 3), SEQ ID NO:236 (HCDR 1), SEQ ID NO:237 (HCDR 2) and SEQ ID NO:238 (HCDR 3);
xiii) SEQ ID NO:250 (LCDR 1), SEQ ID NO:251 (LCDR 2), SEQ ID NO:252 (LCDR 3), SEQ ID NO:256 (HCDR 1), SEQ ID NO:257 (HCDR 2) and SEQ ID NO:258 (HCDR 3);
ix) SEQ ID NO:270 (LCDR 1), SEQ ID NO:271 (LCDR 2), SEQ ID NO:272 (LCDR 3), SEQ ID NO:276 (HCDR 1), SEQ ID NO:277 (HCDR 2) and SEQ ID NO:278 (HCDR 3); and
xv) SEQ ID NO:544 (LCDR 1), SEQ ID NO:545 (LCDR 2), SEQ ID NO:546 (LCDR 3), SEQ ID NO:547 (HCDR 1), SEQ ID NO:548 (HCDR 2) and SEQ ID NO:549 (HCDR 3).
In various embodiments, the anti-TREM-1 antigen binding protein comprises a set of CDR sequences selected from the group consisting of:
i) SEQ ID NO. 10 (LCDR 1), SEQ ID NO. 11 (LCDR 2), SEQ ID NO. 12 (LCDR 3), SEQ ID NO. 16 (HCDR 1), SEQ ID NO. 17 (HCDR 2) and SEQ ID NO. 18 (HCDR 3);
ii) SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3);
iii) SEQ ID NO:50 (LCDR 1), SEQ ID NO:51 (LCDR 2), SEQ ID NO:52 (LCDR 3), SEQ ID NO:56 (HCDR 1), SEQ ID NO:57 (HCDR 2) and SEQ ID NO:58 (HCDR 3);
iv) SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3);
v) SEQ ID NO:130 (LCDR 1), SEQ ID NO:131 (LCDR 2), SEQ ID NO:132 (LCDR 3), SEQ ID NO:136 (HCDR 1), SEQ ID NO:137 (HCDR 2) and SEQ ID NO:138 (HCDR 3);
vi) SEQ ID NO:150 (LCDR 1), SEQ ID NO:151 (LCDR 2), SEQ ID NO:152 (LCDR 3), SEQ ID NO:156 (HCDR 1), SEQ ID NO:157 (HCDR 2) and SEQ ID NO:158 (HCDR 3); or (b)
vii) SEQ ID NO:270 (LCDR 1), SEQ ID NO:271 (LCDR 2), SEQ ID NO:272 (LCDR 3), SEQ ID NO:276 (HCDR 1), SEQ ID NO:277 (HCDR 2) and SEQ ID NO:278 (HCDR 3).
In various embodiments, the anti-TREM-1 antigen binding protein comprises a set of CDR sequences selected from the group consisting of: SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3); or SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3).
In various embodiments, the disclosure provides methods for treating a cardiovascular disease (e.g., atherosclerosis or myocardial infarction) comprising administering an anti-TREM-1 antigen binding protein, wherein the anti-TREM-1 antigen binding protein comprises:
a. a light chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281 and 539;
A sequence encoded by a polynucleotide sequence having at least 80% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279 and 537;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279 and 537; and
b. a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282 and 540;
a sequence encoded by the polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 2184; a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 538.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
a. a light chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 21, 41, 61, 101, 141, 161 and 281;
a sequence encoded by a polynucleotide sequence having at least 80% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 99, 139, 159 and 279;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 99, 139, 159 and 279; and
b. a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 102, 142, 162 and 282;
a sequence encoded by a polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 100, 140, 160 and 280;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 100, 140, 160 and 280.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
a. a light chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 41 and 101;
a sequence encoded by a polynucleotide sequence having at least 80% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs 39 and 99;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 39 and 99; and
b. a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS.42 and 102;
a sequence encoded by a polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 40 and 100;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 40 and 100.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
i) The light chain variable domain sequence set forth in SEQ ID NO. 21 and the heavy chain variable domain sequence set forth in SEQ ID NO. 22;
i) The light chain variable domain sequence set forth in SEQ ID NO. 41 and the heavy chain variable domain sequence set forth in SEQ ID NO. 42;
ii) the light chain variable domain sequence set forth in SEQ ID NO. 61 and the heavy chain variable domain sequence set forth in SEQ ID NO. 62;
iii) The light chain variable domain sequence set forth in SEQ ID NO. 81 and the heavy chain variable domain sequence set forth in SEQ ID NO. 82;
iv) the light chain variable domain sequence set forth in SEQ ID NO. 101 and the heavy chain variable domain sequence set forth in SEQ ID NO. 102;
v) the light chain variable domain sequence set forth in SEQ ID NO. 121 and the heavy chain variable domain sequence set forth in SEQ ID NO. 122;
vi) the light chain variable domain sequence set forth in SEQ ID NO. 161 and the heavy chain variable domain sequence set forth in SEQ ID NO. 162;
vii) the light chain variable domain sequence set forth in SEQ ID NO:181 and the heavy chain variable domain sequence set forth in SEQ ID NO: 182;
viii) the light chain variable domain sequence set forth in SEQ ID NO. 201 and the heavy chain variable domain sequence set forth in SEQ ID NO. 202;
x) the light chain variable domain sequence set forth in SEQ ID NO. 221 and the heavy chain variable domain sequence set forth in SEQ ID NO. 222;
xi) the light chain variable domain set forth in SEQ ID NO. 241 and the heavy chain variable domain set forth in SEQ ID NO. 242;
xii) the light chain variable domain sequence set forth in SEQ ID NO:261 and the heavy chain variable domain sequence set forth in SEQ ID NO: 262;
xiii) the light chain variable domain sequence set forth in SEQ ID NO:281 and the heavy chain variable domain sequence set forth in SEQ ID NO: 282; and
xv) the light chain variable domain sequence set forth in SEQ ID NO. 539 and the heavy chain variable domain sequence set forth in SEQ ID NO. 540.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
i) The light chain variable domain sequence set forth in SEQ ID NO. 21 and the heavy chain variable domain sequence set forth in SEQ ID NO. 22;
ii) the light chain variable domain sequence set forth in SEQ ID NO. 41 and the heavy chain variable domain sequence set forth in SEQ ID NO. 42;
iii) The light chain variable domain sequence set forth in SEQ ID NO. 61 and the heavy chain variable domain sequence set forth in SEQ ID NO. 62;
iv) the light chain variable domain sequence set forth in SEQ ID NO. 101 and the heavy chain variable domain sequence set forth in SEQ ID NO. 102;
v) the light chain variable domain sequence set forth in SEQ ID NO. 141 and the heavy chain variable domain sequence set forth in SEQ ID NO. 142;
vi) the light chain variable domain sequence set forth in SEQ ID NO. 161 and the heavy chain variable domain sequence set forth in SEQ ID NO. 162; or (b)
vii) the light chain variable domain sequence set forth in SEQ ID NO:281 and the heavy chain variable domain sequence set forth in SEQ ID NO: 282.
In various embodiments, the anti-TREM-1 antigen binding protein comprises: the light chain variable domain sequence set forth in SEQ ID NO. 4 and the heavy chain variable domain sequence set forth in SEQ ID NO. 42; or the light chain variable domain sequence set forth in SEQ ID NO. 101 and the heavy chain variable domain sequence set forth in SEQ ID NO. 102.
In various embodiments, the amino acid sequence may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282 and 540, and SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281 and 539.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to the light chain variable region amino acid sequences set forth in SEQ ID NOs.21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 102, 142, 162 and 282. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 41, 61, 101, 141, 161 and 281.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs 42 and 102. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs 41 and 101.
In various embodiments, one or more heavy chain framework amino acids of the anti-antigen binding protein are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence. In various embodiments, one or more light chain framework amino acids of the antigen binding protein are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence.
In some cases, the sequences disclosed herein may contain an N-terminal signal sequence useful for recombinant production. Sequences of anti-TREM-1 antibodies lacking signal sequences are contemplated herein. Exemplary signal sequences include MDMRVPAQLLGLLLLWLRGARC; MAWALLLLTLLTQGTGSWASYEL, and nucleic acids encoding such signal sequences.
In various embodiments, the anti-TREM-1 antigen binding protein further comprises a human light chain constant region linked to the light chain variable region.
In various embodiments, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of IgG, igM, igA, igD, igE, fragments thereof, combinations thereof, and modifications thereof in which one to ten heavy chain framework amino acids are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence.
In various embodiments, an anti-TREM-1 antigen binding protein described herein inhibits the binding of a TREM-1 ligand to TREM-1.
Also contemplated are antigen binding proteins that compete with an anti-TREM-1 antigen binding protein as described herein for binding to a human TREM-1 protein having the sequence of SEQ ID NO. 2.
In various embodiments, the antigen binding protein is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, fab, F (ab') 2, fab2, monovalent IgG, scFv, scFv-Fc, igG1 antibodies, igG2 antibodies, igG3 antibodies, and IgG4 antibodies. In various embodiments, the anti-TREM-1 antigen binding protein is an IgG2 antibody. In various embodiments, the anti-TREM-1 antigen binding protein is an IgG1 antibody. In various embodiments, the IgG1 antibody is an IgG1z or IgG1z-SEFL2 antibody. In various embodiments, the antigen binding protein is a monovalent IgG.
In various embodiments, the antigen binding protein is a human antibody.
Also provided are isolated nucleic acid molecules comprising nucleotide sequences encoding the heavy chain of an anti-TREM-1 antigen binding protein as described herein, isolated nucleic acid molecules comprising nucleotide sequences encoding the light chain of an anti-TREM-1 antigen binding protein as described herein, and isolated nucleic acid molecules comprising nucleotide sequences encoding the heavy and light chains of an anti-TREM-1 antigen binding protein as described herein.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region polynucleotide sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 538. In various embodiments, the antigen binding protein comprises an amino acid sequence that has at least 90% identity to the light chain variable region polynucleotide sequences set forth in SEQ ID NOs 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279 and 537.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region polynucleotide sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 100, 140, 160 and 280. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region polynucleotide sequence selected from the group consisting of SEQ ID NOs: 19, 39, 59, 99, 139, 159 and 279.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region polynucleotide sequence selected from the group consisting of SEQ ID NOs 40 and 100. In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region polynucleotide sequence selected from the group consisting of SEQ ID NOs 39 and 99.
Further contemplated are expression vectors comprising a nucleic acid molecule of an anti-TREM-1 antigen binding protein heavy chain and/or light chain as described herein operably linked to an expression control sequence.
The present disclosure provides recombinant host cells comprising a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of an anti-TREM-1 antigen binding protein or antibody as described herein; or a nucleic acid molecule encoding a light chain or an anti-TREM-1 antibody as described herein; or nucleic acid molecules encoding heavy and light chain nucleic acid molecules of an anti-TREM-1 antibody as described herein; or a vector comprising a nucleic acid molecule encoding the heavy and/or light chain of an anti-TREM-1 antibody as described herein. In various embodiments, the host cell is a mammalian cell. In various embodiments, the host cell is a CHO cell.
Further provided are methods of producing an antigen binding protein using a host cell and antigen binding proteins produced by the method, the method comprising culturing the host cell and recovering the antigen binding protein.
Also provided are sterile pharmaceutical compositions comprising an anti-TREM-1 antigen binding protein as described herein and a pharmaceutically acceptable carrier.
In various embodiments, the antigen binding protein further comprises a human heavy chain constant region linked to the heavy chain variable region of the antigen binding protein. In various embodiments, the antigen binding protein further comprises a human light chain constant region linked to the light chain variable region of the antibody.
In various embodiments, the antigen binding portion is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, fab, F (ab') 2, fab2, monovalent IgG, scFv, scFv-Fc, igG1 antibodies, igG2 antibodies, igG3 antibodies, and IgG4 antibodies. In various embodiments, the antigen binding portion is an IgG. In various embodiments, the antigen binding portion is an IgG2 antibody. In various embodiments, the antigen binding portion is an IgG1 antibody. In various embodiments of the antigen binding protein, the antibody is an IgG1z or IgG1z-SEFL2 antibody. In various embodiments, the antigen binding moiety is a monovalent IgG. In various embodiments, the heavy chain constant region of the antigen binding portion is selected from the group consisting of a heavy chain constant region of IgG, igM, igA, igD, igE, a fragment thereof, a combination thereof, and modifications thereof in which one to ten heavy chain framework amino acids are replaced with a corresponding one or more amino acids from another human antibody constant region.
In various embodiments, the antigen binding proteins have heavy and light chain pairs as listed in tables 7-8, 11, and 12.
The nucleic acid sequence of the TREM-1 variant antibody heavy chain variable region is set forth in SEQ ID NO:284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406 and 408, and also set forth in SEQ ID NO:412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, and 536; and the TREM-1 variant antibody light chain variable region nucleotide sequence is set forth in SEQ ID NO:778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840 are also listed in SEQ ID NO:588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 61, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, and 712.
In various embodiments, one or more heavy chain framework amino acids of the antigen binding protein are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence. In various embodiments, one or more light chain framework amino acids of the antigen binding protein are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence.
In various embodiments, the anti-TREM-1 antigen binding protein further comprises a human light chain constant region linked to the light chain variable region.
In various embodiments, variants of the heavy and/or light chain variable regions of TREM-1 antibodies are provided herein. The TREM-1 antibody heavy chain variable region variant sequence is set forth in SEQ ID NO:283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, tables 7 to 8, 11, and 12.
The TREM-1 antibody light chain variable region variant sequences are set forth in SEQ ID NOs 715-777, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, table 7-8, table 11 and table 12.
In various embodiments, the antigen binding protein is a human antibody.
Also provided are isolated nucleic acid molecules comprising a nucleotide sequence encoding a heavy chain region of an antigen binding protein as described herein, isolated nucleic acid molecules comprising a nucleotide sequence encoding a light chain region of an antigen binding protein as described herein.
The present disclosure provides one or more expression vectors comprising one or more nucleic acid molecules encoding an antigen binding protein as described herein operably linked to an expression control sequence.
Recombinant host cells comprising a nucleic acid molecule comprising an antigen binding protein as described herein, or a vector comprising said nucleic acid are also contemplated. In various embodiments, the host cell is a mammalian cell. In various embodiments, the host cell is a CHO cell. The present disclosure provides methods of producing antigen binding proteins using the host cells described herein, comprising culturing the host cells and recovering the antibodies, and antigen binding proteins produced by the methods.
The present disclosure contemplates methods of treatment comprising administering a pharmaceutical composition comprising an anti-TREM-1 antigen binding protein or antigen binding fragment described herein.
In a related aspect, the disclosure provides a method of treating a cardiovascular disease (e.g., atherosclerosis or myocardial infarction) in a subject in need thereof, the method comprising administering an anti-TREM-1 antigen binding protein as described herein or a composition comprising an anti-TREM-1 antigen binding protein as described herein.
Also contemplated are compositions comprising an anti-TREM-1 antigen binding protein or antigen binding fragment as described herein for use in treating a cardiovascular disease (e.g., atherosclerosis or myocardial infarction). In various embodiments, the disclosure provides the use of a composition comprising an anti-TREM-1 antigen binding protein or antigen binding fragment as described herein in the manufacture of a medicament for treating a cardiovascular disease (e.g., atherosclerosis or myocardial infarction).
In various embodiments, the cardiovascular disease is selected from the group consisting of: myocardial infarction, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), heart failure, stroke (e.g., ischemic, hemorrhagic), atherosclerosis, coronary heart disease, peripheral vascular disease (e.g., peripheral arterial disease), vulnerable plaque, acute coronary syndrome, cerebrovascular disease, cerebrovascular atherosclerosis, and obesity. In various embodiments, the cardiovascular disease is myocardial infarction. In various embodiments, the cardiovascular disease is stroke (ischemic and/or hemorrhagic). In various embodiments, the cardiovascular disease is atherosclerosis. In various embodiments, the cardiovascular disease is NAFLD or NASH. In various embodiments, the cardiovascular disease is heart failure. In various embodiments, the cardiovascular disease is coronary heart disease. In various embodiments, the cardiovascular disease is a peripheral vascular disease. In various embodiments, the cardiovascular disease is peripheral arterial disease. In various embodiments, the cardiovascular disease is vulnerable plaque. In various embodiments, the cardiovascular disease is acute coronary syndrome. In various embodiments, the cardiovascular disease is a cerebrovascular disease. In various embodiments, the cardiovascular disease is cerebrovascular atherosclerosis. In various embodiments, the cardiovascular disease is obesity.
In various embodiments, the treatment is performed by intravenous or subcutaneous administration. In various embodiments, the treatment is administered weekly, biweekly, tricyclically, 4 weekly, monthly, 3 monthly, or six months or yearly.
In various embodiments, TREM-1 is human TREM-1 set forth in SEQ ID NO. 2.
In various embodiments, administration reduces one or more symptoms of cardiovascular disease selected from the group consisting of: inflammatory cell migration to the site of injury, infiltration of myeloid cells into heart tissue, inflammatory cytokines in the microenvironment, tissue damage, reduced foam cell formation, reduced necrotic core size, reduced scarring, reduced endothelial cell dysfunction, and/or reduced thrombosis.
In various embodiments, the methods comprise administering one or two additional therapeutic agents. In various embodiments, the additional therapeutic agent is selected from the group consisting of corticosteroids, NSAIDs, analgesics, immunosuppressives, anti-inflammatory agents, TNFα inhibitors, IL-12/IL-23 inhibitors, IL-17, and IFN- γ. Therapeutic agents (other than antigen binding proteins) useful for cardiovascular disease include, but are not limited to, at least one cholesterol-lowering (serum and/or total internal cholesterol) agent or agents. In some embodiments, agents that increase LDLR expression have been observed to increase serum HDL levels, decrease LDL levels, or decrease triglyceride levels. Exemplary agents include, but are not limited to, statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), PCSK9 inhibitors Nicotinic acid (niacin) (-)>(sustained release of Nick acid), ->(sustained release of niacin)), fibric acid (>(gemfibrozil),>(fenofibrate)), bile acid sequestrant (a. About.)>(cholestyramine), colesevelam->(norbilin)), cholesterol absorption inhibitors (Zetia), combined nicotinic acid with statins (++>(lovastatin and->) Combined statin and absorption inhibitor (>(/>And->) And/or lipid modulators like PCSK9 inhibitors. In some embodiments, the antigen binding protein is combined with: PPARgamma agonist, PPARalpha/gamma agonist, squalene synthetase inhibitor, cholesteryl Ester Transfer Protein (CETP) inhibitor, antihypertensive drug, antithrombotic agent (aspirin), antidiabetic agent (e.g. sulfonylurea, insulin, GLP-1 analogue, DDPIV inhibitor, SGL2 inhibitor), apoB modulators, MTP inhibitors, < > and->(ivabradine) I (f) current inhibitors, omecamicarb cardiac myosin activators, olpasran (AMG 890) (siRNA) that reduces lipoprotein (a), AMG 594 cardiac troponin activators, and/or therapeutic agents for occlusive arteriosclerosis. Combination therapy with AMG 609 is also contemplated, AMG 609 is a small interfering RNA (siRNA) that targets a patatin-like phospholipase domain 3-containing variant (PNPLA 3I 148M). Antigen binding proteins may also be combined with other anti-inflammatory agents.
In various embodiments, the additional therapeutic agent is selected from the group consisting of: one or more cholesterol lowering agents, agents that increase LDLR expression, statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), PCSK9 inhibitorsNicotinic acid (niacin) (-)>(sustained release of Nick acid), ->(sustained release of niacin)), fibric acid (>(gemfibrozil),>(fenofibrate)), bile acid sequestrant (a. About.)>(cholestyramine), colesevelam-> (norbilin)), cholesterol absorption inhibitors (Zetia), combined nicotinic acid with statins (++>(lovastatin and->) Combinations of statins with absorption inhibitors (VYTORIN ()>And->) Lipid modulators like PCSK9 inhibitors, ppary agonists, ppara/y agonists, squalene synthetase inhibitors, cholesteryl Ester Transfer Protein (CETP) inhibitors, antihypertensive agents, antithrombotic agents (aspirin), antidiabetic agents (e.g. sulfonylurea, insulin, GLP-1 analogues, GIPR antagonists, DDPIV inhibitors, SGL2 inhibitors), apoB modulators, MTP inhibitors, SGL2 inhibitors>(ivabradine) I (f) current inhibitors, omecambaryophyllin activators, OLPASIRAN (AMG 890) that reduce lipoprotein (a) (siRNA), AMG 594 cardiac troponin activators, AMG 609, AMG 171 (growth differentiation factor 15 (GDF 15) analogues), AMG 133 (gastric inhibitory peptide receptor (GIPR) antagonists and glucagon-like peptide 1 (GLP-1) receptor agonists), and/or occlusive arteriosclerosis therapeutics.
It is to be understood that each feature or embodiment or combination described herein is a non-limiting, illustrative example of any aspect of the invention, and is therefore intended to be combined with any other feature or embodiment or combination described herein. For example, where features are described using language such as "one embodiment," "some embodiments," "certain embodiments," "additional embodiments," "certain exemplary embodiments," and/or "another embodiment," each of these types of embodiments is intended to be combined with any other feature or combination of features described herein without necessarily listing a non-limiting example of a feature of each possible combination. Such feature or combination of features is applicable to any aspect of the invention. Where examples of values falling within a range are disclosed, any of these examples are considered to be possible endpoints of the range, any and all numbers between such endpoints are considered, and any and all combinations of the upper and lower endpoints are considered.
The headings herein are for the convenience of the reader and are not intended to be limiting. Other aspects, embodiments, and variations of the present invention will become apparent from the detailed description and/or the accompanying drawings and/or the claims.
Drawings
FIGS. 1A-1D show inhibition of ligand (PGLYRP 1/PGN) -mediated TREM-1 signaling by anti-human TREM-1 antibodies in human (FIGS. 1C-1D) or cynomolgus monkey (FIGS. 1A-1B) PBMC as measured by TNFα release.
FIG. 2 shows inhibition of ligand (PGLYRP 1/PGN) mediated signal transduction by anti-human TREM-1Fab in cell lines overexpressing human TREM-1/DAP12 as measured by phosphorylation of spleen tyrosine kinase (pSYK).
FIG. 3 shows the levels of PGLYRP1 in wild type and TREM-1 knockout mice after LPS injection.
Detailed Description
The present disclosure provides TREM-1 antibodies that block TREM-1 ligand binding to a receptor. Antibodies according to the present disclosure have been shown to target immune cells and modulate cellular activity and cytokine responses, and are useful in the treatment of cardiovascular diseases, such as atherosclerosis, stroke, or myocardial infarction.
Definition of the definition
The term "polypeptide binding agent" or "antigen binding protein" refers to a polypeptide that is capable of specifically binding an antigen (e.g., a target or signaling partner thereof), or capable of binding an antigen with a measurable binding affinity. Examples of polypeptide binding agents include antibodies, peptibodies, polypeptides, and peptides, optionally conjugated to other peptide moieties or non-peptide moieties. Antigens to which a polypeptide binding agent can bind include any protein or non-protein molecule capable of eliciting an antibody response or binding to a polypeptide binding agent with a detectable binding affinity that is greater than non-specific binding. Antigens that bind to a regulatory polypeptide binding agent may include a target, a signaling partner of the target, and/or a complex comprising the target and its signaling partner.
The term "antibody" is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind to an antigen (e.g., fab ', F' (ab) 2, fv, single chain antibodies, diabodies), and recombinant peptides comprising the foregoing (so long as they exhibit the desired biological activity). An "immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein consisting of two heavy and two light chains each comprising a variable region and a constant region. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. Antibody fragments or antigen-binding portions include, inter alia, fab ', F (ab') 2, fv, domain antibodies (dabs), complementarity Determining Region (CDR) fragments, CDR-grafted antibodies, single chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies; chelating recombinant antibodies, tri-or diabodies, intracellular antibodies, nanobodies, small Modular Immunopharmaceuticals (SMIPs), antigen binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies, or variants or derivatives thereof, and polypeptides containing at least a portion (e.g., one, two, three, four, five, or six CDR sequences) of an immunoglobulin sufficient to confer antigen-specific binding to the polypeptide (so long as the antibody retains the desired biological activity).
The term "monovalent IgG" as used herein refers to IgG in which a single antigen binding fragment (Fab) is fused to a complete constant domain fragment (Fc) by intra-Fc C H Mutations in the 3 domain are engineered to heterodimerize. Monovalent IgG is also known as "single arm"An antibody.
The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies (i.e., the individual antibodies comprising the population are homogeneous except for possible naturally occurring mutations that may be present in minor amounts).
An "antibody variant" as used herein refers to an antibody polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the variable region of the native antibody variable region domain. The variant may be substantially homologous or substantially identical to the unmodified antibody.
An "isolated" antibody is an antibody that has been identified and isolated and recovered from a component of its natural environment. The contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to an extent of greater than 95% by weight, most preferably greater than 99% by weight of the antibody as measured by the laud method, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence found by using a cup sequencer, or (3) to homogeneity as found by SDS-PAGE under reducing or non-reducing conditions using coomassie blue or preferably silver staining or HPLC methods. Isolated antibodies include in situ antibodies within recombinant cells because at least one component of the natural environment of the antibody will not be present. However, typically, the isolated antibody is prepared by at least one purification step.
As used herein, "heavy chain variable region" refers to an antibody molecular region comprising at least one Complementarity Determining Region (CDR) of the antibody heavy chain variable domain. The heavy chain variable region may comprise one, two, or three CDRs of the antibody heavy chain.
As used herein, "light chain variable region" refers to an antibody molecular region comprising at least one Complementarity Determining Region (CDR) of the antibody light chain variable domain. The light chain variable region may contain one, two, or three CDRs of the antibody light chain, which may be a kappa or lambda light chain, depending on the antibody.
As used herein, an antibody that "specifically binds" is "antigen-specific," "specific for an antigen target," or "immunoreactive with" an antigen refers to an antibody or polypeptide binding agent of the present invention that binds an antigen with greater affinity than other antigens of similar sequence. In one aspect, the antigen binding proteins of the invention, or fragments, variants, or derivatives thereof, will bind with greater affinity to human antigens than their binding affinity to similar antigens of other (i.e., non-human) species, although polypeptide binding agents that recognize and bind to homologs of the targets are also within the scope of the invention.
The term "epitope" refers to a portion of any molecule that is capable of being recognized and bound by a selective binding agent in one or more of the antigen binding regions. Epitopes generally consist of: chemically active surface groupings of molecules such as amino acids or carbohydrate side chains, and have specific three-dimensional structural features, as well as specific charge features. Epitopes as used herein may be continuous or discontinuous.
The term "derivative" when used in connection with the polypeptide binding agents and polypeptides of the invention refers to polypeptides that are chemically modified by the following techniques: ubiquitination, conjugation with therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment, such as pegylation (derivatization with polyethylene glycol), and insertion or substitution of amino acids (e.g., ornithine) by chemical synthesis, which are not typically found in human proteins. The derivatives retain the binding properties of the underivatized molecules of the invention.
As used herein, "linker" refers to a peptide that connects two polypeptides. The linker length may be 1-80 amino acids. In some embodiments, the linker may be 2-40, 3-30, or 3-20 amino acids long. In some embodiments, the linker may be a 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acid long peptide. In other embodiments, the linker may be 3-25, 3-18, 5-20, 6-18, or 10-20 amino acids long. In other embodiments, the linker may be about 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, or 30 amino acids long. In many cases, the linker lacks free cysteine residues (i.e., and therefore does not involve disulfide bonds) and also does not contain an N-glycosylation site (i.e., asn-Xxx-Ser/Thr, where X can be any amino acid other than proline). In certain embodiments, the peptide having the sequence G3SG2 or G4S is a linker between the anti-TREM-1 antigen binding protein and the IL-10 mutein. Examples of other suitable linkers include G2, G3S, G3P, G Q, G5, and the like. Each capital letter in the above-mentioned linker refers to a conventional single letter code for an amino acid, and each number refers to the number of consecutive repeats of the amino acid in the linker. For example, "G3SG2" refers to a linker having the sequence Gly-Gly-Gly-Ser-Gly-Gly. "G4S" refers to a linker having the sequence Gly-Gly-Gly-Gly-Ser.
A "therapeutically effective amount" of a drug for treating a disease refers to an amount that reduces the severity of the disease, reduces the severity of one or more symptoms associated with the disease or its treatment, or delays the onset of more severe symptoms or more severe disease, which may occur at a certain frequency after treatment.
"treating" of any disease as referred to herein encompasses alleviating at least one symptom of the disease, reducing the severity of the disease, or delaying or preventing the disease from progressing to more serious symptoms, which in some cases may accompany the disease or cause at least one other disease. Treatment does not mean that the disease is completely cured. Useful therapeutic agents need only reduce the severity of the disease, reduce the severity of one or more symptoms associated with the disease or treatment thereof, or delay the onset of more severe symptoms or more severe disease, which may occur at a certain frequency after treatment.
"subject" encompasses both mammalian and non-mammalian. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees, and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or gender.
As used herein, "myeloid cells" refers to a subpopulation of immune cells derived from blood progenitor cells of a bone marrow line and includes granulocytes, monocytes, macrophages, dendritic Cells (DCs). Myeloid cells play an important role in protective immunity, often with phagocytic and Antigen Presenting Cell (APC) functions. See, for example, de kler et al,Front.Immunol[ immunology front edge],5:423,2014。
Antigen binding proteins and antibodies
Immunoglobulin variable domains exhibit the same general structure of relatively conserved Framework Regions (FR) connected by three hypervariable regions or CDRs. From the N-terminus to the C-terminus, both the light and heavy chains comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acid allocation for each domain is defined according to the following: kabat Sequences of Proteins of Immunological Interest [ sequence of immune protein of interest ]](National Institutes of Health [ national institutes of health of the United states ]]Besseda, maryland (1987 and 1991)), or Chothia and Lesk,J.Mol.Biol[ molecular biology ]]196:901-917,1987; chothia et al,Nature[ Nature]342:878-883,1989。
The hypervariable region of an antibody refers to the CDR amino acid residues of the antibody responsible for antigen binding. Hypervariable regions comprise amino acid residues from the CDRs [ e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain, and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain, as in Kabat et al, Sequences of Proteins of Immunological Interest[ sequence of immune protein of interest ]]5 th edition Public Health Service [ public health service center of the United states ]]National Institutes of Health [ national institutes of health of the United states ]]Besselda, maryland (1991)]And/or those from hypervariable loops (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain), such as [ Chothia et al,J.Mol.Biol[ molecular biology ]]196:901-917(1987)]Said method. CDRs have also been identified and are based on immunogenetics (ImMunoGenTics, IMGT)) Numbering (Lefranc, m. -p.,TheImmunologist[ immunologist ]]7,132-136 (1999); lefranc, M.—P.et al,Dev.Comp.Immunol[ development and comparative immunology ]]27,55-77 (2003)) which describes the following CDR positions in the light and heavy chain variable domains: CDR1, about residues 27 to 38; CDR2, about residues 56 to 65; and CDR3, about residues 105 to 116 (germline) or residues 105 to 117 (rearrangement). In one embodiment, the CDRs are expected to be located at about residues 26-31 (L1), 49-51 (L2) and 88-98 (L3) in the light chain variable domain and at about residues 26-33 (H1), 51-58 (H2) and 97-110 (H3) in the heavy chain variable domain of a heavy chain or light chain substantially similar to the heavy chain or light chain length of an antibody disclosed herein. However, one skilled in the art will appreciate that when identifying the sequence of a particular antibody, the actual positions of the CDR residues may differ from the predicted residues described above. CDRs assigned sequence identifiers as disclosed herein are defined according to the Kabat method (Kabat and Wu, 1991) and are numbered using the ampoul reference number (amben). Inlet company reference numbers are structure-based numbering systems, established in Honygger and Pluckaphun (J Mol Biol. [ journal of molecular biology ] ]309 (3) 657-70, 2001) based on the Honygger and Pluckthun numbering systems of the variable regions of antibodies. In various embodiments, engineered IgG1 antibodies are contemplated. In various embodiments, the antibody is an IgG1z or IgG1z-SEFL2 antibody as described herein.
In addition, the CDRs may be defined according to Kabat and Chothia accumulation, abM, contact, north, martin, and/or conformational definition or any CDR assay methods known in the art. AbM definition of CDR is a compromise between Kabat and Chothia and uses AbM antibody modeling software from Oxford MolecularThe definition of "contact" of CDRs is based on observed antigen contact and is described in MacCallum et al, 1996, J.mol. Biol. [ journal of molecular biology ]]262:732-745. The definition of "conformation" of CDRs is based on residues that contribute enthalpy to antigen binding (see, e.g., makabe et al, 2008, j. Biol. Chem. [ journal of biochemistry],283:1 156-1 166). North already hasClassical CDR conformations were identified using a different set of preferred CDR definitions (North et al 2011, j. Mol. Biol. [ journal of molecular biology ]]406:228-256). In another approach, referred to herein as "conformational definition" of CDRs, the positions of the CDRs can be identified as residues that contribute enthalpy to antigen binding (Makabe et al, 2008, j biol. Chem. [ journal of biochemistry ]283:1 156-1 166). Martin definition (also called enhanced Chothia definition) combines the Kabat and Chothia definitions and differs only in the heavy chain, wherein CDR-H1 includes all residues of Kabat and Chothia, while CDR-H2 is seven residues shorter than Kabat definition (Martin, bioinformatics tools for antibody engineering [ bioinformatics tools for antibody engineering)]Handbook of Therapeutic Antibodies A therapeutic antibody Manual]Weinheim [ Wei Yinhai ] m]Wiley-VCH Verlag GmbH [ Wili chemical Press ]]The method comprises the steps of carrying out a first treatment on the surface of the (2008) pages 95-117; see also the database maintained by the university of London (University College London) institute of structure and molecular biology (Institute of Structural and Molecular Biology), http:// www.bioinf.org.uk/abs/#cdrid. Other CDR boundary definitions may not strictly follow one of the above methods, but will still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened without significantly affecting antigen binding, as predicted or experimentally found from a particular residue or group of residues or even the entire CDR. For example, "merged" CDRs may also be used. As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR (or other residues of the antibody) may be defined according to any one of Kabat, chothia, north, abM, contact, IMGT, martin, combined Kabat and Chothia, and/or conformational definitions.
For example, the following table shows several commonly used CDR definitions (residues are numbered according to Kabat numbering rather than the security company reference number for convenience).
1. Some of these definitions (especially for the Chothia ring) vary depending on the individual publications under investigation. Some papers describe Chothia CDRs as: CDR-L1: l24-34; CDR-L2: l50-56; CDR-L3: l89-97; CDR-H1: h26-32; CDR-H2: h52-56; CDR-H3: h95-102.
2. Any numbering scheme may be used for these CDR definitions, except that the contact definition uses Chothia or Martin (enhanced Chothia) definitions.
3. When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop. (this is because the Kabat numbering scheme places insertions at H35A and H35B.)
Framework region (or FR) residues are those variable domain residues other than the hypervariable region residues.
As described below, the antibodies contemplated herein (including monoclonal, human, humanized, and other antibodies described herein) are typically recombinant or produced by other methods of manipulating the genetic code in vitro or in vivo and thus do not necessarily reflect the particular antibodies found in nature.
anti-TREM-1 antigen binding proteins
Previous studies characterizing TREM-1 signaling have utilized agonistic antibodies to TREM-1 to mimic ligand activation of the receptor (Tessarz et al, immunol Lett [ immunological communications ]116 (2): 111-6,2008; vandestine et al, jClin Invest [ journal of clinical study ]131 (2): e142468,2021). Decoy peptides have also been tried to modulate TREM-1 activity (see, e.g., international patent publication No. WO 2014037565 a).
The present disclosure encompasses the use of amino acid molecules encoding target-specific antibodies. The anti-TREM-1 antigen binding proteins described herein differentially modulate the interaction of human TREM-1 with its ligand.
In some embodiments, an antigen binding protein is provided comprising a polypeptide having an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable region set forth in SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540, and/or an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region set forth in SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539, and the antibody further comprises at least one, two, three, four, or more LCDRs 1, 3, HCDR, three, or more HCDR. In some embodiments, the amino acid sequence having a percent identity to the light chain variable region may comprise one, two, or three of the light chain CDRs. In other embodiments, the amino acid sequence having a percent identity to the heavy chain variable region may comprise one, two, or three of the heavy chain CDRs.
In another embodiment, an antigen binding protein is provided comprising a polypeptide having an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to all three HCDRs or the following CDRs in the heavy chain variable region of an antibody sequence listed above: the heavy chain CDR1 sequences set forth in SEQ ID NOS 16, 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276 and 550; 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277 and 551; and the heavy chain CDR3 sequences set forth in SEQ ID NOS.18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278 and 552.
In related embodiments, an antigen binding protein is provided comprising a polypeptide having an amino acid sequence that is at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to all three LCDRs or CDRs below in the light chain variable region of an antibody sequence listed above: the light chain CDR1 sequences set forth in SEQ ID nos. 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270 and 544; the light chain CDR2 sequences set forth in SEQ ID NOS 11, 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271 and 545; the light chain CDR3 sequences set forth in SEQ ID Nos. 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272 and 546.
It is contemplated that antibodies of the present disclosure may have one, two, or more amino acid substitutions, e.g., non-conservative or conservative substitutions, in the CDR regions of the antibody. Consensus sequences of the TREM-1 antibody heavy and light chain CDR and/or variable region sequences disclosed herein are also contemplated. For example, the TREM-1 antibody may comprise the following sequence. In various embodiments, a TREM-1 antibody comprises an antigen binding domain comprising a sequence having a light chain variable region comprising an LCDR1 amino acid sequence selected from the group consisting of: x is X 1 ASQSX 2 X 3 X 4 NLA (SEQ ID NO: 553), wherein X 1 Is R or Q, wherein X 2 Is V or I, wherein X 3 Is N or S, and wherein X 4 S, H, I, V or a; QASX 1 DIX 2 X 3 X 4 LN (SEQ ID NO: 558), wherein X 1 Is R or Q, wherein X 2 R, S, N or F, where X 3 Is K or N, and wherein X 4 H, Y or D; RASQSVNSNLA (SEQ ID NO: 566); QASQDIRKHLN (SEQ ID NO: 567); RASQDISSNLN (SEQ ID NO: 568); QASQDIHLN (SEQ ID NO: 569); RASQGIRKWLA (SEQ ID NO: 570) or RASQSVNSNLA (SEQ ID NO: 571) and SGDKLGERVS (SEQ ID NO: 572).
In various embodiments, the TREM-1 antibody comprises an antigen binding domain comprising a sequence having a light chain variable region comprising an LCDR2 amino acid sequence selected from the group consisting of: GAX (GAX) 1 X 2 RAT (SEQ ID NO: 554), wherein X 1 Is S or Y, and wherein X 2 Is T or I; amino acid sequence X 1 X 2 X 3 X 4 LET (SEQ ID NO: 560), wherein X 1 D, G or H, wherein X 2 A, V or T, wherein X 3 S, A or Y, and wherein X 4 Is T or N; GASTRAT (SEQ ID NO: 573); DANLET (SEQ ID NO: 574); and AASRLQS (SEQ ID NO: 575).
In various embodiments, the TREM-1 antibody comprises an antigen binding domain comprising a sequence having a light chain variable region comprising an LCDR3 amino acid sequence selected from the group consisting of: QX (quality control X) 1 X 2 X 3 X 4 X 5 X 6 PX 7 T (SEQ ID NO: 555); wherein X is 1 Q, H or E, wherein X 2 Is F or Y, wherein X 3 K, Y or I, wherein X 4 N, T, L, I, or M; wherein X is 5 W, F, H or Y, wherein X 6 Absence or P; wherein X is 7 W, N, Y, H or L; QX (quality control X) 1 YX 3 X 4 X 5 PX 6 T (SEQ ID NO: 561), wherein X 1 Is Q or H, wherein X 2 D, A or G, wherein X 3 Is N or K; wherein X is 4 Is L or I, and wherein X 5 Is I or L; QQFKNWPPT (SEQ ID NO: 576); QHYDNLPIT (SEQ ID NO: 577); LQAHGFPWT (SEQ ID NO: 578); QQYDNLPLT (SEQ ID NO: 579) and QFWPPWT (SEQ ID NO: 580).
In various embodiments, the TREM-1 antibody comprises an antigen binding domain comprising a sequence having a heavy chain variable region comprising an HCDR1 amino acid sequence selected from the group consisting of: x is X 1 X 2 X 3 MX 4 (SEQ ID NO: 556), wherein X 1 A, R, T or S, wherein X 2 Is Y or N, wherein X 3 Is A or W, and wherein X 4 Is S or N; sequence X 1 YDIN (SEQ ID NO: 563) wherein X 1 Is R or S; GYYX 1 H, where X 1 Is M or I; AYAMS (SEQ ID NO: 581); RYDIN (SEQ ID NO: 582); SYWMS (SEQ ID NO: 583).
In various embodiments, the TREM-1 antibody comprises an antigen binding domain comprising a polypeptide having a polypeptide comprising an HCDR2 selected from the group consisting ofThe sequence of the heavy chain variable region of the amino acid sequence: x is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YYX 1 0 X 11 X 12 VKG (SEQ ID NO: 559), wherein X 1 T, E, or S, wherein X 2 Absence or M, V, or I, wherein X 3 S, R or K, wherein X 4 Is G or Q, wherein X 5 S, D or H, wherein X 6 G, S, L, or A, wherein X 7 Is S, G, or R, wherein X 8 T, S, P or E, wherein X 9 Is T or I, wherein X 10 Is A or V, wherein X 11 Is D or E, and wherein X 12 Is S or A; x is X 1 X 2 NPX 3 X 4 GX 5 X 6 GX 7 X 8 X 9 X 10 FX 11 X 12 (SEQ ID NO: 564), wherein X 1 Is W or R, wherein X 2 Is M or L, wherein X 3 N, Q, or K, wherein X 4 Is S, A, or R, wherein X 5 Is N, or Q, wherein X 6 Is S, A, or T, wherein X 7 S, Q, or Y, wherein X 8 Is V or T, wherein X 9 Q or K, wherein X 10 Is K or N, wherein X 11 Is R or Q, and wherein X 12 Is G or D; TSGSGSTTYYADSVKG (SEQ ID NO: 584); WMNPNSGNSSVQKFRG (SEQ ID NO: 585); NIKQDGSEEYYVDSVKG (SEQ ID NO: 586); and TSGSGTYYADSVKG (SEQ ID NO: 841).
In various embodiments, the TREM-1 antibody comprises an antigen binding domain comprising a sequence having a heavy chain variable region comprising an HCDR3 amino acid sequence selected from the group consisting of: x is X 1 X 2 X 3 X 4 X 5 X 6 X 7 FX 8 YYX 9 (SEQ ID NO: 557) wherein X 1 V, E, A or G, wherein X 2 A, F, Y or G, wherein X 3 G, S, Y or W, wherein X 4 Is S or R, wherein X 5 Absent or N, where X 6 F, S, Y, or is absent, where X 7 Is L or F or is absent, wherein X 8 Is DOr E, and wherein X 9 Y, H or S; x is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 FX 13 X 14 (SEQ ID NO: 565); wherein X is 1 G, L or R, wherein X 2 Is G, I, or R, wherein X 3 Y, R, I, G, or A, wherein X 4 T, S, Y, or V, where X 5 Is S or Y, wherein X 6 Is S, A, I, or R, wherein X 7 W, A, or S, wherein X 8 Absence or S, wherein X 9 Absence or F, W, or Y, wherein X 10 R, S, H, K, or E, wherein X 11 W, H, Y, or F, where X 12 Y, V, A, or S, wherein X 13 Is D or Q, and wherein X 14 L, Y, I, or H; VAGSNFLFDY (SEQ ID NO: 842); GGYTSSWRWYFDL (SEQ ID NO: 843); GGYTSSWSRWYFDL (SEQ ID NO: 844); and DYGDSFDY (SEQ ID NO: 845).
In related embodiments, the residues of the framework are altered. The variable heavy chain framework regions are located within the designated H-FR1, H-FR2, H-FR3 and H-FR4 regions that surround the heavy chain CDR residues and the variable light chain framework regions are located within the designated L-FR1, L-FR2, L-FR3 and L-FR4 regions that surround the light chain CDR residues. Amino acids within the framework regions may be substituted, for example, with any suitable amino acid identified in a human framework or a human consensus framework. It is further contemplated that the framework regions may be altered, but that the antigen binding proteins or antibodies described herein retain the CDRs, LCDR1-3 and/or HCDR-3 of the parent antibody.
The antibody fragment comprises a portion of an intact full length antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2, fcab, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); multispecific antibody fragments, such as bispecific, trispecific, and the like antibodies (e.g., diabodies, triabodies, tetrabodies); a minibody; chelating the recombinant antibody; a tri-or diabody; an inner antibody; a nanobody; binding domain exemption An epidemic globulin fusion protein; camelized antibodies; an antibody comprising a VHH; and other polypeptides formed from antibody fragments. See, e.g., holliger and Hudson,2005Nat.Biotech[ Natural biotechnology]23:1126-36; eyer and Hruska,VeterinarniMedicina[ veterinary medicine]57:439-513,2012。
The antigen binding compounds of the present disclosure preferably retain 10 -8 、10 -9 、10 -10 、10 -11 、10 -12 、10 -13 、10 -14 、10 -15 M or less to TREM-1 as measured by surface plasmon resonance or KinexA. The antigen binding compounds of the present disclosure have a TREM-1 pair of 10 -9 To 10 -12 Or 10 -10 To 10 -13 Or 10 -10 To 10 -15 Binding affinity of M. SPR assays are performed using standard methods, for example, at 25 ℃ (room temperature).
In various embodiments, the antibody is an IgG1 antibody. In certain embodiments, the antibody is an IgG1z antibody.
Examples of full length antibodies that bind TREM-1 having the heavy chain amino acid sequences set forth in SEQ ID NOs 6, 26, 46, 66, 86, 106, 126, 146, 166, 186, 206, 226, 246, 266, and 540 are provided herein; and the light chain amino acid sequences set forth in SEQ ID NOs 5, 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, 245, 265 and 539.
In various embodiments, the TREM-1 antigen binding protein is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, fab, F (ab') 2, fab2, monovalent IgG, scFv, scFv-Fc, igG1 antibodies, igG2 antibodies, igG3 antibodies, and IgG4 antibodies. In various embodiments, the antigen binding portion is an IgG. In various embodiments, the antigen binding portion is an IgG2 antibody. In various embodiments, the antigen binding portion is an IgG1 antibody. In various embodiments, the antibody is an IgG1z antibody. In various embodiments, the antigen binding moiety is a monovalent IgG. In various embodiments, the heavy chain constant region of the antigen binding portion is selected from the group consisting of a heavy chain constant region of IgG, igM, igA, igD, igE, a fragment thereof, a combination thereof, and modifications thereof in which one to ten heavy chain framework amino acids are replaced with a corresponding one or more amino acids from another human antibody constant region.
In various embodiments, the antigen binding protein is present at 10 -8 M to 10 -15 M or 10 -8 M to 10 -12 M, or 10 -8 M、10 -9 M、10 -10 M、10 -11 M、10 -12 M、10 -13 M、10 -14 M, or 10 -15 The binding affinity of M binds its antigen.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
a. a light chain variable domain comprising:
i. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, and 544;
light chain CDR2 comprising an amino acid sequence selected from SEQ ID NOs 11, 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271 and 545;
light chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272 and 546; and
b. comprising a heavy chain variable domain comprising:
i. a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NOs 16, 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276 and 550;
heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277 and 551; and
Heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278 and 552.
In various embodiments of the antigen binding protein,
a. the light chain CDR1 sequences are set forth in SEQ ID NO. 10, 30, 50, 90, 130, 150, or 270;
b. the light chain CDR2 sequences are set forth in SEQ ID NO. 11, 31, 51, 91, 131, 151, or 271;
c. the light chain CDR3 sequences are set forth in SEQ ID NO. 12, 32, 52, 92, 132, 152, or 272;
d. the heavy chain CDR1 sequences are set forth in SEQ ID NO 16, 36, 56, 96, 136, 156, or 276;
e. the heavy chain CDR2 sequences are set forth in SEQ ID NO 17, 37, 57, 97, 137, 157, or 277; and
f. the heavy chain CDR3 sequences are set forth in SEQ ID NO. 18, 38, 58, 98, 138, 158, or 278.
In various embodiments of the antigen binding protein,
a. the light chain CDR1 sequences are set forth in SEQ ID NO. 30 or 90;
b. the light chain CDR2 sequences are set forth in SEQ ID NO. 31 or 91;
c. the light chain CDR3 sequences are set forth in SEQ ID NO. 32 or 92;
d. the heavy chain CDR1 sequences are set forth in SEQ ID NO. 36 or 096;
e. the heavy chain CDR2 sequences are set forth in SEQ ID NO 37 or 97; and
f. the heavy chain CDR3 sequences are set forth in SEQ ID NO. 38 or 98.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 22, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 21. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 42, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 41. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 62, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 61. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 82, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 81. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 102, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 101. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 122, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 121. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO:142, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 141. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 162, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 161. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 182, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 181. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 202, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 201. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO:222, and Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 221. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises the Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 242, and the Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 241. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO:262, and Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 261. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO:282, and Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 281. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the antigen binding protein comprises Heavy Chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO. 540, and Light Chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO. 539. In an exemplary embodiment, the CDRs are defined according to Kabat, chothia, abM, contact or IMGT.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
a. a light chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281 and 539;
a sequence encoded by a polynucleotide sequence having at least 80% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279 and 537;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279 and 537; and
b. a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282 and 540;
a sequence encoded by the polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 538; and
A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 538.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
a. a light chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 21, 41, 61, 101, 141, 161 and 281;
a sequence encoded by a polynucleotide sequence having at least 80% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 99, 139, 159 and 279;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 99, 139, 159 and 279; and
b. a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 102, 142, 162 and 282;
A sequence encoded by a polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 100, 140, 160 and 280;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 100, 140, 160 and 280.
In various embodiments, the anti-TREM-1 antigen binding protein comprises:
a. a light chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 41 and 101;
a sequence encoded by a polynucleotide sequence having at least 80% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs 39 and 99;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 39 and 99; and
b. a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS.42 and 102;
A sequence encoded by a polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 40 and 100;
a sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 40 and 100.
In various embodiments, the amino acid sequence may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539, and SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540.
In various embodiments, the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539.
In various embodiments, the antigen binding protein comprises a heavy chain amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 22, 42, 62, 102, 142, 162, and 282, and a light chain amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 21, 41, 61, 101, 141, 161, and 281.
In various embodiments, the antigen binding protein comprises a heavy chain amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS: 4 and 102, and a light chain amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOS: 41 and 101.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 22; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 21. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 22; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 21. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 22; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 21. When three CDR sequences are combined together and aligned with the sequences disclosed herein, the percent identity is determined from the percent overall identity. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 42; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 41. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 42; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 41. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 42; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 41. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 62; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 61. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 62; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 61. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 62; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 61. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 82; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 81. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 82; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 81. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 82; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 81. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 102; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 101. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 102; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 101. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 102; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 101. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 122; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 121. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 122; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 121. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 122; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 121. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 122; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 121. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 122; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 121. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 122; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 121. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 142; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 141. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO: 142; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 141. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO: 142; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 141. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 162; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 161. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 162; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 161. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 162; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 161. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to three heavy chain CDRs in SEQ ID NO. 182; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO: 181. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO 182; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO: 181. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO 182; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO: 181. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO 202; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 201. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO 202; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 201. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO 202; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 201. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 222; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 221. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 222; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 221. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 222; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 221. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO 242; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 241. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO 242; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 241. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO 242; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 241. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 262; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO: 261. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO: 262; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO: 261. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO: 262; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO: 261. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 282; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 281. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 282; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 281. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 282; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 281. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the antigen binding protein comprises a heavy chain variable domain (VH) comprising three CDRs that in combination have at least 85% identity to the three heavy chain CDRs in SEQ ID NO. 540; and a light chain variable domain (VL) comprising three CDRs which in combination have at least 85% identity to the three light chain CDRs of SEQ ID NO. 539. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 90% identity to the three heavy chain CDRs in SEQ ID NO. 540; and a VL comprising three CDRs which in combination have at least 90% identity to the three light chain CDRs of SEQ ID NO. 539. In various embodiments, the antigen binding protein comprises a VH comprising three CDRs that in combination have at least 95% identity to the three heavy chain CDRs in SEQ ID NO. 540; and a VL comprising three CDRs which in combination have at least 95% identity to the three light chain CDRs of SEQ ID NO. 539. The antigen binding protein may further bind to its target (TREM-1) with a KD value of 100nM or less, or a KD value of 50nM or less.
In various embodiments, the disclosure provides methods for treating a cardiovascular disease (e.g., atherosclerosis or myocardial infarction) comprising administering an antigen binding protein, wherein the antigen binding protein: binds to human myeloid cell trigger receptor 1 (TREM-1) and comprises a set of CDR sequences selected from the group consisting of:
i) SEQ ID NO. 10 (LCDR 1), SEQ ID NO. 11 (LCDR 2), SEQ ID NO. 12 (LCDR 3), SEQ ID NO. 16 (HCDR 1), SEQ ID NO. 17 (HCDR 2) and SEQ ID NO. 18 (HCDR 3);
ii) SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3);
iii) SEQ ID NO:50 (LCDR 1), SEQ ID NO:51 (LCDR 2), SEQ ID NO:52 (LCDR 3), SEQ ID NO:56 (HCDR 1), SEQ ID NO:57 (HCDR 2) and SEQ ID NO:58 (HCDR 3);
iv) SEQ ID NO:70 (LCDR 1), SEQ ID NO:71 (LCDR 2), SEQ ID NO:72 (LCDR 3), SEQ ID NO:76 (HCDR 1), SEQ ID NO:77 (HCDR 2) and SEQ ID NO:78 (HCDR 3);
v) SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3);
vi) SEQ ID NO:110 (LCDR 1), SEQ ID NO:111 (LCDR 2), SEQ ID NO:112 (LCDR 3), SEQ ID NO:116 (HCDR 1), SEQ ID NO:117 (HCDR 2) and SEQ ID NO:118 (HCDR 3);
vii) SEQ ID NO:130 (LCDR 1), SEQ ID NO:131 (LCDR 2), SEQ ID NO:132 (LCDR 3), SEQ ID NO:136 (HCDR 1), SEQ ID NO:137 (HCDR 2) and SEQ ID NO:138 (HCDR 3);
viii) SEQ ID NO:150 (LCDR 1), SEQ ID NO:151 (LCDR 2), SEQ ID NO:152 (LCDR 3), SEQ ID NO:156 (HCDR 1), SEQ ID NO:157 (HCDR 2) and SEQ ID NO:158 (HCDR 3);
ix) SEQ ID NO:170 (LCDR 1), SEQ ID NO:171 (LCDR 2), SEQ ID NO:172 (LCDR 3), SEQ ID NO:176 (HCDR 1), SEQ ID NO:177 (HCDR 2) and SEQ ID NO:178 (HCDR 3);
x) SEQ ID NO. 190 (LCDR 1), SEQ ID NO. 191 (LCDR 2), SEQ ID NO. 192 (LCDR 3), SEQ ID NO. 196 (HCDR 1), SEQ ID NO. 197 (HCDR 2) and SEQ ID NO. 198 (HCDR 3);
xi) SEQ ID NO:210 (LCDR 1), SEQ ID NO:211 (LCDR 2), SEQ ID NO:212 (LCDR 3), SEQ ID NO:216 (HCDR 1), SEQ ID NO:217 (HCDR 2) and SEQ ID NO:218 (HCDR 3);
xii) SEQ ID NO:230 (LCDR 1), SEQ ID NO:231 (LCDR 2), SEQ ID NO:232 (LCDR 3), SEQ ID NO:236 (HCDR 1), SEQ ID NO:237 (HCDR 2) and SEQ ID NO:238 (HCDR 3);
xiii) SEQ ID NO:250 (LCDR 1), SEQ ID NO:251 (LCDR 2), SEQ ID NO:252 (LCDR 3), SEQ ID NO:256 (HCDR 1), SEQ ID NO:257 (HCDR 2) and SEQ ID NO:258 (HCDR 3);
ix) SEQ ID NO:270 (LCDR 1), SEQ ID NO:271 (LCDR 2), SEQ ID NO:272 (LCDR 3), SEQ ID NO:276 (HCDR 1), SEQ ID NO:277 (HCDR 2) and SEQ ID NO:278 (HCDR 3); and
xv) SEQ ID NO:544 (LCDR 1), SEQ ID NO:545 (LCDR 2), SEQ ID NO:546 (LCDR 3), SEQ ID NO:547 (HCDR 1), SEQ ID NO:548 (HCDR 2) and SEQ ID NO:549 (HCDR 3).
In various embodiments, the anti-TREM-1 antigen binding protein comprises a set of CDR sequences selected from the group consisting of:
i) SEQ ID NO. 10 (LCDR 1), SEQ ID NO. 11 (LCDR 2), SEQ ID NO. 12 (LCDR 3), SEQ ID NO. 16 (HCDR 1), SEQ ID NO. 17 (HCDR 2) and SEQ ID NO. 18 (HCDR 3);
ii) SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3);
iii) SEQ ID NO:50 (LCDR 1), SEQ ID NO:51 (LCDR 2), SEQ ID NO:52 (LCDR 3), SEQ ID NO:56 (HCDR 1), SEQ ID NO:57 (HCDR 2) and SEQ ID NO:58 (HCDR 3);
iv) SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3);
v) SEQ ID NO:130 (LCDR 1), SEQ ID NO:131 (LCDR 2), SEQ ID NO:132 (LCDR 3), SEQ ID NO:136 (HCDR 1), SEQ ID NO:137 (HCDR 2) and SEQ ID NO:138 (HCDR 3);
vi) SEQ ID NO:150 (LCDR 1), SEQ ID NO:151 (LCDR 2), SEQ ID NO:152 (LCDR 3), SEQ ID NO:156 (HCDR 1), SEQ ID NO:157 (HCDR 2) and SEQ ID NO:158 (HCDR 3); or (b)
vii) SEQ ID NO:270 (LCDR 1), SEQ ID NO:271 (LCDR 2), SEQ ID NO:272 (LCDR 3), SEQ ID NO:276 (HCDR 1), SEQ ID NO:277 (HCDR 2) and SEQ ID NO:278 (HCDR 3).
In various embodiments, the anti-TREM-1 antigen binding protein comprises a set of CDR sequences selected from the group consisting of: SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3); or SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3).
The binding affinity of the TREM-1 antigen binding protein can be assessed by KD. KD is the equilibrium dissociation constant, k, between an antigen binding protein and its target or antigen off /k on Is a ratio of (2). KD and KA are inversely related. The KD value is related to the concentration of antibody (the amount of antibody required for a particular experiment) and therefore the lower the KD value (the lower the required concentration), the higher the affinity of the antibody. In an exemplary aspect, the TREM-1 antigen binding protein has a KD of about 10 for its target -1 M or less, about 10 -2 M or less, about 10 -3 M or less, about 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M to about 10 -15 M, about 10 -6 M to about 10 -15 M, about 10 -7 M to about 10 -15 M, about 10 -8 M to about 10 -15 M, about 10 -9 M to about 10 -15 M, about 10 -10 M to about 10 -15 M, about 10 -5 M to about 10 -14 M, about 10 -6 M to about 10 -14 M, about 10 -7 M to about 10 -14 M, about 10 -8 M to about 10 -14 M, about 10 -9 M to about 10 -14 M, about 10 -10 M to about 10 -14 M, about 10 -5 M to about 10 -13 M, about 10 -6 M to about 10 -13 M, about 10 -7 M to about 10 -13 M, about 10 -8 M to about 10 -13 M, about 10 -9 M to about 10 -13 M, or about 10 -10 M to about 10 -13 M。
In exemplary aspects, the TREM-1 antigen binding protein has a KD value for its target of micromolar, nanomolar, picomolar, or femtomolar. In an exemplary aspect, the KD is about 10 -4 To 10 -6 M, or 10 -7 To 10 -9 M, or 10 -10 To 10 -12 Or 10 -13 To 10 -15 M is in the range of M. In an exemplary aspect, the TREM-1 antigen binding protein binds to its target with KD values as follows: about 1uM or less, about 900nM or less, about 800nM or less, about 700nM or less, about 600nM or less, about 500nM or less, about 400nM or less, about 300nM or less, about 200nM or less, about 100nM or less, about 90nM or less, about 80nM or less, about 70nM or less, about 60nM or less, about 50nM or less, about 40nM or less, about 30nM or less, about 20nM or less, about 10nM or less, about 5nM or less, about 2nM or less, about 1 nM or lessnM or less, about 900pM or less, about 800pM or less, about 700pM or less, about 600pM or less, about 500pM or less, about 400pM or less, about 300pM or less, about 250pM or less, about 200pM or less, about 150pM or less, about 100pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 25pM or less, about 20pM or less, about 15pM or less, about 10pM or less, about 5pM or less, or about 1pM or less.
KD values can be determined using methods established in the art. One exemplary method for measuring KD is Surface Plasmon Resonance (SPR), a method well known in the art (e.g., nguyen et al Sensors ](Basel)). 5.5.5.2015; 15 (5):10481-510). A biosensor system (e.g.System) KD values were measured by SPR. BIAcore kinetic analysis involves analysis of antigen binding to and dissociation of a chip with immobilized molecules (e.g., molecules comprising epitope binding domains) on the surface. Another well-known method in the art for determining KD of a protein is by using biological layer interferometry (e.g., shah et al J Vis Exp. [ journal of visualization experiments ]]2014; (84):51383). Can use +.>The KD values were measured by bio-layer interferometry by the technique (Octet QKe system, bolfudi biosystems (ForteBio)). Alternatively or additionally, it is also possible to use +.f. available from the company Sa Pi Dien instruments (Sapidyne Instruments) (Boisy, eda.)>(kinetic rejection assay) assay. Any method known in the art for assessing binding affinity between two binding partners is contemplated herein.
In some aspects, KD values are measured by Surface Plasmon Resonance (SPR). The antigen (TREM-1) may be immobilized on, for example, a solid surface. The antigen may be immobilized to the chip, for example, by covalent coupling (e.g., amine coupling). The chip may be a CM5 sensor chip. As the analyte binds to the ligand, the accumulation of protein on the sensor surface results in an increase in refractive index. This refractive index change is measured in real time (sampled every 0.1s in a kinetic analysis experiment) and the results are plotted as Response Units (RU) versus time (referred to as sensorgrams). If there is a difference in refractive index between the running buffer and the sample buffer, a response will also be generated (background response). This background response must be subtracted from the sensorgram to obtain the actual binding response. The background response is recorded by injecting the analyte through a control or reference flow cell that has no ligand immobilized on the sensor surface or no related ligand. Real-time measurement of association and dissociation of binding interactions allows calculation of association and dissociation rate constants and corresponding affinity constants. One RU indicates 1pg of protein bound per square mm. In practice, analyte binding in excess of 50pg per square mm is generally required to produce a good reproducible response.
Dissociation of the antigen binding protein from the antigen can be monitored for about 3600 seconds. SPR analysis may be performed and data collected at about 15 ℃ to about 37 ℃. SPR analysis can be performed and data collected at about 25 ℃ to 37 ℃. SPR analysis can be performed and data collected at about 37 ℃. SPR analysis can be performed and data collected at 37 ℃. K (K) D The values may be measured by SPR using a BIAcore T200 instrument. SPR rates and affinities can be determined by fitting the resulting sensorgram data to a 1:1 model in BIAcore T200 evaluation software version 1.0. The acquisition rate may be about 1Hz.
Another method for determining antibody KD is to use Biological Layer Interferometry (BLI), typically usingTechnology (Octet QKe systems, poerful biosystems). In some embodiments, a biosensor analysis is used. Typically, one interactant is immobilized on the surface of a biosensor (a "ligand", such as an antigen binding protein) and the other is retained in solution (an "analyte", such as an antigen). The measurement starts fromAn initial baseline or equilibration step of assay buffer was used. Next, the ligand (e.g., antigen binding protein) is immobilized (loaded) on the surface of the biosensor by direct immobilization or a capture-based method. After ligand immobilization, the biosensor is immersed in a buffer solution for a baseline step to evaluate assay drift and determine ligand loading levels. After the baseline step, the biosensor is immersed (associated) in a solution containing the binding partner (analyte) of the ligand. In this step, the binding interaction of the analyte with the immobilized ligand is measured. After analyte association, the biosensor is immersed in a buffer solution without analyte and the bound analyte is allowed to dissociate (dissociate) from the ligand. A series of assay steps are then repeated for each analyte being tested on a new or regenerated biosensor. Each binding response was measured and reported on the sensor trace in real time. The instrument may be an Octet QKe system, an Octet RED96 system, an Octet QK384 system, or an RED384 system.
Nucleic acid molecules
The present disclosure also provides isolated nucleic acids encoding the antigen binding proteins described herein (including, for example, antigen binding protein light chains, light chain variable regions, light chain constant regions, antigen binding protein heavy chains, heavy chain variable regions, heavy chain constant regions, linkers, fusion proteins, and any and all components and combinations thereof). The nucleic acids of the present invention include nucleic acids having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% homology to the nucleic acids of the present invention. The terms "percent similarity", "percent identity" and "percent homology" when referring to a particular sequence, such as university of Wisconsin (University of Wisconsin)The use is described in the software program. The nucleic acids of the present disclosure also include complementary nucleic acids. In some cases, the sequences will be perfectly complementary (no mismatches) when aligned. In other cases, up to about 20% mismatches may be present in the sequence. In some embodiments of the invention, nucleic acids encoding both the heavy and light chains of the antibodies of the present disclosure are provided.
The nucleic acids of the present disclosure may be cloned into a vector, such as a plasmid, cosmid, BAC, phage, artificial chromosome (BAC, YAC), or virus, into which another genetic sequence or element (DNA or RNA) may be inserted in order to effect replication of the attached sequence or element. In some embodiments, the expression vector contains a constitutively active promoter segment (such as, but not limited to, CMV, SV40, elongation factor or LTR sequences) or an inducible promoter sequence, for example a steroid inducible pIND vector (Invitrogen), where expression of the nucleic acid may be regulated. The expression vectors of the invention may further comprise regulatory sequences, such as internal ribosome entry sites. For example, the expression vector may be introduced into the cell by transfection.
Also provided are expression vectors comprising the following operably linked elements; a transcription promoter; a first nucleic acid molecule encoding the heavy chain of an antigen binding protein, antibody or antigen binding fragment of the disclosure; a second nucleic acid molecule encoding a light chain of an antigen binding protein, antibody or antigen binding fragment of the disclosure; a transcription terminator. In another embodiment, the present disclosure provides an expression vector comprising the following operably linked elements; a first transcription promoter; a first nucleic acid molecule encoding the heavy chain of an antigen binding protein, antibody or antigen binding fragment of the disclosure; a first transcription terminator; a second transcription promoter, optionally a second nucleic acid molecule encoding the light chain of an antigen binding protein, antibody or antigen binding fragment of the disclosure; and a second transcription terminator.
The secretion signal peptide sequence may also optionally be encoded by an expression vector, operably linked to a coding sequence of interest, such that the expressed polypeptide may be secreted by a recombinant host cell, if desired, to more easily isolate the polypeptide of interest from the cell. For example, in some embodiments, the signal peptide sequence may be appended/fused to the amino terminus of any of the antigen binding proteins, antibodies, or antigen binding fragment polypeptide sequences thereof described herein.
Also provided are heavy and light chains comprising such vectors and expressing antigen binding proteinsIs a recombinant host cell of (a). The recombinant host cell may be a prokaryotic cell (e.g., an E.coli cell), or a eukaryotic cell (e.g., a mammalian cell or a yeast cell). The yeast cells include Saccharomyces cerevisiae (Saccharomyces cerevisiae),Schizosaccharomyces pombe (Schizosaccharomyces pombe)And (d) sumPichia pastoris (Pichia pastoris)) And (3) cells. Mammalian cells include VERO, sea-Law (HeLa), chinese Hamster Ovary (CHO), W138, baby Hamster Kidney (BHK), COS-7, MDCK, human embryonic kidney 293, normal dog kidney cell line, normal cat kidney cell line, monkey kidney cells, african green monkey kidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTK31 cells, mouse sertoli cells (sertoli cells), human cervical cancer cells, buffalo) rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells. The recombinant protein-producing cells of the present disclosure also include any known insect-expressing cell line, such as, for example Spodoptera frugiperda (Spodoptera frugiperda)) And (3) cells. In one embodiment, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a CHO cell.
In certain embodiments, the mammalian cell is a HEK 293 cell.
Protein purification methods are known in the art and are used herein to recover recombinant proteins from cell culture media. For example, methods of protein and antibody purification are known in the art and may be used with the production of antibodies of the present disclosure. In some embodiments, methods for protein and antibody purification include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. The filtration step may comprise ultrafiltration, optionally ultrafiltration and diafiltration. The filtration is preferably performed at least about 5 to 50 times, more preferably 10 to 30 times, most preferably 14 to 27 times. Affinity column chromatography can use, for exampleAffinity chromatography (Milibo Corp., bellica, horse)Salsa Zhu Sai (Millipore, billerica, mass.). In various embodiments, the affinity chromatography step comprises +.>-vA column chromatography. The eluate may be washed in a solvent detergent. The cation exchange chromatography may include, for example, SP-Sepharose cation exchange chromatography. Anion exchange chromatography may include, for example, but is not limited to, Q-Sepharose fast flow anion exchange (Q-Sepharose Fast Flow Anion Exchange). The anion exchange step is preferably non-binding, allowing removal of contaminants including DNA and BSA. The antibody product is preferably nanofiltration, for example, using a Pall DV 20 nanofiltration. Antibody products can be concentrated, for example, using ultrafiltration and diafiltration. The method may further comprise the step of size exclusion chromatography to remove aggregates.
In some embodiments, the different nucleic acid molecules encode the heavy chain variable region and the light chain variable region of the target-specific antibody. In other embodiments, the same nucleic acid molecule encodes the heavy chain variable region and the light chain variable region of the target-specific antibody. In one embodiment, the nucleic acid encodes any of the target-specific antibodies of the disclosure, and polypeptides encoded by the nucleic acids described herein.
In some embodiments, the nucleic acid molecule encodes a VH amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a VH amino acid sequence set forth in SEQ ID nos. 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282 and 540. The nucleic acid molecules of the present disclosure further include nucleic acids that hybridize under high stringency conditions to nucleic acid sequences encoding the heavy chain variable region amino acid sequences of SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540 (as those described herein) or nucleic acids having the heavy chain variable region nucleic acid sequences of any of SEQ ID NOs 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, and 538.
In some embodiments, the nucleic acid molecule encodes a VL amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a VL amino acid sequence set forth in SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539. The nucleic acid molecules of the present disclosure further include nucleic acids that hybridize under high stringency conditions to nucleic acid sequences encoding the light chain variable region amino acid sequences of SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539 (as those described herein) or nucleic acids having the light chain variable region nucleic acid sequences of any of SEQ ID NOs 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, and 537.
In one aspect, the nucleic acid molecules contemplated herein comprise a nucleotide sequence encoding a VL amino acid sequence of an anti-TREM-1 antigen binding protein or portion thereof listed herein. In related aspects, the VL amino acid sequence is a consensus sequence. In some embodiments, the nucleic acid encodes an amino acid sequence of a light chain CDR of the antibody. In some embodiments, the moiety is a contiguous moiety comprising LCDR1-CDR 3. In related aspects, the LCDR1-3 amino acid sequences are consensus sequences. In one embodiment, the portion comprises at least one, two, or three of the light chain CDR1, CDR2, or CDR3 regions, optionally with different humans or human consensus frameworks, and optionally with a total of 1, or up to 2, or up to 3 mutations in the 3 CDRs.
In one aspect, the nucleic acid molecules of the disclosure comprise a nucleotide sequence encoding a VH amino acid sequence of an anti-TREM-1 antigen binding protein or portion thereof listed herein. In related aspects, the VH amino acid sequence is a consensus sequence. In some embodiments, the nucleic acid encodes the amino acid sequence of a heavy chain CDR of the antibody. In some embodiments, the moiety is a contiguous moiety comprising HCDR1-CDR 3. In related aspects, the HCDR1-3 amino acid sequences are consensus sequences. In one embodiment, the portion comprises at least one, two or three of the heavy chain CDR1, CDR2, or CDR3 regions, optionally with different humans or human consensus frameworks, and optionally with a total of 1, or up to 2, or up to 3 mutations in the 3 CDRs.
In exemplary embodiments, the antibodies of the disclosure comprise human kappa (kappa) or human lambda (lambda) light chains or amino acid sequences derived therefrom, or human heavy chains or sequences derived therefrom, or both heavy and light chains together in single chain, dimer, tetramer, or other forms.
Derivatives and their use as inhibitors of viral infection
Suitable detectable molecules may be linked directly or indirectly to the antigen binding proteins of the present disclosure. Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like. For indirect attachment of a detectable molecule or cytotoxic molecule, the detectable molecule or cytotoxic molecule may be conjugated to one member of a complementary/anti-complementary pair, wherein the other member is bound to a binding polypeptide or antibody moiety. For these purposes, biotin/streptavidin is an exemplary complementary/anti-complementary pair.
The antigen binding proteins of the present disclosure also include modified derivatives, for example, by covalently attaching any type of molecule to an antibody such that covalent attachment does not interfere with binding of the antibody to its epitope. Examples of suitable derivatives include, but are not limited to, fucosylated, glycosylated, acetylated, pegylated, phosphorylated, or amidated derivatives. The antigen binding proteins of the present disclosure themselves may be derivatized by known protecting/blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, and the like. In some embodiments of the disclosure, at least one heavy chain of the antigen binding protein is pegylated. In some embodiments, pegylation is N-linked or through a side chain of an amino acid (e.g., lysine).
Glycosylation can promote effector functions of antibodies, particularly IgG1 antibodies. Thus, in some embodiments, the antigen binding proteins of the present disclosure may comprise one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the side chain of the carbohydrate moiety linked to the asparagine residue. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences that enzymatically link a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
In certain embodiments, glycosylation of an antigen binding protein, e.g., as described herein, is increased by adding one or more glycosylation sites to the Fc region of the binding protein. The addition of glycosylation sites to antigen binding proteins can generally be accomplished by altering the amino acid sequence such that it contains one or more of the tripeptide sequences described above (for an N-linked glycosylation site). Alterations may also be made by adding or substituting one or more serine or threonine residues to the starting sequence (for the O-linked glycosylation site). Conveniently, the amino acid sequence of the antigen binding protein may be altered by a change at the DNA level, in particular by mutating the DNA encoding the target polypeptide at preselected bases, thereby generating codons which translate into the desired amino acid.
The invention also encompasses the production of antigen binding proteins with altered carbohydrate structures that result in altered effector activity, including antigen binding proteins exhibiting improved ADCC activity with no or reduced fucosylation. Various methods of reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by binding of an antibody molecule to fcγriii receptor, which is shown to depend on the N-linked glycosylated carbohydrate structure at the N297 residue of the CH2 domain. Non-fucosylated antibodies bind this receptor with increased affinity and trigger fcyriii mediated effector functions more effectively than native, fucosylated antibodies. For example, recombinant production of non-fucosylated antibodies in CHO cells in which the alpha-1, 6-fucosyltransferase has been knocked out gives antibodies with 100-fold increased ADCC activity (see Yamane-Ohnuki et al, Biotechnol Bioeng.[ biotechnology and bioengineering ]]87 (5):614-22,2004). By lowering alphaActivity of 1, 6-fucosyltransferases or other enzymes in the fucosylation pathway, e.g. by siRNA or antisense RNA treatment, engineering cell lines to knock out one or more enzymes, or incubation with selective glycosylation inhibitors can achieve a similar effect (see Rothman et al,Mol Immunol.[ molecular immunology ]]26 (12):1113-23,1989). Some host cell lines, such as Lec13 or rat hybridoma YB2/0 cell lines, naturally produce antibodies with a lower degree of fucosylation (see thields et al,J Biol Chem.[ journal of biochemistry ]]277 (30) 26733-40,2002; shinkawa et al,J Biol Chem.[ journal of biochemistry ]]278 (5):3466-73,2003). It has also been determined that ADCC activity can be increased, for example, by recombinantly producing antibodies in cells that overexpress the GnTIII enzyme to increase the aliquot carbohydrate content (see Umana et al,Nat Biotechnol.[ Natural biotechnology]17(2):176-80,1999)。
In other embodiments, glycosylation of an antigen binding protein is reduced or eliminated by, for example, removing one or more glycosylation sites from the Fc region of the binding protein. Amino acid substitutions that eliminate or alter the N-linked glycosylation site may reduce or eliminate N-linked glycosylation of the antigen binding protein. In certain embodiments, the bispecific antigen binding proteins described herein comprise a mutation at position N297 (EU numbering), e.g., N297Q, N297A or N297G. In a particular embodiment, the bispecific antigen binding proteins of the invention comprise an Fc region with an N297G mutation from a human IgG1 antibody. To improve the stability of the N297 mutation-containing molecules, the Fc region of the molecule may be further engineered. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteines to promote disulfide bond formation in the dimer state. Residues corresponding to V259, a287, R292, V302, L306, V323 or I332 (EU numbering) of the IgG1 Fc region may thus be substituted with cysteine. In one embodiment, specific pairs of residues are substituted with cysteines such that they preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond confusion. In certain embodiments, pairs include, but are not limited to, a287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In particular embodiments, the bispecific antigen binding proteins described herein comprise an Fc region with mutations R292C and V302C from a human IgG1 antibody. In such embodiments, the Fc region may further comprise an N297G mutation.
It may also be desirable to modify the antigen binding proteins of the present disclosure to increase serum half-life, for example by incorporating or adding a salvage receptor binding epitope (e.g. by mutating the appropriate region or by incorporating the epitope into a peptide tag, then fusing it with the antigen binding protein at either end or in between, e.g. by DNA or peptide synthesis; see e.g. WO 96/32478) or adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers. The salvage receptor binding epitope preferably constitutes a region in which either or more amino acid residues from one or both loops in the Fc region are transferred to a similar position in the antigen binding protein. In one embodiment, three or more amino acid residues from one or both loops in the Fc region are transferred. In one embodiment, an epitope is taken from the CH2 domain of an Fc region (e.g., an IgG Fc region) and transferred to the CH1, CH3, or VH region of an antigen binding protein, or more than one such region. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL or VL region, or both, of the antigen binding protein. For a description of Fc variants and their interactions with salvage receptors, see International applications WO 97/34631 and WO 96/32478.
Antigen binding proteins include variants having single or multiple amino acid substitutions, deletions, additions or substitutions that retain their biological properties. One of ordinary skill in the art can produce variants having single or multiple amino acid substitutions, deletions, additions or substitutions. These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acids, (b) variants in which one or more amino acids are added to or deleted from a polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which a polypeptide is fused to another peptide or polypeptide, such as fusion partners, protein tags, or other chemical moieties, which may confer useful properties on the polypeptide, e.g., epitopes of antibodies, polyhistidine sequences, biotin moieties, and the like. Antibodies and bispecific antibodies of the invention may include variants in which amino acid residues from one species are substituted at conserved or non-conserved positions with corresponding residues in another species. In another embodiment, the amino acid residue at a non-conserved position is substituted with a conserved or non-conserved residue. Techniques for obtaining these variants (including genetic (suppression, deletion, mutation, etc.), chemical and enzymatic techniques) are known to those of ordinary skill in the art.
Therapeutic method
TREM-1 is associated with cardiovascular indications. TREM-1 has been shown to be expressed in human atherosclerosis and in Apoe -/- Up-regulation of expression in the circulation and lesions in the murine model infiltrate the dyslipidemia in myeloid cells. Proof of Trem1 -/- Apoe -/- Mice exhibit reduced diet-induced atherosclerosis formation. (Zysset et al, nat. Commun. [ Natural communication)]7:1-16,2016). Boufenzer et al (circle. Res. [ cycle study ]]2015, 116:1772-1782) demonstrated that TREM1 deficiency inhibited recruitment and activation of inflammatory cells to infarcted myocardium, confirming that inhibition of TREM-1 using short peptide or gene knockout reduced myocardial infiltration of neutrophils and improved cardiac function and survival after mice and rats MI. In humans, post-MI TREM-1 activation (as evidenced by elevated levels of sTREM-1) is a predictor of mortality (Boufenzer et al, supra).
Lui et al (nat. Immunol [ natural immunology ]20:1023-1034, 2019) showed that peripheral myeloid cells amplified stroke lesions via activation of TREM 1. Brain-infiltrating neutrophils and macrophages highly express Trem1 (MCAo mouse model) activated by immunogenic cellular components such as risk/injury related molecular patterns (DAMP) (Colonna, m.trends in Immun 2019, 40:781-783). Inhibition of Trem1 by decoy peptide or Trem-1 knockout revealed reduced infiltration of neutrophils/macrophages to the injury site, smaller infarct volume, improved neurological score and increased survival (Lui et al, supra; colonna, supra).
TREM-1 is also associated with the progression of NAFLD/NASH. Rao et al, (J Cell Biochem [ journal of Cell biochemistry ]120:11867-11877,2019) showed that overexpression of Trem1 in mouse hepatocytes resulted in increased lipid accumulation, and that genes involved in lipid uptake (e.g., ldlr, msr 1) were upregulated when Trem1 was overexpressed. In addition, genes involved in cholesterol efflux (e.g., abca1, abcg 1) are down-regulated when Trem1 is overexpressed. Oleic acid treatment of Trem1 overexpressing hepatocytes (HepG 2, PMH) resulted in increased inflammatory cytokine expression. The use of shRNA knockout TREM-1 reduced lipid accumulation in high fat diet mice.
Obesity is characterized by chronic systemic inflammation (elevated tnfα, IL6 and hsCRP), and TREM-1 has been shown to be elevated in obese patients. It is speculated herein that treatment with anti-TREM 1, optionally in combination with anti-obesity drugs, will result in a greater reduction of systemic inflammation.
The antigen binding proteins disclosed herein that are specific for TREM-1 are contemplated herein to be useful in the treatment of cardiovascular disease. Exemplary cardiovascular diseases include myocardial infarction, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), heart failure, stroke (ischemic and ischemic), atherosclerosis, coronary heart disease, peripheral vascular disease (e.g., peripheral arterial disease), vulnerable plaque, acute coronary syndrome, cerebrovascular disease, cerebrovascular atherosclerosis, and obesity.
In certain embodiments, the measurement of TNF- α inhibition is performed using isolated PBMCs. PBMCs are isolated from the blood of a subject (human, mouse, rat, cynomolgus monkey, etc.), stimulated in vitro with LPS or TREM-1 ligand (peptidoglycan recognition protein complexed with bacterial peptidoglycan), and levels of tnfα before and after stimulation are determined using, for example, ELISA, ALPHALISA or MSD Vplex tnfα detection kit in the presence of different test molecules.
In one embodiment, the present disclosure provides a method of inhibiting one or more pro-inflammatory cytokines (e.g., tnfα) in a mammal in need of such treatment, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of an antigen binding protein or antibody of the present disclosure. In a preferred embodiment, the subject is a mammal. In one embodiment, the subject is a human. The method is useful for treating diseases characterized by elevated tnfα expression or activity. The antigen binding proteins or antibodies of the disclosure may be administered with another agent, in the same formulation, or separately.
Additional assays contemplated include measuring the concentration of highly sensitive C-reactive protein, the concentration of soluble TREM-1, ex vivo analysis of phosphorylated spleen tyrosine kinase in isolated PBMCs, and analysis of myeloid cell subsets.
This treatment is expected to reduce the levels of inflammatory cytokines such as TNF- α and IL-6 in the subject.
It is contemplated that treatment with an anti-TREM-1 antigen binding protein of the present disclosure comprising an anti-TREM-1 binding protein can reduce TREM-1 expression in inflammatory cells.
Administration and administration
The method of the present disclosure comprises the steps of: administering a pharmaceutical composition comprising an antigen binding protein as described herein. In certain embodiments, the pharmaceutical composition is a sterile composition.
The amount of the therapeutic composition at a given dose may vary depending on the size of the individual to whom the therapy is being administered and the characteristics of the disorder being treated.
The present disclosure provides compositions comprising an antigen binding protein or antibody of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the antigen binding proteins or antibodies of the present disclosure can be formulated according to known methods to prepare pharmaceutically useful compositions, thereby combining the therapeutic antibodies with a pharmaceutically acceptable carrier as a mixture. If administration of the composition is tolerated by the patient to whom it is administered, it is said to comprise a "pharmaceutically acceptable carrier". Sterile phosphate buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. See, for example, the Getman edit, Remington's Pharmaceutical Sciences [ Lemington pharmaceutical science ]]19 th edition, mack Publishing Company [ Mark publishing Co., ltd](1995)。
For pharmaceutical use, the polypeptides of the present disclosure are formulated for parenteral, particularly intravenous or subcutaneous delivery according to conventional methods. Intravenous administration may be achieved by bolus injection, controlled release (e.g., using micro-infusionA type pump or other suitable technique), or by infusion over a typical period of one to several hours. Generally, pharmaceutical formulations will include an antigen binding protein or antibody of the disclosure in combination with a pharmaceutically acceptable carrier (e.g., saline, buffered saline, 5% dextrose in water, and the like). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent loss of protein at the vial surface, and the like. When such combination therapies are utilized, the antigen binding proteins or antibodies of the disclosure may be combined in a single formulation, or may be administered in separate formulations. Methods of formulation are well known in the art, for example in Gennaro editions,Remington's pharmaceutical Sciences [ Leimgton pharmaceutical science ]]Mack Publishing Co [ Mike publishing Co., ltd ]]Easton Pa. [ Iston, pa.) ](1990) Which is herein incorporated by reference. The therapeutic dose is generally in the range of 0.1 to 100mg/kg of patient body weight per day, preferably 0.5-20mg/kg per day, with the particular dose being determined by the clinician according to accepted criteria, taking into account the nature and severity of the condition to be treated, the characteristics of the patient, etc. Determination of the dosage is well within the level of ordinary skill in the art. More commonly, antibodies will be administered over a period of one week or less, typically over a period of one to three days. Generally, the dosage of antibody administered will vary depending on factors such as the age, weight, height, sex, general physical condition and previous medical history of the patient. Typically, it is desirable to provide the recipient with a dosage of antibody in the range of about 1pg/kg to 10mg/kg (dose/patient weight), although lower or higher dosages may also be administered as appropriate.
Administration of the antigen binding proteins or antibodies of the disclosure to a subject can be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by regional catheter infusion, or by direct intralesional injection. In various embodiments, administration is intravenous or subcutaneous. When the antigen binding protein is administered by injection, administration may be by continuous infusion or by single or multiple bolus injections.
Other routes of administration include oral, mucosal-membrane, pulmonary, and transdermal. Oral delivery is suitable for polyester microsphere, jadeMilkinol-soluble protein microspheres, proteoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, e.g., diBase et al, "Oral Delivery of Microencapsulated Proteins [ oral delivery of microencapsulated proteins ]]"in Sanders et al, editors,protein Delivery: physical Systems [ Protein Delivery: physical system]Pages 255-288, plenum Press [ Proneum Press ]](1997) In (c) a). The feasibility of intranasal delivery is exemplified by the manner in which such insulin is administered (see, e.g., hinchcliffe et al,adv. Drug Deliv. Rev. [ advanced drug delivery comment ]],35:199 (1999)). Dry or liquid particles comprising the antibodies of the invention may be prepared and inhaled by means of a dry powder dispenser, a liquid aerosol generator or a nebulizer (e.g., pettit et al,TIBTECH[ biotechnological trend ]]16:343 (1998); the color of the pattern et al,adv, drug Deliv, rev [ advanced Drug delivery review ]],35:235(1999))。The method is illustrated by a diabetes management system, which is a hand-held electronic inhaler, that delivers aerosolized insulin to the lungs. Studies have shown that proteins up to 48,000kda have been passed through the skin with the aid of low frequency ultrasound at therapeutic concentrations, indicating the feasibility of transdermal administration (mitrogotri et al, Science [ Science ]],269:850(1995))。
For therapeutic purposes, compositions comprising the antigen binding proteins or antibodies of the present disclosure and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. If the amount administered is physiologically significant, it is referred to as a "therapeutically effective amount" of the antigen binding proteins or antibodies of the present disclosure in combination with a pharmaceutically acceptable carrier. An agent is physiologically significant if its presence causes a detectable change in the physiology of the patient receiving treatment. For example, it is physiologically significant if the presence of an agent for treating inflammation can reduce the inflammatory response. Effective treatment can be assessed in a variety of ways. In one embodiment, effective treatment is determined by reduced inflammation. In other embodiments, effective treatment is marked by inhibition of inflammation. In still other embodiments, effective therapy is measured by an increase in the patient's health condition, including signs such as weight gain, strength recovery, pain reduction, physical improvement, and subjective indications from the patient of better physical condition.
It is contemplated that an antigen binding protein, e.g., comprising an anti-TREM-1 antibody sequence, is administered at a dose of 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 125mg, 150mg, 200mg, 250mg, 300mg or more. In various embodiments, the antigen binding protein, e.g., comprising an anti-TREM-1 antibody sequence, is administered at a dose of about 0.05mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1.0mg/kg, 1.25mg/kg, 1.5mg/kg, 2.0mg/kg, 2.5mg/kg, 3.0mg/kg, 3.5mg/kg, 4.0mg/kg, 4.5mg/kg, or 5 mg/kg.
The compositions described herein are administered weekly, twice weekly, biweekly, every three weeks, every 4 weeks, monthly, every 3 months, every six months, or yearly.
Pharmaceutical compositions comprising the antigen binding proteins or antibodies of the present disclosure may be provided in liquid form, aerosol form, or solid form. Liquid forms are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled release forms. The latter form is illustrated by way of example with micropump and implant (Bremer et al,pharm, biotechnol [ pharmaceutical biotechnology]10:239 (1997); ranade, "Implants in Drug Delivery [ implant in drug delivery ]]"in Ranade et al, editorial,drug Delivery Systems [ drug delivery Delivery system]Pages 95-123, CRC Press [ CRC Press ]](1995) In (a) and (b); bremer et al, "Protein Delivery with Infusion Pumps [ delivery of proteins with infusion pumps ]]"in Sanders et al, editors,Protein Delivery: physical Systems [ protein delivery: physical system]Pages 239-254, plenum Press [ Proneum Press ]](1997) In (a) and (b); yewey et al, "Delivery of Proteins from a Controlled Release Injectable Implant [ delivery of proteins from controlled release injection implants ] ]"in Sanders et al, editors,protein Delivery: physical Systems [ Protein Delivery: physical system]Pages 93-117, plenum Press [ Proneum out ]Version society](1997) In (c) a).
The formulations may also contain more than one active compound as necessary for the particular indication being treated, preferably those active compounds having complementary activities without adversely affecting each other. Alternatively, or in addition, the composition may include an agent that enhances its function, e.g., a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.
In one embodiment, the antigen binding proteins or antibodies of the disclosure are administered in combination therapy, i.e., in combination with other agents (e.g., therapeutic agents useful in treating pathological conditions or disorders, such as cardiovascular diseases). In this context, the term "combination" means that the agents are administered substantially contemporaneously, or simultaneously or sequentially. If administered sequentially, the first of the two compounds is preferably still detectable at the treatment site at an effective concentration at the time the second compound begins administration.
It is contemplated that the therapeutic agents of the present disclosure may be administered simultaneously in the same formulation. It is further contemplated that the agents are administered as separate formulations and concurrently, that is, agents administered within 30 minutes of each other. It is further contemplated that the second agent may be administered simultaneously.
In another aspect, the antigen binding protein as described herein is administered prior to administration of the second composition. Prior administration refers to administration of an agent in a range from one week prior to treatment with another agent to 30 minutes prior to administration of the other agent. It is further contemplated that the agent is administered subsequent to the administration of another composition or agent. Subsequent administration refers to administration from 30 minutes after antibody treatment to one week (e.g., 30 minutes, 1 hour, 2 hours, 4 hours, 1 day, 2 days, etc.) after antibody administration. Further consider, a second
For example, combination therapies may include one or more antigen binding proteins or antibodies of the disclosure co-formulated and/or co-administered with, for example, one or more of the following additional therapeutic agents: one or more cholesterol (serum and/or total internal cholesterol) lowering agents, agents which increase LDLR expression, statins (atorvastatin)Statin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), PCSK9 inhibitorsNicotinic acid (niacin) (-)> (sustained release of Nick acid), ->(sustained release of niacin)), fibric acid (>(gemfibrozil),>(fenofibrate)), bile acid sequestrant (a. About.) >(cholestyramine), colesevelam->(norbilin)), cholesterol absorption inhibitors (Zetia), combined nicotinic acid with statins (++>(lovastatin and->) Combinations of statins with absorption inhibitors (VYTORIN ()>And->) Lipid modulators like PCSK9 inhibitors, ppary agonists, ppara/y agonists, squalene synthetase inhibitors, cholesteryl Ester Transfer Protein (CETP) inhibitors, antihypertensive agents, antithrombotic agents (aspirin), antidiabetic agents (e.g. sulfonylurea, insulin, GLP-1 analogues, DDPIV inhibitors, SGL2 inhibitors), apoB modulators, MTP inhibitors,(ivabradine) I (f) current inhibitors, omecambaryophyllin activators, OLPASIRAN (AMG 890) that reduce lipoprotein (a) (siRNA), AMG 594 cardiac troponin activators, AMG 609, AMG 171 (growth differentiation factor 15 (GDF 15) analogues), AMG 133 (gastric inhibitory peptide receptor (GIPR) antagonists and glucagon-like peptide 1 (GLP-1) receptor agonists), and/or occlusive arteriosclerosis therapeutics.
Therapeutic agents used in combination with the antigen binding proteins or antibodies of the present disclosure include agents that interfere at different stages in the inflammatory response. In one embodiment, the antigen binding proteins or antibodies of the disclosure may be co-formulated and/or co-administered with one or more additional agents, such as: other cytokines or growth factor antagonists (e.g., soluble receptors, peptide inhibitors, small molecules, ligand fusions); or an antibody or antigen-binding fragment thereof that binds to other targets (e.g., an antibody that binds to other cytokines or growth factors, receptors thereof, or other cell surface molecules); an anti-inflammatory cytokine or agonist thereof. Non-limiting examples of agents that may be used in combination with the antibodies described herein include, but are not limited to, antagonists of one or more Interleukins (IL) or their receptors, e.g., antagonists of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL17A-F, IL-18, IL-20, IL-21, IL-22, IL-23IL-25, IL-31, IL-32, IL-33; antagonists of cytokines or growth factors or their receptors, such as LT, EMAP-II, GM-CSF, FGF and PDGF. Antibodies of the invention can also be used in combination with, for example, antibodies directed against cell surface molecules (e.g., CD2, CD3, CD4, CD8, CD20 (e.g., CD20 inhibitors) Rituximab) An inhibitor combination of antibodies to CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or a ligand thereof (including CD154 (gp 39 or CD 40L)), or LFA-1/ICAM-1 and VLA-4/VCAM-1 (Yueuf-Makagiansar et al,Med.Res.Rev. [ drug research evaluation ]],22:146-167 (2002)). Exemplary antagonists that may be used in combination include antagonists of IL-1, IL-6, IL-12, TNF alpha, IL-15, IL-18, IL-20, IL-22, IL-23, and IL-31.
In other embodiments, one or more antigen binding proteins of the present disclosure may be co-formulated and/or co-administered with one or more anti-inflammatory agents, immunosuppressants, or metabolic or enzymatic inhibitors. Non-limiting examples of drugs or inhibitors that may be used in combination with the antibodies described herein include, but are not limited to, one or more of the following: one or more non-steroidal anti-inflammatory drugs (NSAIDs), for example, ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids, such as prednisolone; one or more cytokine inhibitory anti-inflammatory drugs (CSAID); inhibitors of nucleotide biosynthesis, for example, inhibitors of purine biosynthesis, folic acid antagonists (e.g., methotrexate (N- [4- [ [ (2, 4-diamino-6-pteridinyl) methyl ] methylamino ] benzoyl ] -glutamic acid), and inhibitors of pyrimidine biosynthesis, for example, dihydroorotate dehydrogenase (DHODH) inhibitors.
Additional inhibitors include one or more of the following: corticosteroids (oral, inhaled and topical injection); immunosuppressants, e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g., sirolimus (rapamycin-)) Or rapamycinA derivative of a hormone, e.g., a soluble rapamycin derivative (e.g., an ester rapamycin derivative, e.g., CCI-779); agents that interfere with signaling through pro-inflammatory cytokines (e.g., IL-1) (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, and variants thereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesterase type IV inhibitor); phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA 2) (e.g., trifluoromethyl ketone analogues); inhibitors of vascular endothelial growth factor or growth factor receptor, for example, VEGF inhibitors and/or VEGF-R inhibitors; and inhibitors of angiogenesis. Preferred therapeutic agents for use in combination with the antigen binding proteins or antibodies of the present disclosure are immunosuppressants, e.g., cyclosporin, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus (rapamycin) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2 inhibitors, e.g., celecoxib and variants thereof; and phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA 2) (e.g., trifluoromethyl ketone analogs).
Kit for detecting a substance in a sample
The present disclosure also contemplates kits comprising one or more containers comprising the antigen binding proteins or antibodies of the disclosure, optimally in a pharmaceutically acceptable carrier or composition. The antigen binding proteins or antibodies of the present disclosure may be provided as unit doses or as sterile powders to be reconstituted prior to injection in the form of single or multiple doses of injectable solutions. Alternatively, such kits may include a dry powder dispenser, a liquid aerosol generator, or a nebulizer for administering one or more therapeutic agents. Such kits may further comprise instructions and written information regarding the indication and use of the pharmaceutical composition.
Syringes, such as single-use or prefilled syringes, sterile sealed containers (e.g., vials, bottles, containers, and/or kits or packages) containing any of the above antigen binding proteins or compositions, optionally with suitable instructions, are also contemplated.
In another embodiment, the invention provides an article of manufacture or unit dosage form comprising: (a) A composition of matter comprising an antigen binding protein or antibody of the disclosure; (b) a container containing the composition; and (c) a label affixed to the container, or a package insert included in the container, relating to the use of the antibody for treating an immune-related disorder.
In another aspect, the composition or kit comprises an additional active ingredient, which may be, for example, an additional antibody or an anti-inflammatory agent, cytotoxic agent, or other agent described herein. Preferably, the composition is sterile.
Examples
Example 1-anti-TREM-1 antibody
High affinity anti-TREM-1 monoclonal antibodies were generated and evaluated for the ability to target immune cells and modulate signaling between TREM-1 and its ligands.
By aligningTransgenic mice were immunized to produce fully human antibodies to human TREM-1. See, for example, U.S. patent No. 6,114,598;6,162,963;6,833,268;7,049,426; and 7,064,244.
Mice were immunized with human and/or cynomolgus TREM1 protein, TREM1 expression vector and/or CHO cells expressing TREM 1. For genetic immunization, the instructions of the manufacturer are followed by the use ofThe gene gun system (BioRad, heracles, calif.) was used to immunize mice 16 times over 8 weeks. Briefly, expression vectors encoding human TREM-1 and DAP12, or cynomolgus TREM-1 and DAP12, were combined, 2 μg of total DNA was coated onto 1.6um gold beads (Berle Corp., heracles, calif.), and delivered into the epithelium of the abdomen of shaved mice. For soluble protein immunization, mice were recombined with human or cynomolgus TREM-1 representing the N-terminal extracellular domain The protein is immunized. Animals were vaccinated with Alum and CpG-ODN or Sigma (Sigma) adjuvant system (Sigma Adjuvant System) adjuvanted recombinant proteins and delivered at two locations along the dorsal midline of mice located in the coccyx and subscapular regions, with 14-17 subcutaneous injections over 10-12 weeks. The initial soluble protein immunization delivered 10 μg followed by a booster of 5 μg. For cell immunization, mice were immunized with 200-400 ten thousand CHO-S cells transiently expressing human TREM-1 or cynomolgus TREM-1 adjuvanted with Alum and CpG-ODN. Animals were given 1 or 2 injections per week for 10 weeks, 13 total injections, alternating intraperitoneal and subcutaneous injections of the tail root. At various time points (from 4 weeks to 10 weeks) during the immunization study, animals were bled and plasma was collected to assess TREM1 specific titers. By ACCURI using transiently transfected 293T cells TM Live cell FACS analysis on a flow cytometer (BD Biosciences) monitored TREM1 specific plasma titers. Animals with the highest antigen-specific plasma titers against human and cynomolgus TREM1 were sacrificed and used for hybridoma production (Kohler and Milstein, 1975).
Hybridoma production: animals exhibiting appropriate antigen-specific serum titers were identified and spleen and/or draining lymph nodes were pooled from selected mice harvested at each time. By grinding in a suitable medium or using a semi-automatic tissue dissociator GENTLEMACS TM The dissociator (Miltenyi Biotec), dissociation of spleen cells and lymphocytes from lymphoid tissue. The IgG-expressing B cells are isolated, expanded, and fused with a suitable cell fusion partner using standard methods. Through CELLINSIGHT TM Hybridoma supernatants were tested for binding to human TREM-1 transiently expressed on HEK293 cells. Briefly, HEK293 cells were transiently co-transfected with mammalian expression constructs encoding human TREM-1 and DAP12 at a 1:1 ratio or with mock vector and DAP12 alone using 293Fectin (invitrogen) according to the manufacturer's protocol. The next day, 15,000 cells/well of transfected HEK293 cells were assayed with an equal volume of spent hybridoma medium and a final concentration of 15 μg/mL of nuclear stain Hoechst 33342 (Pierce) was combined in 384-well FMAT plates (Corning) in a total volume of 30 μl/well. After incubation for 1 hour at room temperature, using The plate washer aspirates the supernatant and washes the wells on AquaMax for 2 cycles, each with 50 μl/well FACS buffer (PBS (hclone), 2% FBS (sigma)). Cells were stained with 5 μg/mL Alexa Fluor 488 goat anti-human IgG Fc (jackson immunoresearch company (Jackson ImmunoResearch)) secondary antibody, shaken on a Big Bear plate shaker, and incubated at room temperature for 20 minutes. Use->The plate washer aspirates the supernatant, washes the wells again for 2 cycles using 50 μl/well FACS buffer, and adds 30 μl of FACS buffer to each well using a multi-point instrument. The plate was placed on a Big Bear plate shaker to evenly distribute the cells in the wells, then at CELLINSIGHT TM The CX7 platform was read using Cell Health Profiling Bio-App.
Cross-reactivity of the TREM-1 specific antibodies identified in the primary screening was evaluated for TREM-1, as well as for TREM-1, but not DAP 12. By FACS (harvests 1-6) or CELLINSIGHT TM (harvest 8-9) test TREM-1 hybridoma supernatants for binding to human or cynomolgus TREM-1 transiently expressed on HEK293 cells. For the TREM1 antibody from harvest 1-6, HEK293 cells were transiently co-transfected with mammalian expression constructs encoding human TREM-1 and DAP12, cynomolgus TREM-1 and DAP12 at a 1:1 ratio or mock vector and human DAP12 using 293 Fectin. The next day, transfected HEK293 cells were transferred into 96-well FACS plates at 50,000 cells/well and incubated with normalized hybridoma supernatant at a final concentration of 2.5 μg/mL for 1 hour at 4 ℃. Cells were then pelleted by centrifugation, the supernatant removed by tumbling, and the wells washed twice with 200 μl/well FACS buffer. mu.g/mL ALEXA was added 647 goat anti-human IgG Fc (Jackson immune research Co.) secondary detection antibody and 2.5 μg/mL 7-amino actinomycin-D (Sigma Co.) viability stain were incubated with cells for 15 min at 4 ℃. Cells were pelleted by centrifugation, supernatant removed by tumbling, and wells were washed once more with 200 μl/well FACS buffer. BD ACCURI with Intellicyt automatic sampler TM The supernatant of the TREM-1 hybridoma showing specific binding of human TREM-1 or cynomolgus monkey TREM-1 was detected by FACS on a C6 flow cytometer. Data are reported as Geometric Mean (GM) fold relative to irrelevant control antibody binding. The binding results for certain anti-TREM-1 antibodies are shown in Table 1.
Table 1: TREM1 antibodies in cross-reactivity and specificity screening of human/cynomolgus monkeys
The ability of TREM1 antibodies with high quality TREM1 specific binding and human/cynomolgus monkey cross-reactivity to block the binding of ligand PGLYRP1 to human TREM1/DAP12 transiently expressed on HEK293 cells was evaluated. Briefly, HEK293 cells were transiently co-transfected with mammalian expression constructs encoding human TREM1 and DAP12 using 293Fectin at a 1:1 ratio or mock vector and human DAP 12. The next day, transfected HEK293 cells were transferred into 96-well FACS plates at 50,000 cells/well and incubated with normalized hybridoma supernatant at a final concentration of 2.5 μg/mL for 1 hour at 4 ℃. Human PGLYRP1-His (R) &D systems Co (R)&Dssystems)) was combined with PGN-ecldss (peptidoglycan, invitogen company) and incubated at room temperature for 15 minutes, and then added to the wells at final concentrations of 7.5 μg/mL PGLYRP1 and 30 μg/mL PGN. The plates were then shaken and incubated at 4℃for 15 minutes. Cells were then pelleted by centrifugation, the supernatant removed by tumbling, and the wells washed with 200 μl FACS buffer. mu.g/mL ALEXA was added647 human anti-His secondary detection antibody and 2.5. Mu.g/mL 7-amino actinomycin-D (Sigma) viability stain were added to the cells, shaken, and incubated at 4℃for 15 min. Cells were washed with FACS buffer, pelleted by centrifugation, supernatant removed by tumbling, and washed once more with FACS buffer. Then in the presence of Intellicyt +.>BD ACCURI of an auto sampler TM Cells were run on a C6 flow cytometer. A total of 518 TREM1 antibodies showed the specific PGLYRP1 blocking activity required. Table 2 summarizes the receptor-ligand inhibition of selected TREM-1 monoclonal antibodies.
Table 2: receptor-ligand inhibition of selected TREM1 antibodies
Relative affinity ranking of TREM-1 antibodies by restriction antigen assay: in useIn the restriction antigen assay performed on FACS with beads (Lu Mingke s (Luminex)), TREM1 hybridoma supernatants were affinity ordered in groups by their binding kinetics to soluble TREM-1. Briefly, internally biotinylated human TREM-1-His antigen (b-huTREM-1-His) was serially diluted in FACS buffer and combined with an equal volume of +. >Bead (each antigen concentration with a different unique barcode coded bead) combination, starting with a final b-huTREM-1-His antigen concentration of 30ng/mL, produced a 5-point 2-fold serial dilution series. Antigen-bead mixtures were plated in 3 wells of a 96-well FACS plate and then incubated at room temperature for 30 minutes in the dark. The beads were then pelleted by centrifugationThe supernatant was removed by flipping and the wells were washed twice with 200 μl/well FACS buffer. The different beads are then resuspended, pooled and +.>Immunoassay stabilizers (SurModics) were diluted to block non-specific binding. Normalized TREM-1 hybridoma supernatants were combined with an equal volume of bead mix in FACS plates at a final concentration of 5. Mu.g/mL test antibody to 0.5. Mu.L beads/well. The plates were then shaken and incubated overnight at room temperature for 18 hours. The beads were then pelleted by centrifugation, the supernatant removed by tumbling, and the wells washed twice with 200 μl/well FACS buffer. ALEXA->A488 goat anti-human IgG F secondary detection antibody (Jackson immune research Co.) was added to the plate at 5 μg/mL, shaken, and incubated at room temperature for 15 minutes in the absence of light. The beads were washed with FACS buffer, precipitated by centrifugation, the supernatant removed by tumbling, and washed once more with FACS buffer. The beads were then resuspended and the beads were then loaded with Intellicyt +. >BD ACCURI of an auto sampler TM C6 flow cytometer.
A TREM-1 hybridoma sample exhibiting at least a double or greater signal compared to a control IgG antibody sample is considered to have a TREM-1 specific binding profile. Antibody binding signals are associated with antibody affinities; the extent of binding of the antibody to the target antigen TREM-1 correlates with the measured fluorescence intensity, so that the affinities of the whole group can be compared relatively. In restriction antigen screening, TREM-1 antibody with better binding than reference antibody 1B2 was advanced to light chain sequencing and human/cynomolgus monkey affinity gap analysis. Table 3 shows the antibody binding data for selected TREM-1 antibodies using representative antigen coating concentrations in the linear range of instrument signal detection.
Table 3: relative affinities of TREM1 antibodies selected in limited antigen binding assays
Relative epitope grouping/analysis of TREM 1: usingBeads (Lu Mingke s) the TREM1 hybridoma supernatants were evaluated on FACS for epitope grouping assays (modified antibody-antibody competition assays) to determine the type of relatively unique epitope groupings in the panel. Briefly, a set of 15 different unique bar code encodedThe beads were each combined with an equal volume of internally biotinylated human TREM-1-His antigen diluted in FACS buffer to a final concentration of 100 ng/mL. Antigen-bead mixtures were plated in 3 wells of a 96-well FACS plate and then incubated at room temperature for 30 minutes in the dark. The beads were then pelleted by centrifugation, the supernatant removed by tumbling, and the wells washed twice with 200 μl/well FACS buffer. 15 different TREM-1 antibodies with different VDJ rearrangements and good quantification were selected, which showed good binding in the restriction antigen assay, as reference antibodies to the pre-coated beads. These 15 antibodies were prepared in FACS buffer at a saturation concentration of 5. Mu.g/mL and were different from 15 +. >Each of the beads was incubated at room temperature for 1 hour in the dark. The beads were pelleted by centrifugation, the supernatant removed by tumbling, and the wells washed three times with 200 μl/well FACS buffer. The different beads are then resuspended, pooled and +.>Immunoassay stabilizers (SurModics) were diluted to block non-specific binding. Normalized TREM-1 hybridoma supernatant (test antibody) and an equal volume of bead mix were placed in FACS platesThe test antibodies were combined at a final concentration of 5. Mu.g/mL to 0.5. Mu.L beads/well. The plates were then shaken and incubated at room temperature for 1 hour in the dark. The beads were then pelleted by centrifugation, the supernatant removed by tumbling, and the wells washed twice with 200 μl/well FACS buffer. ALEXA->A488 goat anti-human IgG F secondary detection antibody (Jackson immune research Co.) was added to the plate at 5 μg/mL, shaken, and incubated at room temperature for 15 minutes in the absence of light. The beads were washed with FACS buffer, precipitated by centrifugation, the supernatant removed by tumbling, and washed once more with FACS buffer. The beads were then resuspended and the beads were then loaded with Intellicyt +.>BD ACCURI of an auto sampler TM C6 flow cytometer.
Test antibodies that compete with the reference antibody for an epitope on the TREM-1 antigen are prevented from binding, while non-competing antibodies are able to bind to the reference antibody to generate an additive signal. The total antibodies were then detected with secondary antibodies. To determine the antibody competition/binding profile of the individual test antibodies, the reference antibody binding signal alone was subtracted from the reference antibody plus test antibody signal for each competition/binding reaction (i.e., the entire reference antibody set). An overview of the relative epitope groupings of the selected TREM-1 antibodies is shown in table 4 below.
Table 4: relative epitope grouping of selected pre-TREM 1 antibodies
TREM1 antibody human/cynomolgus monkey affinity gap determination: the affinity of TREM-1 antibodies with unique CDR3 sequences for human and cynomolgus TREM-1 was analyzed, these antibodies being limitingAntigen screening also showed better binding than the reference antibody. UsingHTX instruments (Boerful Di Biolabs) determined the binding affinity K of 114 TREM-1 antibodies D (M) association Rate constant k a (M -1 s -1- ) Dissociation rate constant k d (s -1 ). In short, by->Assay buffer (10 mM Tris,0.1% Triton X-100, 150mM NaCl,1mM CaCl) 2 TREM-1 hybridoma supernatants normalized to 10 μg/mL in DMEM blank medium (null media) were prepared at 1:10 dilution to a final test concentration of 1 μg/mL in 0.1mg/mL BSA, pH 7.6). Amine reactive second generation AR2G biosensors (molecular instruments (Molecular Devices)) were pre-incubated in 200 μl of nanopore water for at least 10 minutes prior to use. AR2G biosensor was then assayed in 20mM EDC (1-ethyl-3- [ 3-dimethylaminopropyl group)]Carbodiimide hydrochloride) (Poerful Di biological company) was activated for 5 minutes with a solution of 10mM NHS (N-hydroxysulfosuccinimide) (Poerful Di biological company) pre-mixed in nanopore water. The internally generated mouse anti-human Fc monoclonal antibody was coupled to the AR2G biosensor at 10 μg/mL in 10mM sodium acetate buffer at pH 5 for 5 min, quenched with 1M ethanolamine at pH 8.5 for 5 min, and then used to capture the antibody from solution. The TREM-1 test antibody was loaded onto the biosensor for 5 minutes and baseline measurements were taken for 1 minute. Recombinant soluble human TREM-1-His protein was then combined with antibody loaded biosensors in a 3-fold dilution series covering 6 spots from 450nM to 1.85nM or 150nM to 0.62 nM. The association of recombinant human TREM-1 with antibody-loaded sensor was measured for 5 min, followed by +. >Dissociation in buffer for 10 min. The biosensor was then regenerated with 10mM glycine at pH 1.7 and the same TREM-1 antibody was used for the same sensingThe device was reloaded for 5 minutes, and the association and dissociation of cynomolgus TREM-1-His protein were measured in the same way. Data were referenced using an analyte reference sensor of 0 nM. Kinetic analysis was performed in Genedata Screener software using a 1:1 Langmuir mass transfer model. TREM-1 antibodies meeting design goals were identified and showed less than 10-fold difference in affinity between human and cynomolgus TREM-1, with binding affinities shown in table 5.
Table 5: TREM1 antibodies with < 10-fold affinity gap between human and cynomolgus monkey TREM-1
*A<The symbols indicate that less than 10% of the TREM1 dissociates within the assigned 10 minute dissociation time, indicating k d <1.76E-4s -1 . According to k d <1.76E-4s -1 Calculation of<K D (M)。
The selection of antibodies is based on binding to human or cynomolgus TREM-1, lack of binding to TREM2 and their ability to block PGLYRP1 binding to TREM-1. As listed in table 6, 14 antibodies were selected for further study.
Table 6: anti-TREM-1 antibody clones
TABLE 7 full sequence of anti-TREM-1 antibodies
TABLE 8 TREM-1 antibody variable region sequences
TABLE 9 TREM-1 antibody clone light chain CDR sequences
TABLE 10 TREM-1 antibody clone heavy chain CDR sequences
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Example 2 reformatting of anti-TREM-1 antibodies
By fusing the VL domain of the kappa light chain to the CK domain, fusing the VL domain of the lambda light chain to the CL domain, fusing the VH domain to the CH1-CH2-CH3 (221-447) sequence, the light chain will be fused to the VL domain of the kappa light chainThe active lead anti-TREM-1 antibody was converted to an antibody format of IgG1z subtype. The CH2 domain of this antibody isotype was engineered to reduce effector function by introducing an N297G mutation and to improve thermostability by engineered disulfide bonds (R292C, V302C); this antibody isotype was designated IgG1z SEFL2. The lead anti-TREM-1 antibodies were further engineered to remove "hot spots", i.e., residues that negatively affect the expression, purification, thermostability, colloidal stability, long-term storage stability, in vivo pharmacokinetics and/or immunogenicity of the molecule, either computationally predicted or empirically determined. The various amino acid mutations at these hot spots are designed based on prior knowledge of conservation, co-denaturation, chemical similarity, predictions of structural models, and other antibody engineering activities. Engineered antibodies were designed for a small combinatorial theory, including both single mutations and combinations of mutations.
Recombinant expression constructs of rationally designed hot spot engineered variants were produced using the Golden Gate (Golden Gate) cloning method to assemble 1) a construct comprising antibody variable structuresDomain synthesis DNA fragments, 2) previously cloned "partial vectors" containing the necessary constant domains (i.e., CK or CL, CH1-CH2-CH3 (118-447) (R292C, N297G, V C), and 3) mammalian expression vector backbones. The Heavy Chain (HC) was assembled into the carrier backbone with the puromycin selection cassette and the Light Chain (LC) was assembled into the carrier backbone with the hygromycin selection cassette. HC and LC expression vectors were co-transfected in CHO-K1 cells using Lipofectamine LTX (Ji Buke company (Gibco)) at a 1:1 ratio by passaging once every 2-3 days in the presence of 10ug/mL puromycin and 500ug/mL hygromycin until cell viability>90% (Vi-CELL BLU, beckman Coulter) to create a stable pool. The stable pool was inoculated in 2e6 viable cells/mL culture in production medium and at 36℃at 5% CO 2 And incubated for 6 days. Cell supernatants were harvested by centrifugation and AmMag was used TM Protein A magnetic beads (GenScript) or MAG SEPHAROSE TM Prism a (sitova) purified antibodies by magnetic bead affinity chromatography. The identity of each variant was confirmed by complete mass spectrometry. For each variant, protein a sensor was used to pass the company porfudi biology (Bohr life sciences Co., ltd. (Pall Life Sciences)) to measure the expression titer in the conditioned medium. The percentage of high molecular weight (% HMW) species present after protein A affinity chromatography was measured by analytical size exclusion chromatography and by using +.>Non-reducing microcapillary electrophoresis (MCE NR) of GXII (Perkin Elmer) measures% purity of target proteins. The data for the variants prepared by this procedure are shown in table 11.
Table 11: rationally designed panel of hot spot engineered TREM1 antibodies
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Subsets of the three anti-TREM-1 lead antibodies in the campaign (30H 2 (19330), 49A2 (19333) and 46H7 (19332)) were also engineered by yeast display to improve manufacturability and retain binding to TREM-1. For each antibody, a library was generated in which all six possible pairs of residues adjacent to each other in each of the CDRs were simultaneously mutated to all possible amino acids by using the degenerate NNK codon. The library was displayed on the surface of a yeast derivative of BJ5464, wherein the Fd domain was fused to the N-terminus of the α -lectin, and LC was not fused to the yeast surface. />647 binding of conjugated anti-Fab antibodies measures the efficiency exhibited. Libraries were sorted using Fluorescence Activated Cell Sorting (FACS) for high binding to biotin-conjugated recombinant TREM-1ECD using streptavidin PE as secondary fluorescence. The variable domains present in the categorical binding/display double positive pool and display positive pool were amplified with specific primers for framework 1 (FW 1) and FW4 domains of HC and LC and NGS analysis was performed on Illumina MiSeq under 2x 300bp running. Mutations were selected after processing the data by common frequency analysis, in which the ratio of positive binding amino acid frequencies was divided by the positive display amino acid frequency, which was then normalized to the parental sequence ratio. Sequences with enrichment values greater than or equal to the parent sequence are considered to be of interest Beneficial or tolerable diversity, and is used for additional rational antibody engineering after affinity maturation.
The engineered variable domains shown previously were converted to the isotype of IgG1z SEFL2 and cloned using the gold cloning method to assemble 1) synthetic DNA fragments comprising antibody variable domains, 2) previously cloned "partial vectors" containing the necessary constant domains (i.e., CK or CL, CH1-CH2-CH3 (118-447) (R292C, N297G, V C), and 3) mammalian expression vector backbones. The Heavy Chain (HC) was assembled into the carrier backbone with the puromycin selection cassette and the Light Chain (LC) was assembled into the carrier backbone with the hygromycin selection cassette. HC and LC expression vectors were co-transfected in CHO-K1 cells at a 1:1 ratio using Lipofectamine LTX (Ji Buke company), passaged every 2-3 days in the presence of 10ug/mL puromycin and 500ug/mL hygromycin until cell viability>90% (Vi-CELL BLU, beckmann Coulter) to create a stable pool. The stable pool was inoculated in 2e6 viable cells/mL culture in production medium and at 36℃at 5% CO 2 And incubated for 6 days. Using AMMAG TM Protein a magnetic beads (gold srey corporation) purified antibodies by magnetic bead affinity chromatography. The identity of each molecule was confirmed by complete mass spectrometry. The percentage of high molecular weight (% HMW) species present after protein A affinity chromatography was measured by analytical size exclusion chromatography and by using Non-reducing microcapillary electrophoresis (MCE NR) of GXII (Perkin Elmer) measures% purity of target proteins. Data for variants prepared by yeast display hotspot engineering are presented in table 12.
Table 12: yeast display hotspot engineered variant TREM1 antibodies
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By usingThe level of phosphorylation of Syk kinase in cells was analyzed (perkin elmer) and bivalent and monovalent anti-TREM-1 mAb was compared in HEK293 cells expressing TREM-1/DAP 12. Briefly, at 37 ℃/5% CO 2 HEK293 cells stably expressing human TREM1 and DAP12 were cultured in DMEM/F12 Ham medium (Corning) supplemented with 10% dialyzed FBS (Ji Buke), 2mM Glutamax (Ji Buke), 2mM L-glutamine (Sigma), 1% penicillin/streptomycin (Ji Buke) and 0.1mg/mL bleomycin (Ji Buke). The day before the experiment, cells were isolated using trypsin-EDTA and centrifuged at 400x g for 5 minutes. The cell pellet was resuspended in complete medium and then centrifuged again at 400x g for 5 minutes. After centrifugation, the cells were centrifuged at 1X 10 6 Individual cells/mL were resuspended in complete medium and inoculated to +.about.100 μl/well or 50,000 cells/well final volume >96 well transparent flat bottom polystyrene plate (corning). Inoculated plates were incubated at 37℃C/5% CO 2 Incubate overnight for 18 to 24 hours. On the day of the experiment, 70. Mu.L of medium was removed from each well. The TREM1 antibody was diluted to the highest in a 4-fold serial dilution in assay medium (DMEM/F12 HAM medium supplemented with 10% heat-inactivated FBS)3 times the final concentration. Cross-linking reagent protein G (Sigma) was prepared at 3 times the final concentration using assay medium. Titrated TREM1 antibodies were mixed with protein G or assay medium at 1 to 1 and 60 μl of each TREM1 antibody +/-cross-linking reagent was added to each well containing cells. The plates were incubated for 1 hour at room temperature, then all media was removed from the wells and the cells were lysed with 25. Mu.L/well lysis buffer (M-Per mammalian protein extraction reagent and 1 XHalt protease/phosphatase inhibitor). Cells were incubated with lysis buffer for 1 hour on ice, then 5. Mu.L of cell lysate was transferred to each well of 384 Kong Baiban (Perkin Elmer) containing +.>Receptor mixtures (1 nM anti-pSyk, rabbit IgG (anti-phosphorylated Syk (Tyr 525/526) (clone C87C 1)) (cell signaling technique), 1nM biotin-anti-Syk, mouse IgG (clone 4D 10) (BD life sciences Co.), 10. Mu.g/mL anti-rabbit IgG- >Receptor beads (Perkin Elmer), and 1X Hall inhibitor at 1XImmunoassay buffer (perkin elmer). Plates were incubated on ice for a further 2 hours, then 5. Mu.L of +.>Donor mixtures (streptavidin- α -donor beads (perkin elmer) in 1X immunoassay buffer) and incubated for 1 hour at room temperature in the dark. After incubation, & gt>The plate reader (perkin elmer) detects the phosphorylation-Syk (pSyk) signal by FRET (fluorescence resonance energy transfer). Results were calculated by the ratio of the sample pSyk signal to the basal pSyk signal, where the potency and maximum of each antibody were comparedThe signal changes. Treatment with divalent anti-TREM-1 mAb alone induced weak signals in TREM-1/DAP12 expressing cells. No pSyk signal was observed with monovalent anti-TREM-1 mAb, and cross-linking of bivalent and monovalent anti-TREM-1 mAb with protein G resulted in pSyk induction.
Branching and alignment of antibody sequences: VH and VL domains of the input antibody sequences were extracted and aligned with the structure-based IgG numbering system based on honeygger and plurkthun (J Mol Biol journal of molecular biology 309 (3): 657-70, 2001). A distance matrix was generated from the composite multi-sequence alignment using an uncorrected model, where the distance between the two sequences is a fraction of mismatches in the VH and VL domains. Finally, trees were constructed using distance matrices by the UPGMA (non-weighted pairing grouping method with arithmetic mean) method (Sokal and Micheler, university of Kansas Science Bulletin [ Kansas university science bulletin ]38:1409-1438,1958), and the correlation sequences were grouped based on a branch traversal limit of 0.2. Alignment based on chemical similarity of amino acids was manually modified to generate consensus CDR sequences for each branched group, resulting in the sequences shown in table 13.
Table 13: consensus sequences for TREM-1 antigen binding proteins
Example 3 efficacy of TREM-1 antibody to block ligand-mediated Signal transduction in human Peripheral Blood Mononuclear Cells (PBMC)
The ability of TREM-1 antibodies to block signaling was tested by inhibiting ligand binding to TREM-1 receptor in human PBMCs. Frozen human PBMC (IQ Biosciences) were thawed, washed and resuspended in complete cell culture medium (RPMI/10% FBS/2mM GlutaMax/1mM sodium pyruvate/44 uM beta-mercaptoethanol/1 XDNase I). PBMCs were seeded at 100K/well in 96-well cell culture plates and equilibrated at 37 ℃ for at least 30 minutes. Thirteen anti-TREM-1 antibodies were diluted in RPMI/10% FBS (3X serial dilutions), resulting in final antibody concentrations ranging from 0.000017nM to 3nM. Antibody 57F5 was excluded from the analysis because it failed to show any inhibition of signaling with higher concentrations of antibody in the previous iteration of the assay. The antibodies were pre-incubated with PBMCs for 30 minutes prior to the addition of TREM-1 ligand. Peptidoglycan recognition protein 1 (PGLYRP 1), complexed with Peptidoglycan (PGN), is one of several TREM-1 ligands described, whose binding to TREM-1 receptors triggers the production of inflammatory cytokines (e.g., tnfα) and is used in these assays. To complex proteins, human PGLYRP1 (R & D systems) was combined with soluble PGN derived from E.coli (InvivoGen) at a ratio of 1.25:2 and incubated for 45 min at 37 ℃. The complex PGLYRP1/PGN was added to the PBMC/antibody and incubated overnight at 37 ℃. The following day, cell culture medium was collected and tnfα was detected by human tnfα Alphalisa proximity assay (perkin elmer). Light emission at 615nm was measured on an Envision 2103 multi-label plate reader. IC50 values were calculated using GraphPad Prism (v8.4.3) and are listed in table 14 below for two separate assays.
TABLE 14 inhibition of ligand-mediated signaling by TREM-1 antibodies in human PBMC as measured by TNF alpha release
These results indicate that the TREM-1 antibody binds to human TREM-1 and importantly demonstrates inhibition of ligand-induced TREM-1 activation in human primary cells (PBMCs).
Example 4 efficacy of TREM-1 antibodies to block ligand-mediated signaling in cynomolgus monkey Peripheral Blood Mononuclear Cells (PBMCs)
The TREM-1 antibody was assayed to block signaling by inhibiting ligand binding to the TREM-1 receptor in cynomolgus monkey PBMC. Frozen cynomolgus PBMCs (IQ bioscience) were thawed, washed and resuspended in complete cell culture medium (RPMI/10% FBS/2mM GlutaMax/1mM sodium pyruvate/44 uM beta-mercaptoethanol/1 XDNA enzyme I). PBMCs were seeded at 100K/well in 96-well cell culture plates and equilibrated at 37 ℃ for at least 30 minutes. Fourteen anti-TREM-1 antibodies were diluted in RPMI/10% FBS (3X serial dilutions), resulting in final antibody concentrations ranging from 0.00017nM to 30nM. The antibodies were pre-incubated with PBMCs for 30 minutes prior to the addition of TREM-1 ligand. Peptidoglycan recognition protein 1 (PGLYRP 1), complexed with Peptidoglycan (PGN), is one of several TREM-1 ligands described, whose binding to TREM-1 receptor triggers the production of inflammatory cytokines (e.g. tnfα) and is used in this assay. To complex proteins, cynomolgus PGLYRP1 (Creative BioMart) was combined with soluble PGN derived from escherichia coli (invitogen) at a ratio of 1.25:2 and incubated at 37 ℃ for 45 min. The complex PGLYRP1/PGN was added to the PBMC/antibody and incubated overnight at 37 ℃. The following day, cell culture medium was collected and tnfα was detected by cynomolgus monkey tnfα Alphalisa proximity assay (perkin elmer). Light emission at 615nm was measured on an Envision 2103 multi-label plate reader. IC50 values were calculated using GraphPad Prism (v8.4.3) and are listed in table 15 for two separate assays.
TABLE 15 inhibition of ligand-mediated signaling in cynomolgus PBMCs by TREM-1 antibodies as measured by TNF alpha release
These results indicate that the TREM-1 antibody binds to cynomolgus monkey TREM-1 and importantly inhibits ligand-induced TREM-1 activation in cynomolgus monkey primary cells (PBMCs). In addition, figure 1 demonstrates the inhibition of PGLYRP1/PGN mediated TREM1 signaling by TREM1 antibodies in cynomolgus monkey and human PBMCs.
EXAMPLE 5 blocking of spleen tyrosine kinase in human TREM-1/Dap12-HEK293 overexpressing cell lines by TREM-1Fab
Efficacy of (SYK) phosphorylation.
In a cell line overexpressing human TREM-1/Dap12, the ability of TREM-1Fab to block signaling was assayed by inhibiting ligand binding to TREM-1 receptor, and IC50 was determined. Phosphorylation of spleen tyrosine kinase (SYK) is TREM-1The early steps of the signaling cascade were used in this assay for the measurement of TREM-1 signaling. HEK293 cells overexpressing human TREM-1 and its proprietary adapter protein DNAX activator protein (Dap 12) (12 kDa) were seeded at 50K/well in complete medium (DMEM/10% FBS) in CellBIND plates (Corning Co.) and allowed to attach overnight. The inability of bivalent TREM-1 antibodies to inhibit signaling in this system is probably due to technical artefacts that TREM-1 receptors are very abundant in these cells and that bivalent antibodies can crosslink them leading to ligand independent receptor agonism. Thus, in this assay, monovalent Fab is used as a surrogate for whole antibodies. Fourteen anti-TREM-1 Fab were diluted in complete medium (3X serial dilutions), resulting in final antibody concentrations ranging from 0.00017nM to 30nM. Upon addition of human PGLYRP1 (R) complexed with TREM-1 ligand &D systems company) and soluble PGN from escherichia coli (invitofen company), fab was pre-incubated with TREM-1/Dap12-HEK293 for 30 minutes. To complex PGLYRP1 and PGN, proteins were combined at a 1:2 ratio and incubated at 37 ℃ for 45 minutes before addition to the cells/Fab. After one hour incubation at room temperature, the cells were lysed and used with pSYKUltra TM The p-SYK (Tyr 525/526) assay (Perkin Elmer) measures the amount of phosphorylated SYK in cell lysates. Light emission at 615nm was measured on an Envision 2103 multi-label plate reader. IC50 values were calculated using GraphPad Prism (v8.4.3) and listed for two separate assays as shown in table 16.
TABLE 16 inhibition of ligand-mediated Signal transduction by antibodies in cell lines overexpressing human TREM-1/DAP12 as measured by SYK phosphorylation
FIG. 2 is a graph showing inhibition of PGLYRP1/PGN mediated SYK phosphorylation in TREM1/DAP12-HEK293 cells by anti-TREM 1 Fab.
Example 6 in vivo evaluation of TREM-1 antibody in cardiovascular disease
Both acute and disease models were used to determine the blockade of TREM-1 mediated signaling, which would reduce the release of pro-inflammatory cytokines/chemokines (acute response) and improve the disease outcome of atherosclerosis (Ath) and Myocardial Infarction (MI).
Acute model:
lipopolysaccharide (LPS) -induced inflammation model
To determine if blockade of TREM-1 by mouse TREM-1 extracellular domain/Fc (mutem 1-Fc) resulted in a decrease in pro-inflammatory cytokine release in mice that have been systemically challenged with Lipopolysaccharide (LPS), an LPS-induced inflammation model was used. Briefly, female C57Bl/6 mice (10-12 weeks) were randomly grouped (n=6 mice/treatment) and intraperitoneally (i.p.) treated with 3, 10, or 30mg/kg (mpk) mutem 1-Fc or 30mpk muIgG1 (control). After one hour, mice will be administered (i.p.) 100 μg/animal of LPS from e.coli (055: b 5). Blood samples will be collected at 0, 1, 3, 6 and 24 hours. Serum levels of pro-inflammatory cytokines and chemokines (IFN-. Gamma., IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, TNF-. Alpha.) were measured using the V-PLEX Proinflammatory Panel 1 mouse kit (mesoscale discovery Co (Meso Scale Discovery)). Soluble TREM-1 (sTREM-1) levels were measured by ELISA (R & D systems Co.). In addition to mutem 1-Fc analysis in this model, anti-mouse TREM-1 antibodies can be characterized.
TREM-1 ligand mediated inflammation model
Peptidoglycan recognition protein 1 (PGLYRP 1) has been identified as a TREM-1 ligand (Read et al, 2015). PGLYRP1 alone or in combination with its endogenous binding target (bacterial peptidoglycan-PGN) was used to elicit an inflammatory response in C57Bl/6 mice in a dose range of 0.5 to 3mpk (PGLYRP 1) and 0.5 to 5mpk (PGN). Prior to administration of PGLYRP1 or PGLYRP1/PGN, mice were treated with anti-mouse TREM-1 antibody, control antibody or mutTREM 1-Fc (dose range 1-30 mpk). Given the relatively low expression of TREM-1 in mice, LPS (a toll-like receptor 4 ligand known to up-regulate TREM-1 expression) (Zeng et al, 2007) can be administered 24 hours prior to treatment with antibodies/TREM 1-Fc and TREM-1 ligand. Blood samples were collected at 0, 1, 3, 6 and 24 hours. Serum levels of pro-inflammatory cytokines and chemokines (IFN-. Gamma., IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, TNF-. Alpha.) were measured using the V-PLEX Proinflammatory Panel 1 mouse kit (mesoscale discovery). sTREM-1 level was measured by ELISA (R & D systems Co.).
NSG humanized mouse model
NOD Scidγ (NSG) mice (Jackson Labs) are severely immunodeficient mice that can be readily transplanted with human hematopoietic stem cells to create a functional humanized immune system in a mouse model. With this humanized mouse model, an inflammatory response was elicited by LPS treatment, TREM-1 ligand treatment, or both, as described above. Mice were treated with anti-human TREM-1 or isotype control antibodies (dose range 1-30 mpk) prior to eliciting an inflammatory response. Blood samples will be collected at 0, 1, 3, 6 and 24 hours. Serum levels of pro-inflammatory cytokines and chemokines (IFN-. Gamma., IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-. Alpha.) were measured using the V-PLEX Proinflammatory Panel 1 human kit (mesoscale discovery). sTREM-1 level was measured by ELISA (R & D systems Co.).
Macaca fascicularis model
In the LPS challenge model, cynomolgus monkeys (Macaca fascicularis)) were treated with anti-human (cross-reactive with cynomolgus monkeys) TREM-1 antibodies. Following intravenous (i.v.) bolus LPS (10 ug/kg body weight), adult male cynomolgus monkeys received either i.v. or subcutaneous (s.c.) doses of TREM-1 antibody (dose range 1-30 mpk) or isotype control at random. The monkeys of the other control group received vehicle only (no antibody/no LPS). Vital signs were monitored and blood samples were collected at 0, 1, 2, 4 and 8 hours for clinical chemistry, white blood cell count, coagulation parameters, soluble TREM-1 and cytokine plasma concentrations.
Disease model:
ApoE-/-, LDLR-/-, A20 single dose deficiency/ApoE-/-, and combinations thereof
A mouse model of atherosclerosis (e.g., apoE-/-or LDLR-/-mice receiving a high cholesterol high fat diet for 12-16 weeks) is used to determine whether treatment with anti-mouse TREM-1 antibodies would alter disease outcome. Mice were randomized and given s.c. doses (ranging from 1-30 mpk) of control antibody or anti-mouse TREM-1 antibody weekly to these cohorts during the high cholesterol diet feeding phase. Blood was collected at different time points and analyzed for cytokine/chemokine levels, soluble TREM-1 levels, and myeloid cell subsets. After the end of the experiment, the whole aorta will be collected and the surface lesion area quantified by frontal staining with oil red O or sudan IV. In addition, aortic root sections were obtained and stained with oil red O to quantify lesion area, anti-CD 68 antibody to quantify macrophage content, and anti-Smooth Muscle Actin (SMA) antibody to quantify smooth muscle cell content, among other staining. Another useful model is an A20 single dose deficient/ApoE-/-mouse, which shows NF-kb driven increases in the levels of the pro-atherosclerosis/pro-inflammatory target genes, resulting in increased susceptibility to atherosclerosis (Wolfrum et al, 2007). If no suitable anti-mouse TREM-1 surrogate antibodies were identified, a human TREM-1 knock-in mouse in the ApoE-/-background would be generated. This would allow the anti-human TREM-1 antibodies to be tested in a mouse model of atherosclerosis.
NSG humanized mouse model
If NSG mice prove useful in acute settings, they can be developed as disease models by siRNA knockdown of ApoE, ldlr, or both. NSG/siRNA treated mice on a randomized cohort receiving a 12-16 week high fat high cholesterol diet during the high cholesterol diet feeding phase were administered an s.c. dose (ranging from 1-30 mpk) of control antibody or anti-human TREM-1 antibody weekly. Blood was collected at different time points and analyzed for cytokine/chemokine levels, soluble TREM-1 levels, and myeloid cell subsets. After the end of the experiment, the whole aorta was collected and the surface lesion area was quantified by frontal staining and aortic root sections were stained as described above.
Rodent Acute Myocardial Infarction (AMI) model
Rodent MI models can be used to test anti-mouse TREM-1 antibodies and/or mutEM 1-Fc. Briefly, AMI is induced in mice or rats by permanently ligating the Left Anterior Descending (LAD) branch of the coronary artery. Following this procedure, rodents (rat = Sprague Dawley, mouse = C57 Bl/6) will be randomized and receive s.c doses (1-30 mpk) of anti-mouse TREM-1 or isotype control antibody weekly 2-8 weeks after ligation surgery. Cardiac function (e.g., ejection fraction, end diastole volume, end systole volume, and cardiac output) was assessed by non-invasive echocardiography at prescribed times throughout the study. The final cardiac function and contractile force will be measured by the pressure containment loop. Blood samples were periodically drawn to measure blood cell counts, liver and kidney enzymes, troponin, soluble TREM-1 and markers of inflammation (cytokines, hsCRP). At the end of the study, rodents were euthanized and infarct size (IS/AAR) associated with the hazard zone would be determined by ex vivo histological staining. A similar study can be performed in which LAD ligates for 90 minutes and then ligates are removed, a so-called ischemia/reperfusion (I/R) model. The treatment protocol and analysis will be the same as described above.
Miniature pig MI model
Antibodies that are likely to cross-react with porcine TREM-1 were tested in a mini-pig AMI model. Briefly, AMI was induced in a small pig (domestic pig (Sus scrofa domestic)) by inflating an angioplasty balloon in the proximal end of the left anterior descending artery (LAD) for one hour. Animals received a random s.c. dose (1-30 mpk) of TREM-1 or isotype control antibody weekly for 2-4 weeks post-surgery. After the initial dose of antibody is administered at the time of surgery, the angioplasty balloon is deflated. Hemodynamic parameters including heart rate, mean arterial pressure, mean pulmonary arterial pressure, cardiac output, cardiac index and mixed venous oxygen saturation will be monitored throughout the study. Parameters based on the pressure conductance catheter, including left ventricular end diastole and end systole volumes, ejection fraction, maximum and minimum values of the first derivative of ventricular pressure (dP/dtmax, dP/dtmin), time constant of left ventricular pressure decay, and work per beat, will be monitored periodically throughout the study. Pressure/volume data at the time of vena cava occlusion is also collected, including end systole and end diastole pressure volume relationships, maximum ventricular elasticity, arterial elasticity, dp/dtmax versus end diastole volume relationships, preload supplements per beat work and pressure volume area. Blood samples were periodically drawn to measure blood gas, blood cell count, blood lactate, liver and kidney enzymes, troponin, soluble TREM-1 and inflammatory markers (cytokines, hsCRP). At the end of the study, pigs were euthanized and infarct size (IS/AAR) associated with the hazard zone would be determined by ex vivo histological staining.
It is speculated that administration of TREM-1 antibodies that block signaling through the receptor will down-regulate the inflammatory response in the heart model and treat cardiovascular disease by reducing one or more symptoms of cardiovascular disease (e.g., atherosclerosis or myocardial infarction) (e.g., inflammatory cell migration to the site of injury, myeloid cell infiltration to heart tissue, inflammatory cytokines in the microenvironment, tissue damage, reduced foam cell formation, reduced necrotic core size, reduced scar formation, reduced endothelial cell dysfunction, and/or reduced thrombosis).
Example 7 Gene elimination of TREM-1 results in decreased serum peptidoglycan recognition protein 1 (PGLYRP 1) levels following LPS stimulation
TREM-1 knockout (C57 Bl/6 background) and age/background matched mice (n=6) were injected (intraperitoneally) with 100 μg/animal of Lipopolysaccharide (LPS) from escherichia coli (O55: B5) (Sigma Aldrich). 20 microliters of whole blood was withdrawn 0, 1, 3, 6 and 24 hours after LPS administration. Serum from two treatment groups (WT, TREM-1 KO) at each time point was pooled and PGLYRP1 concentration was measured by ELISA (LSBio Inc.). SpectraMax Plus, molecular instruments Co 384 Optical density was measured at 450nM on a spectrophotometer.
FIG. 3 is a graph showing that the TREM-1 ligand PGLYRP1 peaked at 1 hour in wild-type but not TREM-1 knockout mice after LPS administration. This data indicates that TREM-1 is involved in PGLYRP1 modulation, and that antibodies targeting TREM-1 may block both ligand binding to the receptor and the subsequent availability of PGLYRP1 ligand. Blocking the increase in pyglirp 1 with TREM-1 antibodies can prevent subsequent pyglirp 1-mediated inflammation and cardiovascular events.
Any single embodiment herein may be supplemented with one or more elements from any one or more other embodiments herein.
It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover all modifications that are within the spirit and scope of the invention as defined in the following: the appended claims; the above description; the following numbered paragraphs, and/or as illustrated in the accompanying drawings.
It is expected that many modifications and variations of the present invention will come to mind to one skilled in the art to which this invention pertains as set forth in the foregoing illustrative examples. Therefore, only such limitations as appear in the claims are imposed on the present invention.

Claims (47)

1. A method of treating a cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein that binds to myeloid cell trigger receptor 1 (TREM-1);
The antigen binding protein comprises
a. A light chain variable domain comprising:
i. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, and 544;
light chain CDR2 comprising an amino acid sequence selected from SEQ ID NOs 11, 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271 and 545;
light chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272 and 546; and
b. a heavy chain variable domain comprising:
i. a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NOs 16, 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276 and 550;
heavy chain CDR2 comprising an amino acid sequence selected from SEQ ID NOs 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277, 297 and 551; and
heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298 and 552.
2. The method of claim 1, wherein the antigen binding protein comprises:
the light chain CDR1 sequences set forth in SEQ ID NO. 10, 30, 50, 90, 130, 150, or 270;
the light chain CDR2 sequences set forth in SEQ ID NO. 11, 31, 51, 91, 131, 151, or 271;
the light chain CDR3 sequences set forth in SEQ ID NO. 12, 32, 52, 92, 132, 152, or 272;
the heavy chain CDR1 sequences set forth in SEQ ID NOS 16, 36, 56, 96, 136, 156 and 276;
the heavy chain CDR2 sequences set forth in SEQ ID NOs 17, 37, 57, 97, 137, 157 and 277; and
the heavy chain CDR3 sequences set forth in SEQ ID NOS: 18, 38, 58, 98, 138 and 278.
3. The method of claim 1 or 2, wherein the antigen binding protein comprises:
the light chain CDR1 sequence set forth in SEQ ID NO. 30 or 90;
the light chain CDR2 sequences set forth in SEQ ID NO. 31 or 91;
the light chain CDR3 sequences set forth in SEQ ID NO. 32 or 92;
the heavy chain CDR1 sequences set forth in SEQ ID NO. 36 or 96;
e.the heavy chain CDR2 sequences set forth in SEQ ID NO 37 or 97; and
the heavy chain CDR3 sequences set forth in SEQ ID NO. 38 or 98.
4. The method of any one of claims 1-3, wherein the antigen binding protein comprises:
i) SEQ ID NO. 10 (LCDR 1), SEQ ID NO. 11 (LCDR 2), SEQ ID NO. 12 (LCDR 3), SEQ ID NO. 16 (HCDR 1), SEQ ID NO. 17 (HCDR 2) and SEQ ID NO. 18 (HCDR 3);
ii) SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3);
iii) SEQ ID NO:50 (LCDR 1), SEQ ID NO:51 (LCDR 2), SEQ ID NO:52 (LCDR 3), SEQ ID NO:56 (HCDR 1), SEQ ID NO:57 (HCDR 2) and SEQ ID NO:58 (HCDR 3);
iv) SEQ ID NO:70 (LCDR 1), SEQ ID NO:71 (LCDR 2), SEQ ID NO:72 (LCDR 3), SEQ ID NO:76 (HCDR 1), SEQ ID NO:77 (HCDR 2) and SEQ ID NO:78 (HCDR 3);
v) SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3);
vi) SEQ ID NO:110 (LCDR 1), SEQ ID NO:111 (LCDR 2), SEQ ID NO:112 (LCDR 3), SEQ ID NO:116 (HCDR 1), SEQ ID NO:117 (HCDR 2) and SEQ ID NO:118 (HCDR 3);
vii) SEQ ID NO:130 (LCDR 1), SEQ ID NO:131 (LCDR 2), SEQ ID NO:132 (LCDR 3), SEQ ID NO:136 (HCDR 1), SEQ ID NO:137 (HCDR 2) and SEQ ID NO:138 (HCDR 3);
viii) SEQ ID NO:150 (LCDR 1), SEQ ID NO:151 (LCDR 2), SEQ ID NO:152 (LCDR 3), SEQ ID NO:156 (HCDR 1), SEQ ID NO:157 (HCDR 2) and SEQ ID NO:158 (HCDR 3);
ix) SEQ ID NO:170 (LCDR 1), SEQ ID NO:171 (LCDR 2), SEQ ID NO:172 (LCDR 3), SEQ ID NO:176 (HCDR 1), SEQ ID NO:177 (HCDR 2) and SEQ ID NO:178 (HCDR 3);
x) SEQ ID NO. 190 (LCDR 1), SEQ ID NO. 191 (LCDR 2), SEQ ID NO. 192 (LCDR 3), SEQ ID NO. 196 (HCDR 1), SEQ ID NO. 197 (HCDR 2) and SEQ ID NO. 198 (HCDR 3);
xi) SEQ ID NO:210 (LCDR 1), SEQ ID NO:211 (LCDR 2), SEQ ID NO:212 (LCDR 3), SEQ ID NO:216 (HCDR 1), SEQ ID NO:217 (HCDR 2) and SEQ ID NO:218 (HCDR 3);
xii) SEQ ID NO:230 (LCDR 1), SEQ ID NO:231 (LCDR 2), SEQ ID NO:232 (LCDR 3), SEQ ID NO:236 (HCDR 1), SEQ ID NO:237 (HCDR 2) and SEQ ID NO:238 (HCDR 3);
xiii) SEQ ID NO:250 (LCDR 1), SEQ ID NO:251 (LCDR 2), SEQ ID NO:252 (LCDR 3), SEQ ID NO:256 (HCDR 1), SEQ ID NO:257 (HCDR 2) and SEQ ID NO:258 (HCDR 3);
xiv) SEQ ID NO:270 (LCDR 1), SEQ ID NO:271 (LCDR 2), SEQ ID NO:272 (LCDR 3), SEQ ID NO:276 (HCDR 1), SEQ ID NO:277 (HCDR 2) and SEQ ID NO:278 (HCDR 3); or (b)
xv) SEQ ID NO:544 (LCDR 1), SEQ ID NO:545 (LCDR 2), SEQ ID NO:546 (LCDR 3), SEQ ID NO:550 (HCDR 1), SEQ ID NO:551 (HCDR 2) and SEQ ID NO:552 (HCDR 3).
5. The method of any one of claims 1-4, wherein the antigen binding protein comprises a set of CDRs selected from the group consisting of:
i) SEQ ID NO. 10 (LCDR 1), SEQ ID NO. 11 (LCDR 2), SEQ ID NO. 12 (LCDR 3), SEQ ID NO. 16 (HCDR 1), SEQ ID NO. 17 (HCDR 2) and SEQ ID NO. 18 (HCDR 3);
ii) SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3);
iii) SEQ ID NO:50 (LCDR 1), SEQ ID NO:51 (LCDR 2), SEQ ID NO:52 (LCDR 3), SEQ ID NO:56 (HCDR 1), SEQ ID NO:57 (HCDR 2) and SEQ ID NO:58 (HCDR 3);
iv) SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3);
v) SEQ ID NO:130 (LCDR 1), SEQ ID NO:131 (LCDR 2), SEQ ID NO:132 (LCDR 3), SEQ ID NO:136 (HCDR 1), SEQ ID NO:137 (HCDR 2) and SEQ ID NO:138 (HCDR 3);
vi) SEQ ID NO:150 (LCDR 1), SEQ ID NO:151 (LCDR 2), SEQ ID NO:152 (LCDR 3), SEQ ID NO:156 (HCDR 1), SEQ ID NO:157 (HCDR 2) and SEQ ID NO:158 (HCDR 3); or (b)
vii) SEQ ID NO:270 (LCDR 1), SEQ ID NO:271 (LCDR 2), SEQ ID NO:272 (LCDR 3), SEQ ID NO:276 (HCDR 1), SEQ ID NO:277 (HCDR 2) and SEQ ID NO:278 (HCDR 3).
6. The method of any one of claims 1-5, wherein the antigen binding protein comprises:
a. A light chain comprising the amino acid sequences SEQ ID NO:30 (LCDR 1), SEQ ID NO:31 (LCDR 2), SEQ ID NO:32 (LCDR 3), and a heavy chain comprising the amino acid sequences SEQ ID NO:36 (HCDR 1), SEQ ID NO:37 (HCDR 2) and SEQ ID NO:38 (HCDR 3); or (b)
b. A light chain comprising the amino acid sequences SEQ ID NO:90 (LCDR 1), SEQ ID NO:91 (LCDR 2), SEQ ID NO:92 (LCDR 3), and a heavy chain comprising the amino acid sequences SEQ ID NO:96 (HCDR 1), SEQ ID NO:97 (HCDR 2) and SEQ ID NO:98 (HCDR 3).
7. The method of any one of claims 1-6, wherein the antigen binding protein comprises:
a. a light chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281 and 539;
a sequence encoded by the polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281 and 539; or (b)
A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 19, 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279 and 537; and
b. A heavy chain variable domain comprising an amino acid sequence selected from the group consisting of:
i. a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282 and 540;
a sequence encoded by the polynucleotide sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282 and 540; or (b)
A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 2184.
8. The method of any one of claims 1 to 7, wherein the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region amino acid sequence selected from the group consisting of seq id nos: 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540.
9. The method of any one of claims 1 to 8, wherein the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOS.22, 42, 62, 102, 142, 162 and 282.
10. The method of any one of claims 1 to 9, wherein the antigen binding protein comprises an amino acid sequence having at least 90% identity to a heavy chain variable region amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOS.42 and 102.
11. The method of any one of claims 1 to 10, wherein the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region amino acid sequence selected from the group consisting of seq id nos: 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281 and 539.
12. The method of any one of claims 1 to 11, wherein the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOS.21, 41, 61, 101, 141, 161 and 281.
13. The method of any one of claims 1 to 12, wherein the antigen binding protein comprises an amino acid sequence having at least 90% identity to a light chain variable region amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOS: 41 and 101.
14. The method of any one of claims 1 to 13, wherein the antigen binding protein comprises:
i) The light chain variable domain sequence set forth in SEQ ID NO. 21 and the heavy chain variable domain sequence set forth in SEQ ID NO. 22;
ii) the light chain variable domain sequence set forth in SEQ ID NO. 41 and the heavy chain variable domain sequence set forth in SEQ ID NO. 42;
iii) The light chain variable domain sequence set forth in SEQ ID NO. 61 and the heavy chain variable domain sequence set forth in SEQ ID NO. 62;
iv) the light chain variable domain sequence set forth in SEQ ID NO. 81 and the heavy chain variable domain sequence set forth in SEQ ID NO. 82;
v) the light chain variable domain sequence set forth in SEQ ID NO. 101 and the heavy chain variable domain sequence set forth in SEQ ID NO. 102;
vi) the light chain variable domain sequence set forth in SEQ ID NO. 121 and the heavy chain variable domain sequence set forth in SEQ ID NO. 122;
vii) the light chain variable domain sequence set forth in SEQ ID NO. 141 and the heavy chain variable domain sequence set forth in SEQ ID NO. 142;
viii) the light chain variable domain sequence set forth in SEQ ID NO. 161 and the heavy chain variable domain sequence set forth in SEQ ID NO. 162;
ix) the light chain variable domain sequence set forth in SEQ ID NO:181 and the heavy chain variable domain sequence set forth in SEQ ID NO: 182;
x) the light chain variable domain set forth in SEQ ID NO. 201 and the heavy chain variable domain set forth in SEQ ID NO. 202;
xi) the light chain variable domain sequence set forth in SEQ ID NO. 221 and the heavy chain variable domain sequence set forth in SEQ ID NO. 222;
xii) the light chain variable domain sequence set forth in SEQ ID NO. 241 and the heavy chain variable domain sequence set forth in SEQ ID NO. 242;
xiii) the light chain variable domain sequence set forth in SEQ ID NO:261 and the heavy chain variable domain sequence set forth in SEQ ID NO: 262;
xiv) the light chain variable domain sequence set forth in SEQ ID NO. 281 and the heavy chain variable domain sequence set forth in SEQ ID NO. 282; or (b)
xv) the light chain variable domain sequence set forth in SEQ ID NO. 539 and the heavy chain variable domain sequence set forth in SEQ ID NO. 540.
15. The method of any one of claims 1 to 14, wherein the antigen binding protein comprises:
i) The light chain variable domain sequence set forth in SEQ ID NO. 21 and the heavy chain variable domain sequence set forth in SEQ ID NO. 22;
ii) the light chain variable domain sequence set forth in SEQ ID NO. 41 and the heavy chain variable domain sequence set forth in SEQ ID NO. 42;
iii) The light chain variable domain sequence set forth in SEQ ID NO. 61 and the heavy chain variable domain sequence set forth in SEQ ID NO. 62;
iv) the light chain variable domain sequence set forth in SEQ ID NO. 101 and the heavy chain variable domain sequence set forth in SEQ ID NO. 82;
v) the light chain variable domain sequence set forth in SEQ ID NO. 141 and the heavy chain variable domain sequence set forth in SEQ ID NO. 142;
vi) the light chain variable domain sequence set forth in SEQ ID NO. 161 and the heavy chain variable domain sequence set forth in SEQ ID NO. 162;
vii) the light chain variable domain sequence set forth in SEQ ID NO:281 and the heavy chain variable domain sequence set forth in SEQ ID NO: 282.
16. The method of any one of claims 1 to 15, wherein the antigen binding protein comprises: the light chain variable domain sequence set forth in SEQ ID NO. 41 and the heavy chain variable domain sequence set forth in SEQ ID NO. 42; or the light chain variable domain sequence set forth in SEQ ID NO. 101 and the heavy chain variable domain sequence set forth in SEQ ID NO. 102.
17. The method of any one of claims 1-16, wherein one or more heavy chain framework amino acids of the anti-antigen binding protein are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence.
18. The method of any one of claims 1 to 17, wherein one or more light chain framework amino acids of the antigen binding protein are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence.
19. The method of any one of claims 1 to 18, wherein the heavy chain comprises a constant region selected from the group consisting of: igG, igM, igA, igD, igE, fragments thereof, combinations thereof, and modifications thereof in which one to ten heavy chain framework amino acids are replaced with a corresponding one or more amino acids from another human antibody amino acid sequence.
20. A method of treating a cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein that competes with the antigen binding protein of any one of claims 1-19 for binding to TREM-1.
21. The method of any one of claims 1 to 20, wherein the antigen binding protein is selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, fab, F (ab') 2, fab2, monovalent IgG, scFv, scFv-Fc, igG1 antibodies, igG2 antibodies, igG3 antibodies, and IgG4 antibodies.
22. The method of any one of claims 1 to 21, wherein the antigen binding protein is an IgG1 antibody.
23. The method of any one of claims 1 to 22, wherein the antigen binding protein is a monovalent IgG.
24. The method of any one of claims 1-23, wherein the antigen binding protein is a human antibody.
25. The method of any one of claims 1 to 24, wherein the antigen binding protein comprises a heavy chain amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540, and a light chain amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539.
26. The method of any one of claims 1 to 25, wherein the antigen binding protein has a heavy chain amino acid sequence selected from SEQ ID NOs 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, and 540, and a light chain amino acid sequence selected from SEQ ID NOs 21, 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, and 539.
27. The method of any one of claims 1 to 26, wherein the antigen binding protein has a heavy chain amino acid sequence selected from SEQ ID NOs 22, 42, 62, 102, 142, 162 and 282.
28. The method of any one of claims 1 to 27, wherein the antigen binding protein has a heavy chain amino acid sequence selected from SEQ ID NOs 42 and 102.
29. The method of any one of claims 1 to 27, wherein the antigen binding protein has a light chain amino acid sequence selected from SEQ ID NOs 21, 41, 61, 81, 101, 141, 161 and 281.
30. The method of any one of claims 1 to 27, wherein the antigen binding protein has a light chain amino acid sequence selected from SEQ ID NOs 41 and 101.
31. The method of any one of claims 1-30, wherein the antigen binding protein further comprises a pharmaceutically acceptable carrier.
32. The method of any one of claims 1-31, wherein the cardiovascular disease is selected from the group consisting of: myocardial infarction, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), heart failure, stroke (ischemic and ischemic), atherosclerosis, coronary heart disease, peripheral vascular disease (e.g., peripheral arterial disease), vulnerable plaque, acute coronary syndrome, cerebrovascular disease, cerebrovascular atherosclerosis, and obesity.
33. The method of any one of claims 1 to 32, wherein the cardiovascular disease is atherosclerosis.
34. The method of any one of claims 1 to 32, wherein the cardiovascular disease is myocardial infarction.
35. The method of any one of claims 1-34, wherein the treatment is performed by intravenous or subcutaneous administration.
36. The method of any one of claims 1-35, wherein the treatment is performed by once weekly, once every two weeks, once every three weeks, once every 4 weeks, once monthly, once every 3 months, once every six months, or once annually.
37. The method of any one of claims 1-36, further comprising administering one or two additional therapeutic agents.
38. The method of claim 37, wherein the additional therapeutic agents are selected from the group consisting of: a first partOne or more cholesterol lowering agents, agents that increase LDLR expression, statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), PCSK9 inhibitorsNicotinic acid (niacin) (-)> (sustained release of Nick acid), ->(sustained release of niacin)), fibric acid (>(gemfibrozil),>(fenofibrate)), bile acid sequestrant (a. About.)>(cholestyramine), colesevelam- >(norbilin)), cholesterol absorption inhibitors (Zetia), combined nicotinic acid with statins (++>(lovastatin and->) Combinations of statins with absorption inhibitors (VYTORIN ()>And->) Lipid modulators like PCSK9 inhibitors, ppary agonists, ppara/y agonists, squalene synthetase inhibitors, cholesteryl Ester Transfer Protein (CETP) inhibitors, antihypertensive agents, antithrombotic agents (aspirin), antidiabetic agents (e.g. sulfonylurea, insulin, GLP-1 analogues, DDPIV inhibitors, SGL2 inhibitors), apoB modulators, MTP inhibitors,(ivabradine) I (f) current inhibitors, omecambaryophyllin activators, OLPASIRAN (AMG 890) that reduce lipoprotein (a) (siRNA), AMG 594 cardiac troponin activators, AMG 609, AMG 171 (growth differentiation factor 15 (GDF 15) analogues), AMG 133 (gastric inhibitory peptide receptor (GIPR) antagonists and glucagon-like peptide 1 (GLP-1) receptor agonists), and/or occlusive arteriosclerosis therapeutics.
39. The method of any one of claims 1 to 38, wherein the TREM-1 is human TREM-1 set forth in SEQ ID No. 2.
40. The method of any one of claims 1 to 39, wherein the administration reduces one or more symptoms of a cardiovascular disease selected from the group consisting of: inflammatory cell migration to the site of injury, infiltration of myeloid cells into heart tissue, inflammatory cytokines in the microenvironment, tissue damage, reduced foam cell formation, reduced necrotic core size, reduced scarring, reduced endothelial cell dysfunction, and/or reduced thrombosis.
41. A composition comprising an antigen binding protein that binds to myeloid cell trigger receptor 1 (TREM-1) for use in the treatment of a cardiovascular disease, wherein the antigen binding protein comprises:
a. a light chain variable domain comprising:
i. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, and 544;
light chain CDR2 comprising an amino acid sequence selected from SEQ ID NOs 11, 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271 and 545;
light chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272 and 546; and
b. a heavy chain variable domain comprising:
i. a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NOs 16, 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276 and 550;
heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277 and 551; and
Heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278 and 552.
42. Use of a composition comprising an antigen binding protein that binds to myeloid cell trigger receptor 1 (TREM-1) for the manufacture of a medicament for the treatment of cardiovascular diseases, the antigen binding protein comprising
a. A light chain variable domain comprising:
i. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, and 544;
light chain CDR2 comprising an amino acid sequence selected from SEQ ID NOs 11, 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271 and 545;
light chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 12, 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272 and 546; and
b. a heavy chain variable domain comprising:
i. a heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NOs 16, 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276 and 550;
heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 17, 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277 and 551; and
Heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NOs 18, 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278 and 552.
43. The use of claim 41 or 42, wherein the cardiovascular disease is selected from the group consisting of: myocardial infarction, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), heart failure, stroke (ischemic and ischemic), atherosclerosis, coronary heart disease, peripheral vascular disease (e.g., peripheral arterial disease), vulnerable plaque, acute coronary syndrome, cerebrovascular disease, cerebrovascular atherosclerosis, and obesity.
44. A method of treating a cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein that binds to myeloid cell-triggering receptor 1 (TREM-1), the antigen binding protein comprising:
a. a light chain variable domain comprising:
i. comprising the amino acid sequence X 1 ASQSX 2 X 3 X 4 Light chain CDR1 of NLA (SEQ ID NO: 553), wherein X 1 Is R or Q, wherein X 2 Is V or I, wherein X 3 Is N or S, and wherein X 4 S, H, I, V or a;
comprising the amino acid sequence GAX 1 X 2 Light weight of RAT (SEQ ID NO: 554) Chain CDR2, wherein X 1 Is S or Y, and wherein X 2 Is T or I; and
comprising the amino acid sequence QX 1 X 2 X 3 X 4 X 5 X 6 PX 7 T (SEQ ID NO: 555) light chain CDR3; wherein X is 1 Q, H or E, wherein X 2 Is F or Y, wherein X 3 K, Y or I, wherein X 4 N, T, L, I, or M; wherein X is 5 W, F, H or Y, wherein X 6 Absence or P; wherein X is 7 W, N, Y, H or L; and
b. a heavy chain variable domain comprising:
i. comprising the amino acid sequence X 1 X 2 X 3 MX 4 (SEQ ID NO: 556) heavy chain CDR1, wherein X 1 A, R, T or S, wherein X 2 Is Y or N, wherein X 3 Is A or W, and wherein X 4 Is S or N;
comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 YYX 10 X 11 X 12 VKG (SEQ ID NO: 559), wherein X is a heavy chain CDR2 1 T, E, or S, wherein X 2 Absence or M, V, or I, wherein X 3 S, R or K, wherein X 4 Is G or Q, wherein X 5 S, D or H, wherein X 6 G, S, L, or A, wherein X 7 Is S, G, or R, wherein X 8 T, S, P or E, wherein X 9 Is T or I, wherein X 10 Is A or V, wherein X 11 Is D or E, and wherein X 12 Is S or A; and
comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 FX 8 YYX 9 (SEQ ID NO: 557) heavy chain CDR3, wherein X 1 V, E, A or G, wherein X 2 A, F, Y or G, wherein X 3 G, S, Y or W, wherein X 4 Is S or R, wherein X 5 Absent or N, where X 6 F, S, Y, or is absent, where X 7 Is L orF or not present, where X 8 Is D or E, and wherein X 9 Y, H or S.
45. The method of claim 44, wherein the antigen binding protein comprises:
a. a light chain variable domain comprising:
i. a light chain CDR1 comprising amino acid sequence RASQSVNSNLA (SEQ ID NO: 556);
light chain CDR2 comprising the amino acid sequence GASTRAT (SEQ ID NO: 573);
light chain CDR3 comprising amino acid sequence QQFKNWPPT (SEQ ID NO: 576); and
b. a heavy chain variable domain comprising:
i. a heavy chain CDR1 comprising the amino acid sequence AYAMS (SEQ ID NO: 581);
heavy chain CDR2 comprising amino acid sequence TSGSGSTTYYADSVKG (SEQ ID NO: 584); and
heavy chain CDR3 comprising amino acid sequence VAGSNFLFDY (SEQ ID NO: 842).
46. A method of treating a cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein that binds to myeloid cell-triggering receptor 1 (TREM-1), wherein the antigen binding protein comprises:
a. a light chain variable domain comprising:
i. comprising the amino acid sequence QASX 1 DIX 2 X 3 X 4 LN (SEQ ID NO: 558) light chain CDR1, wherein X 1 Is R or Q, wherein X 2 R, S, N or F, where X 3 Is K or N, and wherein X 4 H, Y or D;
comprising the amino acid sequence X 1 X 2 X 3 X 4 Light chain CDR2 of LET (SEQ ID NO: 560), wherein X 1 D, G or H, wherein X 2 A, V or T, wherein X 3 S, A or Y, and wherein X 4 Is T or N;
comprising the amino acid sequence QX 1 YX 3 X 4 X 5 PX 6 T (SEQ ID NO: 561) light chain CDR3, wherein X 1 Is Q or H, wherein X 2 D, A or G, wherein X 3 Is N or K; wherein X is 4 Is L or I, and wherein X 5 Is I or L; and
b. a heavy chain variable domain comprising:
i. comprising the amino acid sequence X 1 Heavy chain CDR1 of YDIN (SEQ ID NO: 563) wherein X 1 Is R or S;
comprising the amino acid sequence X 1 X 2 NPX 3 X 4 GX 5 X 6 GX 7 X 8 X 9 X 10 FX 11 X 12 Heavy chain CDR2 of (SEQ ID NO: 564), wherein X 1 Is W or R, wherein X 2 Is M or L, wherein X 3 N, Q, or K, wherein X 4 Is S, A, or R, wherein X 5 Is N, or Q, wherein X 6 Is S, A, or T, wherein X 7 S, Q, or Y, wherein X 8 Is V or T, wherein X 9 Q or K, wherein X 10 Is K or N, wherein X 11 Is R or Q, and wherein X 12 Is G or D; and
comprising the amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 FX 13 X 14 (SEQ ID NO: 565) a heavy chain CDR3; wherein X is 1 G, L or R, wherein X 2 Is G, I, or R, wherein X 3 Y, R, I, G, or A, wherein X 4 T, S, Y, or V, where X 5 Is S or Y, wherein X 6 Is S, A, I, or R, wherein X 7 W, A, or S, wherein X 8 Absence or S, wherein X 9 Absence or F, W, or Y, and wherein X 10 R, S, H, K, or E, wherein X 11 W, H, Y, or F, where X 12 Y, V, A, or S, wherein X 13 Is D or Q, and wherein X 14 L, Y, I, or H.
47. The method of claim 46, wherein the antigen binding protein comprises:
a. a light chain variable domain comprising:
i. a light chain CDR1 comprising amino acid sequence QASQDIRKHLN (SEQ ID NO: 567);
light chain CDR2 comprising the amino acid sequence DASLET (SEQ ID NO: 574); and
light chain CDR3 comprising amino acid sequence QHYDNLPIT (SEQ ID NO: 577); and
b. a heavy chain variable domain comprising:
i. heavy chain CDR1 comprising the amino acid sequence RYDIN (SEQ ID NO: 582);
heavy chain CDR2 comprising amino acid sequence WMNPNSGNSSVQKFRG (SEQ ID NO: 585); and
heavy chain CDR3 comprising amino acid sequence GGYTSSWRWYFDL (SEQ ID NO: 843) or GGYTSSWSRWYFDL (SEQ ID NO: 844).
CN202280053372.3A 2021-06-25 2022-06-24 Treatment of cardiovascular diseases with TREM-1 antigen binding proteins Pending CN117751142A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US63/215,260 2021-06-25
US202263353223P 2022-06-17 2022-06-17
US63/353,223 2022-06-17
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