IL-10 mutant proteins and their fusion proteins

Abstract

This provides a mutant protein of IL-10. [Solution] This disclosure provides a monomeric mutant protein of IL-10 useful for the treatment of inflammatory diseases. In one embodiment, a human interleukin-10 (IL-10) mutant protein having a specific sequence is provided, having at least one mutation selected from mutations in helix loop AB, helix loop CD, helix loop DE, helix A, helix B, helix C, helix D, helix E and / or helix F.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 045,041, filed on 26 June 2020, and U.S. Provisional Patent Application No. 63 / 199,218, filed on 14 December 2020, which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to mutant interleukin-10 proteins and antigen-binding proteins comprising IL-10 and antigen-binding moieties, such as anti-TREM-1 or anti-PD-1 antibodies, for the treatment of inflammatory diseases such as inflammatory bowel disease.

[0003] Reference to electronically submitted materials The sequence listing, which is part of this disclosure, is submitted together with this specification as a text file. The name of the text file containing the sequence listing is "55384_Seqlisting.txt", which was created on June 22, 2021, and is 5,726,194 bytes in size. The contents of the sequence listing are incorporated herein by reference in their entirety. [Background technology]

[0004] Interleukin-10 (IL-10) is an important cytokine involved in various immunomodulatory processes (see Ouyang and O'Garra, Immunity, 50(4):871-891 2019). In particular, it has either immunosuppressive or immunostimulatory effects depending on the immune system cell type. IL-10 suppresses the upregulation of inflammatory cytokines, MHCII, CD86, and ICAM in monocytes and tissue macrophages. IL-10 has also been reported to promote Treg cell suppression activity. However, IL-10 also stimulates CD8+ T cell activation and B cell activation. There is a strong genetic link between IL-10 and inflammatory bowel disease (IBD). Patients with homozygous loss-of-function mutations in IL-10, IL-10R1, or IL-10R2 developed severe IBD in infants (Kotlarz, Gastroenterology, 2012; Glocker, NEM, 2009). IL10 rs3024505 has also been reported to be associated with IBD in GWAS, with an RAF of 0.16, an OR of 1.46, and a p-value of 10. -42 It has been shown to have this property (Jostins L, 2012). PEGylated IL-10 has shown low efficacy in Crohn's disease studies, possibly due to dose-limiting toxicity (Schreiber et al., Gastroenterology 119:1461, 2000). Previous studies using mice with IL-10R1 knockout in bone marrow cells have shown that the anti-inflammatory activity of IL-10 in bone marrow cells is important in controlling the development of colitis (Zigmond et al., Immunity, 40(5):720-33, 2014). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Ouyang and O'Garra,Immunity,50(4):871-891 2019 [Non-Patent Document 2] Kotlarz,Gastroenterology,2012;Glocker,NEM,2009 [Non-Patent Document 3] Schreiber et al., Gastroenterology 119:1461,2000 [Non-Patent Document 4] Zigmond et al.,Immunity,40(5):720-33,2014 [Overview of the project] [Means for solving the problem]

[0006] This disclosure provides monomeric mutant proteins of IL-10 useful for the treatment of inflammatory diseases. The IL-10 mutant proteins are thought to retain / restore IL-10 inhibitory activity in monocytes and macrophages and reduce IL-10 stimulation of CD8+ T cells and B cells. This disclosure also provides antibodies against TREM-1 useful for the treatment of inflammatory diseases. In a further embodiment, this disclosure provides antigen-binding proteins containing IL-10 mutant proteins and antigen-binding moieties, such as anti-TREM-1 or anti-PD-1 antibodies, useful for the treatment of inflammatory diseases.

[0007] Provided herein are human interleukin-10 (IL-10) mutant proteins having an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 2, wherein the IL-10 mutant protein has at least one mutation selected from mutations in helix loop AB, helix loop CD, helix loop DE, helix A, helix B, helix C, helix D, helix E and / or helix F. In various embodiments, the IL-10 mutant protein of this disclosure includes at least one mutation in helix A, helix F or helix loop AB. In various embodiments, the IL-10 mutant protein is at least 95% identical to the amino acid sequence described in SEQ ID NO: 2. In various embodiments, the IL-10 mutant protein amino acid sequence is 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO: 2.

[0008] In various embodiments, the IL-10 mutant protein is derived from residues N10, H14, F15, P20, M22, L23, R24, R27, D28, K34, T35, Q38, M39, K40, D41, Q42, L43, D44, N45, L46, L47, L48, K49, F56, K57, Y59, L60, Q63, E67, Q70, M77, Q79, N82, Q83, D84, P85 , including mutations in one or more of D86, I87, A89, H90, S93, T100, L103, H109, ​​R110, L112, E115, N116, A127, K130, I136, Y137, K138, S141, E142, D144, I145, E151, M154, M156, K157, or N160 and / or the addition of 4 to 8 amino acids between helix D and helix E. In various embodiments, the mutations are R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, or I87A of SEQ ID NO: 2, which optionally include the addition of 6 amino acids between helix D and helix E. In various embodiments, the mutations optionally include 4 to 8 amino acids between helix D and helix E, such as N10Q, N10I, N10K, R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, I87A, H14Q, F15Y, M22V, K49T, K49S, F56Y, K57N, Y59T, L60Q, Q63E of SEQ ID NO: 2 These are Q63L, E67C, Q70E, Q70K, M77R, M77V, Q79R, Q79C, D84R, A89P, H90E, H90Q, S93E, S93Q, T100R, L103E, H109D, R110P, R110Q, L112V, E115K, N116D, N116Q, A127M, K130Q, I136C, Y137C, M154V, M156C, K157N, or N160D. In various embodiments, the IL10 mutant protein encompasses the combinations of IL10 mutants described in Table 16 or Table 17 or Table 21 and the disclosures herein. In various embodiments, the amino acid between helix D and helix E is GGGSGG (SEQ ID NO: 2676).In various embodiments, the amino acid sequences of specific IL-10 mutant proteins are as follows: SEQ ID NOs. 3-10 and SEQ ID NOs. 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 24 18, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480 This is described in 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2540 and 2777-2791.

[0009] In various embodiments, this disclosure relates to Sequence IDs 3-10 or Sequence IDs 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2360, 2362, 2364, 2366, 236 8, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 24 36, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2500, 2502, 2504, 2 The present invention provides a human interleukin-10 (IL-10) mutant protein containing an amino acid sequence that is at least 90% identical to the amino acid sequences described in 506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538 or 2540 and 2777-2791. In various embodiments, the mutant protein contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the mutant protein sequences described herein.

[0010] In various embodiments, IL-10 mutant proteins reduce the suppression of TNF-α production in myeloid cells, decrease the level of CD8+ T cell stimulation, and / or decrease the level of B cell stimulation compared to wild-type (wt) IL-10. In various embodiments, anti-TREM-1 / IL-10 mutant protein antigen-binding proteins suppress TNF-α production in myeloid cells. In various embodiments, anti-TREM-1 / IL-10 mutant protein antigen-binding proteins suppress TNF-α production in myeloid cells while reducing CD8+ T cell and B cell activation.

[0011] In various embodiments, the IL-10 mutant protein is fused to a binding site that targets an antigen-binding protein to bone marrow cells, B cells, or T cells. In various embodiments, the protein binds to cell surface proteins in bone marrow cells, CD8+ T cells, CD4+ T cells, or B cells.

[0012] In various embodiments, the IL-10 mutant protein further comprises a half-life extension region. In various embodiments, the half-life extension region is an Fc domain. In various embodiments, the half-life extension region is polyethylene glycol (PEG), etc.

[0013] In various embodiments, the mutant protein is a dimer. In various embodiments, the mutant protein dimer contains two different mutant protein monomers. In various embodiments, the mutant protein dimer contains the same mutant protein monomer.

[0014] Isolated nucleic acid molecules containing nucleotide sequences encoding the IL-10 mutant proteins described herein are further provided. In various embodiments, the polynucleotide sequences of the IL-10 mutant proteins are described in SEQ ID NOs: 11-18.

[0015] This disclosure also envisions an expression vector comprising a nucleic acid molecule containing a nucleotide sequence encoding an IL-10 mutant protein operably linked to an expression regulatory sequence.

[0016] Recombinant host cells comprising nucleic acids or vectors containing a nucleotide sequence encoding an IL-10 mutant protein are provided herein. In various embodiments, the host cell is a mammalian cell. In various embodiments, the host cell is a CHO cell. A method for generating an IL-10 mutant protein using a host cell, comprising culturing the host cell and recovering the IL-10 mutant protein, and the IL-10 mutant protein produced by this method are also provided herein.

[0017] This disclosure also provides pharmaceutical compositions comprising the IL-10 mutant protein described herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be sterile pharmaceutical compositions.

[0018] In another aspect, the present disclosure relates to an isolated antigen-binding protein, a. An antibody or antibody fragment; b. Binds to the human trigger receptor (TREM-1) expressed on myeloid cells 1, having the amino acid sequence described in Sequence ID No. 20; c. Light chain variable domain, i. Light chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290 and 2190; ii. Light chain CDR2 containing an amino acid sequence selected from SEQ ID NOs: 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271, 291 and 2191; iii. Light chain CDR3 containing an amino acid sequence selected from SEQ ID NOs: 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292 and 2192. Includes a light chain variable domain; and d. Heavy chain variable domain, i. Heavy chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276, 296, and 2196; ii. Heavy chain CDR2 containing an amino acid sequence selected from SEQ ID NOs: 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277, 297 and 2197; and iii. Heavy chain CDR3 containing an amino acid sequence selected from SEQ ID NOs: 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298 and 2198. This provides an isolated antigen-binding protein containing a heavy chain variable domain.

[0019] In various embodiments, a. Light chain CDR1 sequences are described in SEQ ID NOs: 30, 50, 70, 110, 150, 170, or 290; b. Light chain CDR2 sequences are described in SEQ ID NOs: 31, 51, 71, 111, 151, 171, or 291; c. Light chain CDR3 sequences are described in SEQ ID NOs: 32, 52, 72, 112, 152, 172, or 292. d. Heavy chain CDR1 sequences are described in SEQ ID NOs: 36, 56, 76, 116, 156, 176, or 296; e. Heavy chain CDR2 sequences are described in SEQ ID NOs: 37, 57, 77, 117, 157, 177, or 297; f. The heavy chain CDR3 sequence is described in SEQ ID NOs: 38, 58, 78, 118, 158, 178, or 298.

[0020] In various embodiments, a. The light chain CDR1 sequence is described in SEQ ID NO: 50 or 110; b. The light chain CDR2 sequence is described in SEQ ID NO: 51 or 111; c. The light chain CDR3 sequence is described in sequence number 52 or 112; d. The heavy chain CDR1 sequence is described in SEQ ID NO: 56 or 116; e. The heavy chain CDR2 sequence is described in SEQ ID NO: 57 or 117; f. The heavy chain CDR3 sequence is described in SEQ ID NO: 58 or 118.

[0021] Consensus sequences of the TREM-1 antibody heavy chain and light chain CDR and / or variable region sequences disclosed herein are also possible. For example, in various embodiments, the TREM-1 antibody is X1ASQSX2X3X4NLA (Sequence ID 2199) (where X1 is R or Q, X2 is V or I, X3 is N or S, and X4 is S, H, I, V, or A); QASX1DIX2X3X4LN (Sequence ID 2204) (where X1 is R or Q, X2 is R, S, N, or F, X3 is K or N, and X4 is H, Y, or D); RASQSVNSNLA(sequence code 2212); Q ASQDIRKHLN(Sequence ID 2213); RASQDISSNLN(sequence code 2214); QASQDIHLN(sequence code 2215); RASQGIRKWLA (Sequence ID 2216) RASQSVNSNLA (Sequence ID 2217) and SGDKLGERVS (Sequence ID 2218) It contains an antigen-binding domain that includes a sequence having a light chain variable region containing an LCDR1 amino acid sequence selected from the group consisting of the above.

[0022] In various embodiments, the TREM-1 antibody is GAX1X2RAT(sequence number 2200)(where X1 is S or Y, and X2 is T or I); X1X2X3X4LET(sequence code 2206)(where X1 is D, G, or H; X2 is A, V, or T; X3 is S, A, or Y; and X4 is T or N); GASTRAT(SEQ ID NO: 2219); DASNLET(sequence number 2220); and AASRLQS (Sequence ID 2221) It contains an antigen-binding domain that includes a sequence having a light chain variable region containing an LCDR2 amino acid sequence selected from the group consisting of the above.

[0023] In various embodiments, the TREM-1 antibody is QX1X2X3X4X5X6PX7T (Sequence ID 2201) (where X1 is Q, H, or E; X2 is F or Y; X3 is K, Y, or I; X4 is N, T, L, I, or M; X5 is W, F, H, or Y; X6 is nonexistent or P; X7 is W, N, Y, H, or L); QX1YX3X4X5PX6T (Sequence ID 2207) (where X1 is Q or H, X2 is D, A or G, X3 is N or K; X4 is L or I, and X5 is I or L); QQFKNWPPT(sequence code 2222); QHYDNLPIT(sequence number 2223); LQAHGFPWT(Sequence ID 2224); QQYDNLPLT (Sequence ID 2225) and QFWPPWT (Sequence ID 2226) It contains an antigen-binding domain that includes a sequence having a light chain variable region containing an LCDR3 amino acid sequence selected from the group consisting of the above.

[0024] In various embodiments, the TREM-1 antibody is X1X2X3MX4 (Sequence ID 2202) (where X1 is A, R, T, or S; X2 is Y or N; X3 is A or W; and X4 is S or N); X1YDIN (sequence number 2208) (where X1 is R or S); GYYX1H (sequence number 2723) (where X1 is M or I); AYAMS(sequence code 2227); RYDIN(sequence key 2228); and SYWMS (Sequence ID 2229) An antigen-binding domain comprising an array having a heavy-chain variable region comprising an HCDR1 amino acid sequence selected from the group consisting of

[0025] In various embodiments, the TREM-1 antibody is X1X2X3X4X5X6X7X8X9YYX 10 X 11 X 12 VKG (SEQ ID NO: 2205) (where X1 is T, E or S; X2 is absent or M, V or I; X3 is S, R or K; X4 is G or Q; X5 is S, D or H; X6 is G, S, L or A; X7 is S, G or R; X8 is T, S, P or E; X9 is T or I; X 10 is A or V; X 11 is D or E; X 12 is S or A); X1X2NPX3X4GX5X6GX7X8X9X 10 FX 11 X 12 (SEQ ID NO: 2209) (where X1 is W or R; X2 is M or L; X3 is N, Q or K; X4 is S, A or R; X5 is N or Q; X6 is S, A or T; X7 is S, Q or Y; X8 is V or T; X9 is Q or K; X 10 is K or N; X 11 is R or Q; X 12 is G or D); TSGSGSTTYYADSVKG (SEQ ID NO: 2230); WMNPNSGNSSVQKFRG (SEQ ID NO: 2231); NIKQDGSEEYYVDSVKG (SEQ ID NO: 2232); and TSGSGTYYADSVKG (SEQ ID NO: 2669) An antigen-binding domain comprising an array having a heavy-chain variable region comprising an HCDR2 amino acid sequence selected from the group consisting of

[0026] In various embodiments, the TREM-1 antibody is X1X2X3X4X5X6X7FX8YYX9 (Sequence ID 2203) (where X1 is V, E, A, or G; X2 is A, F, Y, or G; X3 is G, S, Y, or W; X4 is S or R; X5 is nonexistent or N; X6 is F, S, Y, or nonexistent; X7 is L, F, or nonexistent; X8 is D or E; and X9 is Y, H, or S); X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 FX 13 X 14 (Sequence ID 2210) (where X1 is G, L or R, X2 is G, I or R, X3 is Y, R, I, G or A, X4 is T, S, Y or V, X5 is S or Y, X6 is S, A, I or R, X7 is W, A or S, X8 is nonexistent or S, X9 is nonexistent or F, W or Y, X 10 is R, S, H, K, or E, and X 11 is W, H, Y, or F, and X 12 is Y, V, A, or S, and X 13 is D or Q, and X 14 is L, Y, I, or H; VAGSNFLFDY(Sequence ID 2670); GGYTSSWRWYFDL(Sequence ID 2671); GGYTSSWSRWYFDL(sequence number 2672); and DYGDSFDY (Sequence ID 2673) It contains an antigen-binding domain that includes a sequence having a heavy chain variable region containing an HCDR3 amino acid sequence selected from the group consisting of the above.

[0027] In various embodiments, an isolated antigen-binding protein, a. An antibody or antibody fragment; b. Binds to human TREM-1 having the amino acid sequence described in Sequence ID No. 20; c. Light chain variable domain, i. Light chain CDR1 containing the amino acid sequence X1ASQSX2X3X4NLA (SEQ ID NO: 2199) (where X1 is R or Q, X2 is V or I, X3 is N or S, and X4 is S, H, I, V, or A); ii. Light chain CDR2 containing the amino acid sequence GAX1X2RAT ​​(SEQ ID NO: 2200) (where X1 is S or Y and X2 is T or I); and iii. Light chain CDR3 containing the amino acid sequence QX1X2X3X4X5X6PX7T (SEQ ID NO: 2201) (where X1 is Q, H, or E; X2 is F, or Y; X3 is K, Y, or I; X4 is N, T, L, I, or M; X5 is W, F, H, or Y; X6 is absent or P; X7 is W, N, Y, H, or L) Includes a light chain variable domain; and d. Heavy chain variable domain, i. Heavy chain CDR1 containing the amino acid sequence X1X2X3MX4 (SEQ ID NO: 2202) (where X1 is A, R, T, or S; X2 is Y or N; X3 is A or W; and X4 is S or N); ii. Amino acid sequence X1X2X3X4X5X6X7X8X9YYX 10 X 11 X 12 VKG (Sequence ID 2205) (where X1 is T, E, or S; X2 is nonexistent or M, V, or I; X3 is S, R, or K; X4 is G, or Q; X5 is S, D, or H; X6 is G, S, L, or A; X7 is S, G, or R; X8 is T, S, P, or E; X9 is T, I, and X 10 is A or V, and X 11 is D or E, and X 12 heavy chain CDR2 (which is S or A); and iii. Heavy chain CDR3 containing amino acid sequence X1X2X3X4X5X6X7FX8YYX9 (SEQ ID NO: 2203) (where X1 is V, E, A or G; X2 is A, F, Y or G; X3 is G, S, Y or W; X4 is S or R; X5 is absent or N; X6 is F, S, Y or absent; X7 is L or F or absent; X8 is D or E; X9 is Y, H or S) Heavy chain variable domains including Isolated antigen-binding proteins, including the above, are provided herein.

[0028] In various embodiments, the antigen-binding protein is a. Light chain variable domain, i. Light chain CDR1 containing the amino acid sequence RASQSVNSNLA (SEQ ID NO: 2212); ii. Light chain CDR2 containing the amino acid sequence GASTRAT (SEQ ID NO: 2219); iii. Light chain CDR3 containing amino acid sequence QQFKNWPPT (SEQ ID NO: 2222) Light chain variable domains including; and b. Heavy chain variable domain, i. Heavy chain CDR1 containing the amino acid sequence AYAMS (SEQ ID NO: 2227); ii. Heavy chain CDR2 containing the amino acid sequence TSGSGSTTYYADSVKG (SEQ ID NO: 2230); and iii. Heavy chain CDR3 containing the amino acid sequence VAGSNFLFDY (SEQ ID NO: 2670) Heavy chain variable domains including Includes.

[0029] In various embodiments, the present disclosure relates to an isolated antigen-binding protein, a. An antibody or antibody fragment; b. Binds to human TREM-1 having the amino acid sequence described in Sequence ID No. 20; c. Light chain variable domain, i. Light chain CDR1 containing the amino acid sequence QASX1DIX2X3X4LN (SEQ ID NO: 2204) (where X1 is R or Q, X2 is R, S, N, or F, X3 is K or N, and X4 is H, Y, or D); ii. Light chain CDR2 containing the amino acid sequence X1X2X3X4LET (SEQ ID NO: 2206) (where X1 is D, G, or H; X2 is A, V, or T; X3 is S, A, or Y; and X4 is T or N); iii. Light chain CDR3 containing the amino acid sequence QX1YX3X4X5PX6T (SEQ ID NO: 2207) (where X1 is Q or H, X2 is D, A or G, X3 is N or K; X4 is L or I, and X5 is I or L) Includes a light chain variable domain; and d. Heavy chain variable domain, i. Heavy chain CDR1 containing the amino acid sequence X1YDIN (SEQ ID NO: 2208) (where X1 is R or S); ii. Amino acid sequence X1X2NPX3X4GX5X6GX7X8X9X 10 FX 11 X 12 (Sequence ID 2209) (where X1 is W or R, X2 is M or L, X3 is N, Q or K, X4 is S, A or R, X5 is N or Q, X6 is S, A or T, X7 is S, Q or Y, X8 is V or T, X9 is Q or K, X 10 is K or N, and X 11 is R or Q, and X 12 (is G or D) heavy chain CDR2; and iii. Amino acid sequence X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 FX 13 X 14(Sequence ID 2210) (where X1 is G, L or R, X2 is G, I or R, X3 is Y, R, I, G or A, X4 is T, S, Y or V, X5 is S or Y, X6 is S, A, I or R, X7 is W, A or S, X8 is nonexistent or S, X9 is nonexistent or F, W or Y, X 10 is R, S, H, K, or E, and X 11 is W, H, Y, or F, and X 12 is Y, V, A, or S, and X 13 is D or Q, and X 14 Heavy chain CDR3 containing L, Y, I, or H This provides an isolated antigen-binding protein containing a heavy chain variable domain.

[0030] In various embodiments, the antigen-binding protein is a. Light chain variable domain, i. Light chain CDR1 containing the amino acid sequence QASQDIRKHLN (SEQ ID NO: 2213); ii. Light chain CDR2 containing the amino acid sequence DASNLET (SEQ ID NO: 2220); and iii. Light chain CDR3 containing amino acid sequence QHYDNLPIT (SEQ ID NO: 2223) Light chain variable domains including; and b. Heavy chain variable domain, i. Heavy chain CDR1 containing the amino acid sequence RYDIN (SEQ ID NO: 2228); ii. Heavy chain CDR2 containing the amino acid sequence WMNPNSGNSSVQKFRG (SEQ ID NO: 2231); and iii. Heavy chain CDR3 containing the amino acid sequence GGYTSSWRWYFDL (SEQ ID NO: 2671) or GGGYTSSWSRWYFDL (SEQ ID NO: 2672) Heavy chain variable domains including Includes.

[0031] In various embodiments, the present disclosure relates to an isolated antigen-binding protein, a. An antibody or antibody fragment; b. Binds to the human trigger receptor (TREM-1) expressed on myeloid cells 1, having the amino acid sequence described in Sequence ID No. 20; c. Select a set of CDR sequences from the following: i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 90 (LCDR1), 91 (LCDR2), 92 (LCDR3), 96 (HCDR1), 97 (HCDR2), and 98 (HCDR3); v) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); vi) Sequence IDs 130 (LCDR1), 131 (LCDR2), 132 (LCDR3), 136 (HCDR1), 137 (HCDR2), and 138 (HCDR3); vii) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); viii) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); ix) Sequence IDs 190 (LCDR1), 191 (LCDR2), 192 (LCDR3), 196 (HCDR1), 197 (HCDR2), and 198 (HCDR3); x) Sequence IDs 210 (LCDR1), 211 (LCDR2), 212 (LCDR3), 216 (HCDR1), 217 (HCDR2), and 218 (HCDR3); xi) Sequence IDs 230 (LCDR1), 231 (LCDR2), 232 (LCDR3), 236 (HCDR1), 237 (HCDR2), and 238 (HCDR3); xii) Sequence IDs 250(LCDR1), 251(LCDR2), 252(LCDR3), 256(HCDR1), 257(HCDR2), and 258(HCDR3); xiii) Sequence IDs 270 (LCDR1), 271 (LCDR2), 272 (LCDR3), 276 (HCDR1), 277 (HCDR2), and 278 (HCDR3); xiv) Sequence IDs 290(LCDR1), 291(LCDR2), 292(LCDR3), 296(HCDR1), 297(HCDR2), and 298(HCDR3); or xv) Sequence IDs 2190 (LCDR1), 2191 (LCDR2), 2192 (LCDR3), 2196 (HCDR1), 2197 (HCDR2), and 2198 (HCDR3) This provides isolated antigen-binding proteins, including [specific proteins].

[0032] In various embodiments, the anti-TREM-1 antigen-binding protein is i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); v) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); vi) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); or vii) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3) Includes a set of CDR sequences selected from.

[0033] In various embodiments, the anti-TREM-1 antigen-binding protein comprises a set of CDR sequences selected from SEQ ID NOs. 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); or SEQ ID NOs. 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3).

[0034] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299, and 2183; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299 and 2183. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186; ii. Sequences encoded by polynucleotide sequences that are at least 80% identical to amino acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0035] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 121, 161, 181, and 301; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 39, 59, 79, 119, 159, 179, and 299; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a nucleic acid sequence selected from SEQ ID NOs: 39, 59, 79, 119, 159, 179, and 299. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 42, 62, 82, 122, 162, 182, and 302; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 40, 60, 80, 120, 160, 180, and 300; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 120, 160, 180, and 300. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0036] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 61 and 121; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 59 and 119; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs. 59 and 119. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs. 62 and 122; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs. 60 and 120; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs. 60 and 120. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0037] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) The light chain variable domain described in Sequence ID No. 41 and the heavy chain variable domain described in Sequence ID No. 42; ii) The light chain variable domain described in Sequence ID No. 61 and the heavy chain variable domain described in Sequence ID No. 62; iii) The light chain variable domain described in Sequence ID No. 81 and the heavy chain variable domain described in Sequence ID No. 82; iv) The light chain variable domain described in Sequence ID No. 101 and the heavy chain variable domain described in Sequence ID No. 102; v) The light chain variable domain described in Sequence ID No. 121 and the heavy chain variable domain described in Sequence ID No. 122; vi) The light chain variable domain described in Sequence ID No. 161 and the heavy chain variable domain described in Sequence ID No. 162; vii) The light chain variable domain described in Sequence ID No. 181 and the heavy chain variable domain described in Sequence ID No. 182; viii) The light chain variable domain described in Sequence ID No. 201 and the heavy chain variable domain described in Sequence ID No. 202; x) The light chain variable domain described in Sequence ID No. 221 and the heavy chain variable domain described in Sequence ID No. 222; xi) The light chain variable domain described in Sequence ID No. 241 and the heavy chain variable domain described in Sequence ID No. 242; xii) The light chain variable domain described in Sequence ID No. 261 and the heavy chain variable domain described in Sequence ID No. 262; xiii) The light chain variable domain described in Sequence ID No. 281 and the heavy chain variable domain described in Sequence ID No. 282; xiv) The light chain variable domain described in Sequence ID No. 301 and the heavy chain variable domain described in Sequence ID No. 302; or xv) Light chain variable domain described in Sequence ID No. 2185 and heavy chain variable domain described in Sequence ID No. 2186 Includes.

[0038] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) The light chain variable domain described in Sequence ID No. 41 and the heavy chain variable domain described in Sequence ID No. 42; ii) The light chain variable domain described in Sequence ID No. 61 and the heavy chain variable domain described in Sequence ID No. 62; iii) The light chain variable domain described in Sequence ID No. 81 and the heavy chain variable domain described in Sequence ID No. 82; iv) The light chain variable domain described in Sequence ID No. 121 and the heavy chain variable domain described in Sequence ID No. 122; v) The light chain variable domain described in Sequence ID No. 161 and the heavy chain variable domain described in Sequence ID No. 162; vi) The light chain variable domain described in Sequence ID No. 181 and the heavy chain variable domain described in Sequence ID No. 182; or vii) Light chain variable domain described in Sequence ID No. 301 and heavy chain variable domain described in Sequence ID No. 302 Includes.

[0039] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein includes the light chain variable domain described in SEQ ID NO: 61 and the heavy chain variable domain described in SEQ ID NO: 62; or the light chain variable domain described in SEQ ID NO: 121 and the heavy chain variable domain described in SEQ ID NO: 122.

[0040] In various embodiments, the amino acid sequence may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence numbers 42, 62, 82, 102, 122, 142, 162, 181, 202, 222, 242, 262, 282, 302, and 2186, as well as sequence numbers 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185.

[0041] In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to the heavy chain variable region amino acid sequence selected from SEQ ID NOs: 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186. In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to the light chain variable region amino acid sequence described in SEQ ID NOs: 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185.

[0042] In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to the heavy chain variable region amino acid sequence selected from SEQ ID NOs: 42, 62, 82, 122, 162, 182, and 302. In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to the light chain variable region amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 121, 161, 181, and 301.

[0043] In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to the heavy chain variable region amino acid sequence selected from SEQ ID NOs. 62 and 122. In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to the light chain variable region amino acid sequence selected from SEQ ID NOs. 61 and 121.

[0044] In various embodiments, one or more heavy chain framework amino acids of the antigen-binding protein are substituted with corresponding 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 substituted with corresponding amino acids from another human antibody amino acid sequence.

[0045] In some cases, the sequences disclosed herein may contain N-terminal signal sequences useful for recombinant production. Sequences of anti-TREM-1 antibodies or antigen-binding proteins lacking signal sequences are assumed herein. Exemplary signal sequences include MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 2674); MAWALLLLTLLTQGTGSWASYEL (SEQ ID NO: 2675) and nucleic acids encoding such signal sequences.

[0046] In various embodiments, the anti-TREM-1 antigen-binding protein further comprises a human light chain constant region bound to the light chain variable region.

[0047] In various embodiments, the heavy chain constant region is selected from the heavy chain constant regions of IgG, IgM, IgA, IgD, IgE, their fragments, combinations thereof, and modifications thereof in which 1 to 10 heavy chain framework amino acids are substituted with corresponding amino acids from another human antibody amino acid sequence.

[0048] In various embodiments, the anti-TREM-1 antigen-binding proteins described herein inhibit the binding of TREM-1 ligands to TREM-1.

[0049] Regarding binding to the human TREM-1 protein having the sequence of SEQ ID NO: 20, antigen-binding proteins that compete with the anti-TREM-1 antigen-binding proteins described herein are also conceivable.

[0050] 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 antibody, IgG2 antibody, IgG3 antibody, and IgG4 antibody. 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.

[0051] In various embodiments, the antigen-binding protein is a human antibody.

[0052] Isolated nucleic acid molecules containing nucleotide sequences encoding the heavy chain of the anti-TREM-1 antigen-binding protein described herein, isolated nucleic acid molecules containing nucleotide sequences encoding the light chain of the anti-TREM-1 antigen-binding protein described herein, and isolated nucleic acid molecules containing nucleotide sequences encoding the heavy and light chains of the anti-TREM-1 antigen-binding protein described herein are also provided.

[0053] In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to a heavy chain variable region polynucleotide sequence selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184. In various embodiments, the antigen-binding protein includes an amino acid sequence that is at least 90% identical to a light chain variable region polynucleotide sequence described in SEQ ID NOs: 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299, and 2183.

[0054] In various embodiments, the antigen-binding protein comprises an amino acid sequence that is at least 90% identical to a heavy chain variable region polynucleotide sequence selected from SEQ ID NOs. 40, 60, 80, 120, 160, 180, and 300. In various embodiments, the antigen-binding protein comprises an amino acid sequence that is at least 90% identical to a light chain variable region polynucleotide sequence selected from SEQ ID NOs. 39, 59, 79, 119, 159, 179, and 299.

[0055] In various embodiments, the antigen-binding protein comprises an amino acid sequence that is at least 90% identical to a heavy chain variable region polynucleotide sequence selected from SEQ ID NOs. 60 and 120. In various embodiments, the antigen-binding protein comprises an amino acid sequence that is at least 90% identical to a light chain variable region polynucleotide sequence selected from SEQ ID NOs. 59 and 119.

[0056] Further considerations include expression vectors comprising nucleic acid molecules of the anti-TREM-1 antigen-binding protein heavy and / or light chain described herein, operably linked to an expression control sequence.

[0057] This disclosure provides recombinant host cells comprising a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of an anti-TREM-1 antigen-binding protein or antibody as described herein; or a nucleic acid molecule encoding the light chain of an anti-TREM-1 antibody as described herein; or a nucleic acid molecule encoding the 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.

[0058] A method for generating an antigen-binding protein using host cells, comprising culturing the host cells and recovering the antigen-binding protein, and an antigen-binding protein produced by this method are further provided.

[0059] Sterile pharmaceutical compositions comprising the anti-TREM-1 antigen-binding protein described herein and a pharmaceutically acceptable carrier are also provided.

[0060] An antigen-binding protein comprising an antigen-binding moiety and one or two IL-10 moieties, a. The antigen-binding portion is an antibody or antibody fragment. b. Each IL-10 moiety is independently monovalent or divalent. c. Each IL-10 moiety was independently selected from one or more human IL-10 mutant proteins having a sequence that is 90% identical to SEQ ID NO: 2. d. Further considerations include antigen-binding proteins in which at least one IL-10 moiety is covalently bound to the antigen-binding portion.

[0061] In various embodiments, at least one IL-10 moiety is fused to the C-terminus of the antigen-binding moiety. In various embodiments, at least one IL-10 moiety is fused to the N-terminus of the antigen-binding moiety. In various embodiments, at least one IL-10 moiety is fused to both the N and C-terminuses of the antigen-binding moiety. In various embodiments, the IL-10 moiety is fused to an internal site in the antigen-binding moiety. In various embodiments, at least one IL-10 moiety is fused to the heavy chain and / or light chain of the antigen-binding moiety. In various embodiments, the heavy chain and / or light chain are modified or manipulated heavy or light chains.

[0062] (a) A polypeptide sequence having formula ALM or MLA, i) A is the immunoglobulin heavy chain of the IgG antibody that binds to the TREM-1 protein as described in Sequence ID No. 20, ii) L is a linker peptide containing 4 to 20 amino acids, and iii) M is a polypeptide sequence of IL-10 mutant protein having at least 90% sequence identity with wt IL-10 described in Sequence ID No. 2; and (b) Immunoglobulin light chain of IgG antibody that binds to TREM-1 protein as described in Sequence ID No. 20 An antigen-binding protein containing, The immunoglobulin heavy chain of (a) and the immunoglobulin light chain of (b) form an IgG antibody moiety that binds to TREM-1, and the protein comprises one or two molecules of polypeptide of (a) and one or two molecules of light chain of (b), and optionally, an antigen-binding protein is also provided in which only one polypeptide of (a) comprises the M moiety.

[0063] In various embodiments, antigen-binding proteins reduce the suppression of TNF-α production in myeloid cells, decrease the level of CD8+ T cell stimulation, and / or decrease the level of B cell stimulation compared to wt IL-10.

[0064] In various embodiments of antigen-binding proteins, a. The antigen-binding portion is an antibody. b. The IL-10 moiety is fused to each heavy chain of the antibody.

[0065] In various embodiments of the antigen-binding protein, each IL-10 moiety is a monomer. In various embodiments, the antigen-binding protein contains two different mutant protein monomers. In various embodiments, the antigen-binding protein contains two identical mutant protein monomers.

[0066] In various embodiments of the antigen-binding protein, each IL-10 moiety comprises an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 2, and each IL-10 moiety independently comprises at least one mutation selected from mutations in helix loops AB, CD, DE, helix A, B, C, D, E, and / or F.

[0067] In various embodiments of the antigen-binding protein, at least one IL-10 moiety contains at least one mutation in helix A. In various embodiments of the antigen-binding protein, at least one IL-10 moiety contains at least one mutation in helix F. In various embodiments of the antigen-binding protein, at least one IL-10 moiety contains at least one mutation in helix loop AB.

[0068] In various embodiments, anti-TREM-1 / IL-10 mutant protein antigen-binding proteins lack CD8+ T cell stimulation and / or B cell stimulation in human subjects, compared to, for example, wt IL-10.

[0069] In various embodiments, IL-10 mutant proteins reduce the suppression of TNF-α production in myeloid cells, decrease the level of CD8+ T cell stimulation, and / or decrease the level of B cell stimulation compared to wt IL-10.

[0070] In various embodiments, IL-10 mutant protein antigen-binding proteins, such as anti-TREM-1 / IL-10 mutant protein antigen-binding proteins, suppress TNF-α production in bone marrow cells. In various embodiments, IL-10 mutant protein antigen-binding proteins, such as anti-TREM-1 / IL-10 mutant protein antigen-binding proteins, suppress TNF-α production in bone marrow cells, but still lack CD8+ T cell and B cell activation.

[0071] In various embodiments, antigen-binding proteins having an IL-10 mutant protein fused to an antigen-binding partner for a cell surface protein in CD8+ cells and / or B cells enhance the level of CD8+ T cell stimulation and / or B cell stimulation in human subjects.

[0072] In various embodiments of the antigen-binding protein, each IL-10 mutant protein independently corresponds to residues N10, H14, F15, P20, M22, L23, R24, R27, D28, K34, T35, Q38, M39, K40, D41, Q42, L43, D44, N45, L46, L47, L48, K49, F56, K57, Y59, L60, Q63, E67, Q70, M77, Q79, N82, Q This includes mutations in one or more of the following helices and / or the addition of 4 to 8 amino acids between helix D and helix E: 83, D84, P85, D86, I87, A89, H90, S93, T100, L103, H109, ​​R110, L112, E115, N116, A127, K130, I136, Y137, K138, S141, E142, D144, I145, E151, M154, M156, K157, N160.

[0073] In various embodiments, each IL-10 mutant protein independently contains one or more mutations selected from the group consisting of R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D or I87A of SEQ ID NO: 2, which independently contain R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, I87A, H14Q, F15Y, M22V, K49T, K4 9S, F56Y, K57N, Y59T, L60Q, Q63E, Q63L, E67C, Q70E, Q70K, M77R, M77V, Q79R, Q79C, D84R, A8 It contains one or more mutations selected from the group consisting of 9P, H90E, H90Q, S93E, S93Q, T100R, L103E, H109D, R110P, R110Q, L112V, E115K, N116D, N116Q, A127M, K130Q, I136C, Y137C, M154V, M156C, K157N, or N160D.

[0074] In various embodiments, each IL-10 mutant protein independently contains one or more mutations selected from the group consisting of R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, or I87A of SEQ ID NO: 2, which optionally include the addition of six amino acids between helix D and helix E. In various embodiments, the amino acids between helix D and helix E are GGGSGG (SEQ ID NO: 2676). In various embodiments, each IL-10 mutant protein independently and optionally includes the addition of six amino acids between helix D and helix E, such as N10Q, N10I, N10K, R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, I87A, H14Q, F15Y, M22V, K49T, K49S, F56Y, K57N, Y59T, L60Q, Q63E, Q It includes one or more mutations selected from the group consisting of 63L, E67C, Q70E, Q70K, M77R, M77V, Q79R, Q79C, D84R, A89P, H90E, H90Q, S93E, S93Q, T100R, L103E, H109D, R110P, R110Q, L112V, E115K, N116D, N116Q, A127M, K130Q, I136C, Y137C, M154V, M156C, K157N, or N160D. In various embodiments, the amino acid between helix D and helix E is GGGSGG (SEQ ID NO: 2676).

[0075] In various embodiments of the antigen-binding protein, each IL-10 mutant protein is at least 95% identical to the amino acid sequence described in SEQ ID NO: 2. In various embodiments, the amino acid sequences of the IL-10 mutant proteins are described in SEQ ID NOs: 3-10. In various embodiments, the amino acid sequences of the IL-10 mutant proteins are as described in SEQ ID NOs: 3-10 and SEQ ID NOs: 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 218 0, 2182, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2 420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482 This is described in 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540 and 2777-2791.

[0076] In various embodiments, the antigen-binding protein includes at least one linker fused to at least one C-terminus of the antigen-binding moiety, and the IL-10 moiety is covalently bonded to the C-terminus of each linker. In various embodiments, the linker is 4 to 18 amino acids long. In various embodiments, the linker is 6 amino acids long. In various embodiments, the linker is a trimer, tetramer, pentamer, hexamer, heptamer, or octamer peptide. In various embodiments, the linker contains a GS residue.

[0077] In various embodiments, the antigen-binding protein comprises an "embedded" IL-10 mutant protein. In various embodiments, the antigen-binding protein comprises: (a) a Fab portion of the anti-TREM1 binding moiety; (b) at least one G4 linker fused to the C-terminus of the Fab portion of the TREM1 binding moiety; (c) an IL-10 portion covalently bonded to the C-terminus of each linker; another G4 linker fused to the C-terminus of the IL-10 portion; and an Fc region covalently bonded to the C-terminus of this second G4 linker. Further hypothetical linkers will be described in more detail.

[0078] In various embodiments, the antigen-binding portion of the antigen-binding protein further comprises a human heavy chain constant region bound to the heavy chain variable region of the antibody portion of the antigen-binding protein. In various embodiments, the antigen-binding protein further comprises a human light chain constant region bound to the light chain variable region of the antibody portion of the antigen-binding protein.

[0079] In various embodiments of the antigen-binding protein, the antigen-binding moiety 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 antibody, IgG2 antibody, IgG3 antibody, and IgG4 antibody. In various embodiments, the antigen-binding moiety is IgG. In various embodiments, the antigen-binding moiety is IgG2 antibody. In various embodiments, the antigen-binding moiety is IgG1 antibody. In various embodiments of the antigen-binding protein, the antibody is IgG1z or IgG1z-SEFL2 antibody. In various embodiments, the antigen-binding moiety is monovalent IgG. In various embodiments, the heavy chain constant region of the antigen-binding moiety is selected from the heavy chain constant regions of IgG, IgM, IgA, IgD, IgE, their fragments, combinations thereof, and modifications thereof in which 1 to 10 heavy chain framework amino acids are substituted with corresponding amino acids from another human antibody constant region.

[0080] In various embodiments of the antigen-binding protein, the antigen-binding moiety binds to human PD-1 having the amino acid sequence described in SEQ ID NO: 22. In various embodiments, the antigen-binding moiety binds to human TREM-1 having the amino acid sequence described in SEQ ID NO: 20. In various embodiments, the antigen-binding moiety has at least 10 -8 It binds to human TREM-1 with binding affinity M. In various embodiments, the antigen-binding moiety is at least 10 -8 M~10 -15 M or 10 -8 M~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 Binds to the antigen due to its binding affinity for M.

[0081] In various embodiments of antigen-binding proteins, the anti-TREM-1 antigen-binding moiety is a. Light chain variable domain, i. Light chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290 and 2190; ii. A light chain CDR2 sequence containing an amino acid sequence selected from SEQ ID NOs: 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271, 291, and 2191; iii. Light chain CDR3 sequence containing an amino acid sequence selected from SEQ ID NOs: 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292 and 2192 Light chain variable domains including; and b. Heavy chain variable domain, i. Heavy chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276, 296, and 2196; ii. Heavy chain CDR2 sequences containing amino acid sequences selected from SEQ ID NOs: 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277, 297 and 2197; and iii. Heavy chain CDR3 sequences containing amino acid sequences selected from SEQ ID NOs: 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298 and 2198. Heavy chain variable domains including Includes.

[0082] In various embodiments of the antigen-binding protein, the anti-TREM-1 antigen-binding moiety includes the following: a. Light chain CDR1 sequences are described in SEQ ID NOs: 30, 50, 70, 110, 150, 170, or 290; b. Light chain CDR2 sequences are described in SEQ ID NOs: 31, 51, 71, 111, 151, 171, or 291; c. Light chain CDR3 sequences are described in SEQ ID NOs: 32, 52, 72, 112, 152, 172, or 292. d. Heavy chain CDR1 sequences are described in SEQ ID NOs: 36, 56, 76, 116, 156, 176, or 296; e. Heavy chain CDR2 sequences are described in SEQ ID NOs: 37, 57, 77, 117, 157, 177, or 297; f. The heavy chain CDR3 sequence is described in SEQ ID NOs: 38, 58, 78, 118, 158, 178, or 298.

[0083] In various embodiments of the antigen-binding protein, the anti-TREM-1 antigen-binding moiety includes the following: a. The light chain CDR1 sequence is described in SEQ ID NO: 50 or 110; b. The light chain CDR2 sequence is described in SEQ ID NO: 51 or 111; c. The light chain CDR3 sequence is described in sequence number 52 or 112; d. The heavy chain CDR1 sequence is described in SEQ ID NO: 56 or 116; e. The heavy chain CDR2 sequence is described in SEQ ID NO: 57 or 117; f. The heavy chain CDR3 sequence is described in SEQ ID NO: 58 or 118.

[0084] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 90 (LCDR1), 91 (LCDR2), 92 (LCDR3), 96 (HCDR1), 97 (HCDR2), and 98 (HCDR3); v) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); vi) Sequence IDs 130 (LCDR1), 131 (LCDR2), 132 (LCDR3), 136 (HCDR1), 137 (HCDR2), and 138 (HCDR3); vii) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); viii) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); ix) Sequence IDs 190 (LCDR1), 191 (LCDR2), 192 (LCDR3), 196 (HCDR1), 197 (HCDR2), and 198 (HCDR3); x) Sequence IDs 210 (LCDR1), 211 (LCDR2), 212 (LCDR3), 216 (HCDR1), 217 (HCDR2), and 218 (HCDR3); xi) Sequence IDs 230 (LCDR1), 231 (LCDR2), 232 (LCDR3), 236 (HCDR1), 237 (HCDR2), and 238 (HCDR3); xii) Sequence IDs 250(LCDR1), 251(LCDR2), 252(LCDR3), 256(HCDR1), 257(HCDR2), and 258(HCDR3); xiii) Sequence IDs 270 (LCDR1), 271 (LCDR2), 272 (LCDR3), 276 (HCDR1), 277 (HCDR2), and 278 (HCDR3); xiv) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3); and xv) Sequence IDs 2190 (LCDR1), 2191 (LCDR2), 2192 (LCDR3), 2196 (HCDR1), 2197 (HCDR2), and 2198 (HCDR3) Includes a set of CDR sequences selected from.

[0085] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); v) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); vi) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); and vii) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3) Includes a set of CDR sequences selected from.

[0086] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein includes a set of CDR sequences selected from SEQ ID NOs: 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); and SEQ ID NOs: 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3).

[0087] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299, and 2183; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299 and 2183. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186; ii. Sequences encoded by polynucleotide sequences that are at least 80% identical to amino acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0088] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 121, 161, 181, and 301; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 39, 59, 79, 119, 159, 179, and 299; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a nucleic acid sequence selected from SEQ ID NOs: 39, 59, 79, 119, 159, 179, and 299. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 42, 62, 82, 122, 162, 182, and 302; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 40, 60, 80, 120, 160, 180, and 300; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 120, 160, 180, and 300. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0089] In various embodiments of antigen-binding proteins, the anti-TREM-1 antigen-binding moiety is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 61 and 121; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 59 and 119; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs. 59 and 119. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs. 62 and 122; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs. 60 and 120; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs. 60 and 120. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0090] In various embodiments, the amino acid sequence may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence numbers 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185 and sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186.

[0091] In various embodiments of the antigen-binding protein, the antigen-binding moiety includes an amino acid sequence that is at least 90% identical to the heavy chain variable region amino acid sequence selected from SEQ ID NOs: 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186.

[0092] In various embodiments of the antigen-binding protein, the antigen-binding portion includes an amino acid sequence that is at least 90% identical to the light chain variable region amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185.

[0093] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) The light chain variable domain described in Sequence ID No. 41 and the heavy chain variable domain described in Sequence ID No. 42; ii) The light chain variable domain described in Sequence ID No. 61 and the heavy chain variable domain described in Sequence ID No. 62; iii) The light chain variable domain described in Sequence ID No. 81 and the heavy chain variable domain described in Sequence ID No. 82; iv) The light chain variable domain described in Sequence ID No. 101 and the heavy chain variable domain described in Sequence ID No. 102; v) The light chain variable domain described in Sequence ID No. 121 and the heavy chain variable domain described in Sequence ID No. 122; vi) The light chain variable domain described in Sequence ID No. 141 and the heavy chain variable domain described in Sequence ID No. 142; vii) The light chain variable domain described in Sequence ID No. 161 and the heavy chain variable domain described in Sequence ID No. 162; viii) The light chain variable domain described in Sequence ID No. 181 and the heavy chain variable domain described in Sequence ID No. 182; ix) The light chain variable domain described in Sequence ID No. 201 and the heavy chain variable domain described in Sequence ID No. 202; x) The light chain variable domain described in Sequence ID No. 221 and the heavy chain variable domain described in Sequence ID No. 222; xi) The light chain variable domain described in Sequence ID No. 241 and the heavy chain variable domain described in Sequence ID No. 242; xii) The light chain variable domain described in Sequence ID No. 261 and the heavy chain variable domain described in Sequence ID No. 262; xiii) The light chain variable domain described in Sequence ID No. 281 and the heavy chain variable domain described in Sequence ID No. 282; xiv) The light chain variable domain described in Sequence ID No. 301 and the heavy chain variable domain described in Sequence ID No. 302; or xv) Light chain variable domain described in Sequence ID No. 2185 and heavy chain variable domain described in Sequence ID No. 2186 Includes.

[0094] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) The light chain variable domain described in Sequence ID No. 41 and the heavy chain variable domain described in Sequence ID No. 42; ii) The light chain variable domain described in Sequence ID No. 61 and the heavy chain variable domain described in Sequence ID No. 62; iii) The light chain variable domain described in Sequence ID No. 81 and the heavy chain variable domain described in Sequence ID No. 82; iv) The light chain variable domain described in Sequence ID No. 121 and the heavy chain variable domain described in Sequence ID No. 122; v) The light chain variable domain described in Sequence ID No. 161 and the heavy chain variable domain described in Sequence ID No. 162; vi) The light chain variable domain described in Sequence ID No. 181 and the heavy chain variable domain described in Sequence ID No. 182; or vii) Light chain variable domain described in Sequence ID No. 301 and heavy chain variable domain described in Sequence ID No. 302 Includes.

[0095] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein includes the light chain variable domain described in SEQ ID NO: 61 and the heavy chain variable domain described in SEQ ID NO: 62; or the light chain variable domain described in SEQ ID NO: 121 and the heavy chain variable domain described in SEQ ID NO: 122.

[0096] In various embodiments, the antigen-binding protein is sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 9 18, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 9 86, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042 It has a light chain amino acid sequence described in any one of the following: 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, and 1086.

[0097] In various embodiments, the TREM-1 antibody or antigen-binding protein is sequence numbers 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2 It has a light chain amino acid sequence described in any one of the following: 573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, or 2605.

[0098] In various embodiments, the TREM-1 antibody or antigen-binding protein is SEQ ID NOs: 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 22 It has a light chain amino acid sequence described in any one of 93, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, and 2357.

[0099] In various embodiments, the antigen-binding protein is SEQ ID NOs: 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 9 17, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 9 85, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, It has a heavy chain amino acid sequence described in any one of the following: 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, and 1085.

[0100] In various embodiments, the TREM-1 antibody or antigen-binding protein is SEQ ID NOs: 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 19 It has a heavy chain amino acid sequence described in any one of the following: 43, 1945, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005, and 2007.

[0101] In various embodiments, the TREM-1 antibody or antigen-binding protein is SEQ ID NOs. 2011, 2013, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 20 It has a heavy chain amino acid sequence described in any one of the following: 71, 2073, 2075, 2077, 2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131, 2133, and 2135.

[0102] In various embodiments, the antigen-binding protein has a heavy chain amino acid sequence described in any one of sequence numbers 2726 to 2776.

[0103] In various embodiments, the antigen-binding protein is SEQ ID NOs: 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 97 3, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1 029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1 The heavy chain amino acid sequences described in any one of 079, 1081, 1083, and 1085, as well as the sequences 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 96 4, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070,It has the corresponding light chain amino acid sequences described in 1072, 1074, 1076, 1078, 1080, 1082, 1084, and 1086.

[0104] In various embodiments, the TREM-1 antibody or antigen-binding protein is SEQ ID NOs: 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 19 One of the following: 39, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005, and 2007 The heavy chain amino acid sequences described in and SEQ ID NOs. 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 257 It has the corresponding light chain amino acid sequence described in 4, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604 and 2605.

[0105] In various embodiments, the TREM-1 antibody or antigen-binding protein is SEQ ID NOs. 2011, 2013, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 20 One of the following: 67, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131, 2133, and 2135 The heavy chain amino acid sequences described in and SEQ ID NOs. 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293, 229 It has the corresponding light chain amino acid sequence described in 5, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355 and 2357.

[0106] In various embodiments, the antigen-binding protein may have a heavy-chain and light-chain pair as described in Table 10, Table 11, Table 13A, or Table 13B, or a heavy-chain antigen-binding protein as described in Table 16, Table 17, or Table 22 may be used.

[0107] The nucleic acid sequences of the heavy and light chains of specific antigen-binding proteins are described in SEQ ID NOs. 303-526 (bivalent) and SEQ ID NOs. 527-862 (monovalent). The nucleic acid sequences of the variable region of the heavy chain of the TREM-1 mutant antibody are described in SEQ ID NOs. 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002, 2004, 2006 And described in 2008, with sequence numbers 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 207 Also described in 2, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134 and 2136;The nucleotide sequences of the light chain variable region of the TREM-1 mutant antibody are as follows: SEQ ID NOs. 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 263 3, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 26 Listed in 67, 2668, sequence numbers 2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290, 2292, 22 This is described in sections 94, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, and 2358.

[0108] In various embodiments of antigen-binding proteins, one or more heavy chain framework amino acids of the antigen-binding protein are substituted with corresponding 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 substituted with corresponding amino acids from another human antibody amino acid sequence.

[0109] In various embodiments, the anti-TREM-1 antigen-binding protein further comprises a human light chain constant region bound to the light chain variable region.

[0110] In various embodiments of the antigen-binding protein, the antigen-binding protein comprises two light chains and two heavy chains, each heavy chain comprising an IL-10 moiety attached to the C-terminus of the heavy chain; each heavy chain IL-10 moiety antigen-binding protein is represented by SEQ ID NOs: 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 88 9, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967 ,969,971,973,975,977,979,981,983,985,987,989,991,993,995,997,999,1001,1003,1005,1007,1009,1011,1013,1015,1017,1019,1021,1023,1025,1027,1029,1031,1033,1035,1 It comprises an amino acid sequence that is at least 90% identical to a sequence selected from 037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083 and 1085;And each light chain is sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924 ,926,928,930,932,934,936,938,940,942,944,946,948,950,952,954,956,958,960,962,964,966,968,970,972,974,976,978,980,982,984,986,988,990,9 92, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, and 1086.

[0111] In various embodiments, variants of the TREM-1 antibody heavy chain and / or light chain variable region are provided herein. TREM-1 antibody heavy chain variable region variant sequences include SEQ ID NOs: 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 194 1, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005, 2007, 2011, 20 13, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 2 These are listed as 083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131, 2133, 2135, 2726-2776 and Tables 10, 11, 13A, 13B, and 22.

[0112] The TREM-1 antibody light chain variable region mutant sequences are SEQ ID NOs: 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 25 71, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293, 2295, 22 See Tables 10 and 11 for 97, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357. The TREM-1-IL-10 antigen-binding protein is thought to contain the TREM-1 antibody variant heavy chain and / or light chain sequences disclosed herein.

[0113] In various embodiments, the antigen-binding protein may be monovalent. In various embodiments, the heavy chain IL-10 partial antigen-binding protein may be SEQ ID NOs: 1087, 1090, 1093, 1096, 1105, 1108, 1111, 1114, 1117, 1123, 1126, 1129, 1132, 1138, 1141, 1147, 1150, 1153, 1156, 1159, 116 2, 1165, 1168, 1171, 1174, 1177, 1180, 1183, 1186, 1189, 1192, 1195, 1198, 1201, 1204, 1207, 1210, 1213, 1216, 1219, 1222, 1225, 1228, 1231, 1237, 1240, 1243, 1246, 125 2, 1255, 1258, 1261, 1264, 1267, 1270, 1273, 1276, 1279, 1285, 1288, 1294, 1297, 1300, 1303, 1309, 1312, 1315, 1318, 1321, 1324, 1333, 1336, 1342, 1345, 1348, 1351, 135 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 4, 1357, 1360, 1363, 1366, 1369, 1372, 1375, 1378, 1381, 1384, 1387, 1390, 1393, 1396, 1399, 1402, 1408, 1411, 1414, 1417, and 1420.In various embodiments, the light chain is sequence numbers 1088, 1091, 1094, 1097, 1106, 1109, 1112, 1115, 1118, 1124, 1127, 1130, 1133, 1139, 1142, 1148, 1151, 1154, 1157, 1160, 1163, 1166, 1169, 1172, 1175, 1178, 1181, 1184, 1187, 1190, 1193, 1196, 1199, 1202, 1205, 1208, 1211, 1214, 1217, 1220, 1223, 1226, 1229, 1232, 1238, 1241, 1244, 1247, 1253, 1256, 1 259, 1262, 1265, 1268, 1271, 1274, 1277, 1280, 1286, 1289, 1295, 1298, 1301, 1304, 1310, 1313, 1316, 1319, 1322, 1325, 1334, 1337, 1343, 1346, 1349, 1352, 1355, 13 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 58, 1361, 1364, 1367, 1370, 1373, 1376, 1379, 1382, 1385, 1388, 1391, 1394, 1397, 1400, 1403, 1409, 1412, 1415, 1418, and 1421.In various embodiments, the monovalent antigen-binding protein is, for example, SEQ ID NOs: 1089, 1092, 1095, 1098, 1107, 1110, 1113, 1116, 1119, 1125, 1128, 1131, 1134, 1140, 1143, 1149, 1152, 1155, 1158, 1161, 11 64, 1167, 1170, 1173, 1176, 1179, 1182, 1185, 1188, 1191, 1194, 1197, 1200, 1203, 1206, 1209, 1212, 1215, 1218, 1221, 1224, 1227, 1230, 1233, 1239, 1242, 1245, 12 48, 1254, 1257, 1260, 1263, 1266, 1269, 1272, 1275, 1278, 1281, 1287, 1290, 1296, 1299, 1302, 1305, 1311, 1314, 1317, 1320, 1323, 1326, 1335, 1338, 1344, 1347, 13 The Fc regions described in 50, 1353, 1356, 1359, 1362, 1365, 1368, 1371, 1374, 1377, 1380, 1383, 1386, 1389, 1392, 1395, 1398, 1401, 1404, 1410, 1413, 1416, 1419, and 1422 are further included. The monovalent antigen-binding protein and the Fc regions may be assembled or matched as described in Table 13B.

[0114] In various embodiments of the antigen-binding protein, the antigen-binding protein comprises one or two light chains and one or two heavy chains, each heavy chain comprising an IL-10 moiety attached to the C-terminus of the heavy chain; each heavy chain IL-10 moiety antigen-binding protein is sequence numbers 2137, 2139, 2141, 2143, 2145, 2147, 2149, 2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2359, 2361, 2363, 2365, 2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2143, 2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485, 2487, 2489, 2491, 2493, 2495, The sequence includes sequences selected from 2497, 2498, 2499, 2501, 2503, 2505, 2507, 2509, 2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533, 2535, 2537, 2539 and 2726-2776, as well as amino acid sequences that are at least 90% identical to sequences listed in Table 16, Table 17, Table 21, or Table 22.

[0115] In various embodiments, the antigen-binding protein is sequence numbers 2137, 2139, 2141, 2143, 2145, 2147, 2149, 2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2359, 2361, 23 63, 2365, 2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2143, 2415, 2417, 2419, 2421, 2423, 24 25, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485, 24 It contains an amino acid sequence selected from 87, 2489, 2491, 2493, 2495, 2497, 2498, 2499, 2501, 2503, 2505, 2507, 2509, 2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533, 2535, 2537, and 2539. In various embodiments, the heavy chain portion of the antigen-binding protein is paired with a corresponding light chain clone, such as antibody 61B12 (sequence number 105, but lacking a signal sequence, or sequence number 2552, TREM1_61B12.001_huIgGz SEFL2 light chain, or sequences numbers 1042, 1044, 1046, 1048, 1052, 1054), or sequences 2233-2358, as described in Table 10 or Table 11.

[0116] In various embodiments, the antigen-binding protein has a heavy chain amino acid sequence that is at least 90% identical to the sequence described in any one of SEQ ID NOs. 2726-2776. In various embodiments, the antigen-binding protein has a heavy chain amino acid sequence described in any one of SEQ ID NOs. 2726-2776. In various embodiments, the antigen-binding protein has a heavy chain amino acid sequence described in any one of SEQ ID NOs. 2727-2732. In various embodiments, the heavy chain pairs with the corresponding light chain clone, for example, SEQ ID NOs. 976 or SEQ ID NOs. 2554 (without a signal sequence) for clone 63F8.001 and SEQ ID NOs. 992 or SEQ ID NOs. 2555 (without a signal sequence) for 64D7.001. In various embodiments, the antigen-binding protein includes the heavy chain amino acid sequence of SEQ ID NOs. 2727 or 2728 and the light chain amino acid sequence described in SEQ ID NOs. 976 or SEQ ID NOs. 2554 or other light chain sequences for clone 63F8 or 63F8.001 as described herein. In various embodiments, the antigen-binding protein comprises the heavy chain amino acid sequence of SEQ ID NO: 2729, 2730, 2731, or 2732 and the light chain amino acid sequence described in SEQ ID NO: 992 or SEQ ID NO: 2555, or other light chain sequences of 64D7 or 64D7.001 as described herein.

[0117] In various embodiments of antigen-binding proteins, the antigen-binding moiety inhibits the binding of the TREM-1 ligand to TREM-1.

[0118] In various embodiments, the antigen-binding protein comprises an antigen-binding moiety and one or two IL-10 mutant protein moieties, where, a. The antigen-binding portion is an antibody or antibody fragment. b. Each IL-10 moiety is independently monovalent or divalent. c. Each IL-10 moiety was independently selected from one or more human IL-10 mutant proteins having a sequence that is 90% identical to SEQ ID NO: 2. d. At least one IL-10 moiety is covalently bound to the antigen-binding moiety, e. The antigen-binding moiety competes with the anti-TREM-1 antigen-binding moiety described herein for binding to the human TREM-1 protein.

[0119] In various embodiments of antigen-binding proteins, the antigen-binding portion is a human antibody.

[0120] Isolated nucleic acid molecules comprising a nucleotide sequence encoding the heavy chain region of an antigen-binding protein described herein, isolated nucleic acid molecules comprising a nucleotide sequence encoding the light chain region of an antigen-binding protein described herein, and isolated nucleic acid molecules comprising a nucleotide sequence encoding the heavy chain region of an antigen-binding protein, and further comprising a nucleotide sequence encoding the light chain region of an antigen-binding protein described herein.

[0121] This disclosure provides one or more expression vectors comprising molecules encoding antigen-binding proteins described herein, which are operably linked to nucleic acid molecules or expression regulatory sequences.

[0122] Recombinant host cells comprising nucleic acid molecules containing the antigen-binding proteins described herein or vectors containing the nucleic acids are also envisioned. In various embodiments, the host cells are mammalian cells. In various embodiments, the host cells are CHO cells. This disclosure provides a method for producing antigen-binding proteins using the host cells described herein, comprising culturing the host cells and recovering the antibodies, and provides the antigen-binding proteins produced by this method.

[0123] This disclosure envisions a pharmaceutical composition comprising an antigen-binding protein and a pharmaceutically acceptable carrier as described herein. A pharmaceutical composition comprising the IL-10 mutant protein of this disclosure is also provided. A pharmaceutical composition comprising the anti-TREM-1 antibody or antibody fragment of this disclosure is further provided. The pharmaceutical composition is assumed to be a sterile pharmaceutical composition.

[0124] In one embodiment, the present disclosure provides a method for treating an inflammatory disease in a person in need thereof, comprising administering an IL-10 mutant protein or a composition comprising an IL-10 mutant protein as described herein.

[0125] In a related embodiment, the Disclosure provides a method for treating an inflammatory disease in a subject in need thereof, 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. In various embodiments, the method comprises administering an anti-IL-10 mutant protein as described herein in combination with an anti-TREM-1 antigen-binding protein. In various embodiments, the anti-TREM-1 antigen-binding protein and the IL-10 mutant protein are administered in the same composition or in different compositions.

[0126] A method for treating an inflammatory disease in a person in need thereof is also provided, comprising administering an antigen-binding protein described herein or a composition comprising an antigen-binding protein described herein. In various embodiments, a method for treating an inflammatory disease in a person in need thereof is provided, comprising administering an anti-TREM-1 / IL-10 mutant protein antigen-binding protein described herein or a composition comprising said antigen-binding protein.

[0127] In various embodiments, the inflammatory disease is selected from the group consisting of inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis, or cytokine release syndrome (CRS). In one embodiment, the inflammatory disease is inflammatory bowel disease. In one embodiment, the inflammatory disease is ulcerative colitis. In one embodiment, the inflammatory disease is Crohn's disease. In one embodiment, the inflammatory disease is irritable bowel syndrome. In one embodiment, the inflammatory disease is rheumatoid arthritis. In one embodiment, the inflammatory disease is psoriasis. In one embodiment, the inflammatory disease is psoriatic arthritis. In one embodiment, the inflammatory disease is cytokine release syndrome.

[0128] In various embodiments, treatment with IL-10 mutant protein reduces the suppression of TNF-α production in bone marrow cells, reduces the level of CD8+ T cell stimulation, and / or B cell stimulation, compared to wt IL-10. In various embodiments, treatment with IL-10 mutant protein antigen-binding proteins, such as anti-TREM-1 / IL-10 mutant protein antigen-binding proteins, suppresses TNF-α production in bone marrow cells. In various embodiments, treatment with IL-10 mutant protein antigen-binding proteins, such as anti-TREM-1 / IL-10 mutant protein antigen-binding proteins, suppresses TNF-α production in bone marrow cells while reducing CD8+ T cell and B cell activation. In various embodiments, the treatment inhibits TNF-α production in bone marrow cells without CD8+ T cell stimulation and / or B cell stimulation in the subject. In various embodiments, the treatment reduces the level of TNF-α in the subject.

[0129] In various embodiments, the treatment is administered intravenously or subcutaneously. In various embodiments, the treatment is administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every six months.

[0130] In various embodiments, the method includes administering one or two further therapeutic agents. In various embodiments, the further therapeutic agents are selected from corticosteroids, NSAIDs, analgesics, immunosuppressants, anti-inflammatory agents, TNFα inhibitors, IL-12 / IL-23 inhibitors, IL-17, and IFN-γ.

[0131] This disclosure also provides compositions comprising the IL-10 mutant protein described herein for use in treating inflammatory diseases. In certain embodiments, this disclosure provides the use of compositions comprising the IL-10 mutant protein described herein in the preparation of agents for treating inflammatory diseases.

[0132] Further considerations include compositions comprising the anti-TREM-1 antibody or antigen-binding fragment described herein for use in treating inflammatory diseases. In certain embodiments, this disclosure provides the use of compositions comprising the anti-TREM-1 antibody or antigen-binding fragment described herein in the preparation of agents for treating inflammatory diseases.

[0133] Compositions comprising the anti-TREM-1 antibody or antigen-binding fragment described herein, combined with the anti-IL-10 mutant protein described herein, for use in treating inflammatory diseases are also envisioned. In various embodiments, this disclosure provides the use of compositions comprising the anti-TREM-1 antibody or antigen-binding fragment described herein, combined with the anti-IL-10 mutant protein described herein, in the preparation of agents for treating inflammatory diseases.

[0134] Compositions comprising the antigen-binding proteins described herein, such as anti-TREM-1 / IL-10 mutant protein antigen-binding proteins, for use in treating inflammatory diseases are also envisioned. Further use of compositions comprising the antigen-binding proteins described herein, such as anti-TREM-1 / IL-10 mutant protein antigen-binding proteins, in the preparation of agents for treating inflammatory diseases is also envisioned.

[0135] In various embodiments, the inflammatory disease is inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis or cytokine release syndrome (CRS). In one embodiment, the inflammatory disease is ulcerative colitis. In one embodiment, the inflammatory disease is Crohn's disease. In one embodiment, the inflammatory disease is irritable bowel syndrome. In one embodiment, the inflammatory disease is rheumatoid arthritis. In one embodiment, the inflammatory disease is psoriasis. In one embodiment, the inflammatory disease is psoriatic arthritis. In one embodiment, the inflammatory disease is cytokine release syndrome. [[ID="]]

[0136] Each feature or embodiment or combination described herein is a non-limiting, exemplary example of any aspect of the invention, and thus is intended to be combinable with any other feature or embodiment or combination described herein. For example, when a feature is described using terms such as "one embodiment", "an embodiment", "a particular embodiment", "a further embodiment", "a particular exemplary embodiment" and / or "another embodiment", each of these types of embodiments is a non-limiting example of a feature intended to be combinable with any other feature or combination of features described herein without having to list every possible combination. Such features or combinations of features are applicable to any aspect of the invention. When examples of values within a range are disclosed, any of these examples are considered as possible endpoints of the range, and any numerical value between such endpoints is envisioned, and any combination of upper and lower endpoints is envisioned.

[0137] The headings herein are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments and variations of the invention will become apparent from the detailed description and / or the drawings and / or the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] [Figure 1A]Schematic diagrams illustrating bivalent and monovalent anti-TREM1 mAb / IL-10 mutant protein antigen-binding proteins are shown. [Figure 1B] Schematic diagrams illustrating bivalent and monovalent anti-TREM1 mAb / IL-10 mutant protein antigen-binding proteins are shown. [Figure 1C] Schematic diagrams illustrating bivalent and monovalent anti-TREM1 mAb / IL-10 mutant protein antigen-binding proteins are shown. [Figure 1D] This shows the binding site of the IL-10 mutant protein to the antigen-binding protein. [Figure 2] This shows that both bivalent and monovalent TREM-1 binding significantly promoted the suppression of IL-10 mutant proteins in monocyte activation. [Figure 3A] This shows that the anti-TREM-1 mAb / IL-10 mutant protein antigen-binding protein did not stimulate the activation of CD8+ T cells (Figure 3A) and B cells (Figure 3B). [Figure 3B] This shows that the anti-TREM-1 mAb / IL-10 mutant protein antigen-binding protein did not stimulate the activation of CD8+ T cells (Figure 3A) and B cells (Figure 3B). [Figure 4A] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 4B] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 4C] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 4D] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 4E] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 4F] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 4G] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 4H] This study demonstrates that TREM-1 binding significantly enhances the suppression of IL-10 mutant proteins in monocyte activation for various fusion protein constructs. [Figure 5A] This shows that the efficacy of IL-10 mutant proteins in human (Figure 5A) or mouse (Figure 5B) monocytes, as demonstrated by TNF levels, is improved by TREM-1 binding of the fusion protein compared to the antibody or IL-10 mutant protein alone. [Figure 5B] This shows that the efficacy of IL-10 mutant proteins in human (Figure 5A) or mouse (Figure 5B) monocytes, as demonstrated by TNF levels, is improved by TREM-1 binding of the fusion protein compared to the antibody or IL-10 mutant protein alone. [Figure 6A] This shows the inhibition of ligand (PGLYRP1 / PGN)-mediated TREM-1 signaling in human (Figure 6C-6D) or cynomolgus monkey (Figure 6A-6B) PBMCs by anti-human TREM-1 antibodies, as measured by TNF-α release. [Figure 6B] This shows the inhibition of ligand (PGLYRP1 / PGN)-mediated TREM-1 signaling in human (Figure 6C-6D) or cynomolgus monkey (Figure 6A-6B) PBMCs by anti-human TREM-1 antibodies, as measured by TNF-α release. [Figure 6C] This shows the inhibition of ligand (PGLYRP1 / PGN)-mediated TREM-1 signaling in human (Figure 6C-6D) or cynomolgus monkey (Figure 6A-6B) PBMCs by anti-human TREM-1 antibodies, as measured by TNF-α release. [Figure 6D]This shows the inhibition of ligand (PGLYRP1 / PGN)-mediated TREM-1 signaling in human (Figure 6C-6D) or cynomolgus monkey (Figure 6A-6B) PBMCs by anti-human TREM-1 antibodies, as measured by TNF-α release. [Figure 7] This study demonstrates the inhibition of ligand (PGLYRP1 / PGN)-mediated signaling by anti-human TREM-1 Fab in cell lines overexpressing human TREM-1 / DAP12, as measured by phosphorylation of spleen tyrosine kinase (pSYK). [Modes for carrying out the invention]

[0139] This disclosure provides IL-10 mutant proteins and antigen-binding proteins containing IL-10 mutant proteins. The antigen-binding proteins / fusion proteins according to this disclosure exhibit improved targeting of immune cells and modulation of cellular activity and cytokine responses, which are useful for treating inflammatory diseases such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, or rheumatoid arthritis. Therefore, targeting IL-10 inhibitory activity in bone marrow cells may provide an effective treatment for inflammatory bowel disease (IBD) without causing side effects. The IL-10 mutant protein antigen-binding proteins envisioned herein include an antigen-binding moiety capable of targeting the IL-10 mutant protein to inflammatory cells. Preferred antigen-binding moieties include anti-TREM-1 antibodies and anti-PD-1 antibodies.

[0140] definition The terms “polypeptide conjugate” or “antigen-binding protein” refer to polypeptides that are capable of specifically binding to an antigen, such as a target or its signaling partner, or that are capable of binding to an antigen with a measurable binding affinity. Examples of polypeptide conjugates include antibodies, peptide bodies, polypeptides, and peptides that are optionally conjugated to other peptide or non-peptide portions. Antigens to which polypeptide conjugates can bind include any protein or non-protein molecule that is capable of eliciting an antibody response or that is capable of binding to the polypeptide conjugate with a detectable binding affinity higher than nonspecific binding. Antigens to which modulated polypeptide conjugates can bind may include a target, a signaling partner of the target, and / or a complex containing the target and its signaling partner. Antigen-binding proteins as envisioned herein may further include heterologous portions as part of another polypeptide, such as as part of a fusion protein. In this case, the antigen-binding protein may include IL-10 mutant proteins such as antibodies or their antigen-binding fragments, fused to, linked to, or continuous with the antigen-binding portion or its fragment, and the antigen-binding portion.

[0141] 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 capable of binding to an antigen (e.g., Fab’, F(ab)2, Fv, single-chain antibodies, diabodies), and recombinant peptides including those above as long as they exhibit the desired biological activity. An "immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein consisting of two heavy chains and two light chains, each containing a variable region and a constant region. The antigen-binding portion can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, in particular, Fab, Fab’, F(ab’)2, Fv, domain antibodies (dAb), complementarity-determining region (CDR) fragments, CDR grafted antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies; chelated recombinant antibodies, tribodies or biobodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIP), antigen-binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies or variants or derivatives thereof and polypeptides containing at least a portion of an immunoglobulin sufficient to confer a specific antigen binding to a polypeptide, such as 1, 2, 3, 4, 5 or 6 CDR sequences as long as the antibody retains the desired biological activity.

[0142] As used herein, the term "monovalent IgG" refers to an IgG in which a single antigen-binding fragment (Fab) is fused to a complete constant domain fragment (Fc) engineered to heterodimerize by mutations in the C H 3 domain. Monovalent IgG is also known as a "one-armed" antibody.

[0143] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0144] As used herein, "antibody variant" refers to an antibody polypeptide sequence comprising at least one amino acid substitution, deletion, or insertion in the variable region of the variable region domain of a native antibody. The variant may be substantially homologous or substantially identical to the unmodified antibody.

[0145] "Isolated" antibodies are those identified, separated, and removed from components of their natural environment. These contaminant components of the natural environment are materials that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. In preferred embodiments, the antibody is purified (1) to more than 95% by weight, most preferably more than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) to homogenize by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining or an HPLC method. Since isolated antibodies lack at least one component of the antibody's natural environment, they include antibodies in situ within recombinant cells. However, typically, isolated antibodies are prepared by at least one purification step.

[0146] As used herein, the term "heavy chain variable region" refers to a region of an antibody molecule that includes at least one complementarity-determining region (CDR) of the antibody heavy chain variable domain. The heavy chain variable region may contain one, two, or three CDRs of the antibody heavy chain.

[0147] As used herein, the term "light chain variable region" refers to a region of an antibody molecule that includes 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 either a κ or λ light chain depending on the antibody.

[0148] As used herein, "specifically binding" means "antigen-specific," "specific to" an antigen target, or "immunoreactive" to an antigen, and refers to the antibody or polypeptide conjugate of the present invention that binds to an antigen with a higher affinity than other antigens of similar sequences. In one embodiment, a polypeptide conjugate of the present invention that binds to a human antigen with a higher affinity than its binding affinity to similar antigens of other, i.e., non-human species, but recognizes and binds to the target ortholog, is within the scope of the present invention.

[0149] The term "epitope" refers to any portion of a molecule that can be recognized and bound by a selective binder at one or more antigen-binding regions. Epitopes typically consist of a group of chemically active surfaces of molecules, such as amino acids or carbohydrate side chains, and possess specific three-dimensional structural and charge properties. As used herein, epitopes may be continuous or discontinuous.

[0150] The term "derivative" as used in relation to the polypeptide binder and polypeptide of the present invention refers to a polypeptide that has been chemically modified by techniques such as ubiquitination, conjugation to therapeutic or diagnostic agents, labeling (e.g., using radionuclides or various enzymes), PEGylation (derivativeation using polyethylene glycol), and chemically synthesized insertion or substitution of amino acids such as ornithine, which do not normally occur in human proteins. The derivative retains the binding properties of the non-derivativeated molecule of the present invention.

[0151] As used herein, “linker” refers to a peptide that links two polypeptides. The linker may be 1 to 80 amino acid long. In one embodiment, the linker may be 2 to 40, 3 to 40, 3 to 30, or 3 to 20 amino acid long. In one embodiment, the linker may be a peptide with 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acid long. In another embodiment, the linker may be 3 to 25, 3 to 18, 5 to 20, 6 to 18, or 10 to 20 amino acid long. In yet another embodiment, 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 acid long. In many cases, the linker lacks a free cysteine ​​residue (i.e., is not involved in disulfide bonding) and 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, peptides having the sequence G3SG2 (SEQ ID NO: 2676) or G4S (SEQ ID NO: 2725) are linkers between the anti-TREM-1 antigen-binding protein and the IL-10 mutant protein. There are many other suitable linker examples, but in particular, G2, G3, G3S (SEQ ID NO: 2705), G3P (SEQ ID NO: 2706), G3Q (SEQ ID NO: 2707), and G5 (SEQ ID NO: 2705). In the above linkers, each initial letter refers to the conventional single-letter code of the amino acid, and each number refers to the number of tandem repeats of the amino acid in the linker. For example, "G3SG2 (SEQ ID NO: 2676)" refers to a linker with the sequence Gly-Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 2676). "G4S" refers to a linker with the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2725).

[0152] The "therapeutic effective dose" of a drug used to treat a disease is the 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 serious symptoms or a more serious disease that may occur with a certain frequency after treatment of the condition.

[0153] "Treatment" of any disease referred to herein includes alleviation of at least one symptom of the disease, reduction of the severity of the disease, or delay or prevention of the progression of the disease to more serious symptoms that may accompany the disease or may cause at least one other disease. Treatment does not necessarily mean that the disease is completely cured. An effective therapeutic agent only needs to reduce the severity of the disease, reduce the severity of one or more symptoms associated with the disease or its treatment, or delay the onset of more serious symptoms or more serious diseases that may occur with a certain frequency after treatment of the condition. For example, if the disease is inflammatory bowel disease, a therapeutic agent used as treatment may reduce the number of individual sites of inflammation in the intestine or the entire extent of the affected intestine. It may reduce pain and / or swelling, alleviate symptoms such as diarrhea, constipation or vomiting, and / or prevent intestinal perforation.

[0154] The term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian species: humans, non-human primates such as chimpanzees and other apes and monkey species; domestic animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals such as rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. This term does not indicate a specific age or sex.

[0155] As used herein, "bone marrow cells" refers to a subset of immune cells derived from hematopoietic progenitor cells of the myeloid lineage, including granulocytes, monocytes, macrophages, and dendritic cells (DCs). Bone marrow cells often possess phagocytic and antigen-presenting cell (APC) functions and play important roles in protective immunity. See, for example, de Kleer et al., Front.Immunol., 5:423, 2014.

[0156] Interleukin-10 Interleukin-10 (IL-10) is an inflammatory cytokine involved in various immunomodulatory processes. IL-10 signals through a receptor complex consisting of two IL-10 receptor-1 (IL-10R1) and two IL-10 receptor-2 (IL-10R2) proteins. The biologically active form of IL-10 is a homodimer that binds to IL-10R1 / IL-10R2 and signals through the Jak1 / Tyk2 and stat3 pathways. IL-10 has a higher affinity for IL-10R1 compared to IL-10R2. IL-10 is expressed as a 178-amino acid protein containing an 18-amino acid signal peptide. The IL-10 homodimer secondary structure exhibits a helix-turn-helix domain swap, containing six α-helices, A, B, C, D, E, and F helices within each domain. The nucleotide and amino acid sequences of human IL-10 are described in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Sequence ID 2 refers to mature human IL-10 lacking a signal sequence.

[0157] Human IL-10 (hIL-10) and Epstein-Barr virus IL-10 (vIL-10) share approximately 83% sequence identity and similar secondary structures. vIL-10 suppresses inflammatory cytokine production, inhibits MHC II expression in macrophages, and prevents T cell proliferation. vIL-10 does not co-stimulate thymocyte and mast cell proliferation, does not induce B cell MHC class II expression, and has a lower affinity for cell surface IL-10R1. Ala87 (Ile in human) is a key residue involved in the reduced immunostimulatory activity of vIL-10.

[0158] Human IL-10 and IL-10 in cynomolgus monkeys (cynomolgus monkey IL-10) share approximately 96% sequence identity, while human and cynomolgus monkey IL-10R1 share approximately 93% sequence identity. There is a 4-amino acid difference at the binding interface between human and cynomolgus monkey IL-10R1. I45T and E46G are on the loop that interacts with the AB loop of IL-10, but the interaction is mainly on the main chain. A189T and S192T are on the loop that interacts with helix A but does not have a direct interaction with IL-10. In IL-10, there is a 2-amino acid difference near the IL-10R1 binding interface for human and cynomolgus monkey IL-10, with N21 and L46 in human both being adjacent to the IL-10 / IL-10R1 interaction surface.

[0159] Previous studies have shown that IL-10 inhibition of myeloid cells is sufficient for the treatment of inflammatory bowel disease (IBD). However, the clinical efficacy of IL-10 has been hampered by dose-limiting toxicity. To circumvent this problem, mutant IL-10 proteins are provided herein that have reduced activity to minimize systemic adverse side effects while still retaining immunosuppressive properties for the treatment of IBD. Treatment with IL-10 mutant proteins with reduced activity may mitigate some of the immunostimulatory effects of cytokines. To address the possible reduction in the desirable immunosuppressive effect, the IL-10 mutant protein is fused to a target moiety (e.g., an antibody) to increase its local concentration in monocytes, regulatory T cells, and CD4+ T cells.

[0160] Mutant IL-10 proteins were targeted in different sections of the IL-10 protein, including site Ia, which interacts with IL-10R1 in helix A, helix F, and AB loop, and site Ib, which interacts with IL-10R1 in helix A, helix F, AB loop, helix B, helix C, CD loop, and helix E. Mutant proteins include changes in one or more of the following residues: site Ia (helix A), K34, T35, Q38; site Ia (helix F), I136, Y137, K138, S141, E142, D144, I145; site Ib (helix A), H14, F15, M22, P20, L23, R24, R27, D28; site Ib (F), E151, M154, M156, K157, N160; site Ia (AB loop) , M39, K40, D41, Q42, L43, D44, N45, L46, L47, L48, K49; Helix B, F56, K57, Y59; Helix C, L60, E67, Q70, M77, M79; CD loop, N82, Q83, D84, P85, D86, I87; Helix D, A89, H90, S93, T100, L103, H109, ​​R110, L112, E115, N116; or Helix E, A127, K130.

[0161] The amino acid sequences of specific IL-10 mutant proteins are as follows: SEQ ID NOs. 3-10 and SEQ ID NOs. 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, These are listed in 2490, 2492, 2494, 2496, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540 and 2777-2791, as well as in Tables 1, 16, 17, and 21.

[0162] The IL-10 mutant proteins and antigen-binding proteins described herein may be screened for binding affinity by methods known in the art. For example, gel shift assays, Western blotting, injection labeling competition assays, chromatographic co-fractionation, coprecipitation, crosslinking, ELISA, surface plasmon resonance (SPR), and KinExA may be used, as described, for example, in Current Protocols in Molecular Biology (1999), John Wiley & Sons, NY, which is incorporated herein by reference in whole.

[0163] The IL-10 mutant proteins of this disclosure preferably have reduced potency compared to wt IL-10 activity. In various embodiments, the IL-10 mutant proteins have potency reduced by about 10 to about 5000 times compared to wt IL-10. In various embodiments, the IL-10 mutant proteins have potency reduced by about 10 to 1000 times, about 50 to 500 times, about 100 to 500 times or more. In various embodiments, potency is measured in an LPS-stimulated assay. In one embodiment, the assay read is LPS-induced TNFα production in monocyte / myeloid cells.

[0164] Antigen-binding proteins and antibodies Immunoglobulin variable domains exhibit the same general structure of a relatively conserved framework region (FR) linked by three hypervariable regions or CDRs. From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain follow the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989.

[0165] The hypervariable region of an antibody refers to the CDR amino acid residues of the antibody involved in antigen binding. The hypervariable region consists of amino acid residues from the CDR [e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] and / or the hypervariable loop (e.g., [Chothia et al. The CDR includes residues from 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, as described by al., J. Mol. Biol. 196: 901-917 (1987), and residues from 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain. The CDR is numbered using ImMunoGenTics (IMGT) (Lefranc, M.-P., the Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al.). The CDRs are also identified and numbered according to al., Dev. Comp. Immunol., 27, 55-77 (2003), which describes the CDR positions in the light and heavy chain variable domains as follows: CDR1, approximately residues 27-38; CDR2, approximately residues 56-65; and CDR3, approximately residues 105-116 (germline) or residues 105-117 (rearrangement). In one embodiment, the CDRs are thought to be located approximately residues 26-31 (L1), 49-51 (L2), and 88-98 (L3) in the light chain variable domain and approximately residues 26-33 (H1), 51-58 (H2), and 97-110 (H3) in the heavy chain variable domain of an antibody heavy or light chain of substantially the same length as disclosed herein. However, those skilled in the art will understand that when the sequence of a particular antibody is identified, the actual positions of the CDR residues may differ from the expected residues described above.The CDRs disclosed herein are defined according to the Kabat method (Kabat and Wu, 1991) and numbered according to Amgen reference numbering. Amgen reference numbering is a structure-based numbering system based on the Honegger and Plueckthun numbering system for antibody variable regions described by Honegger and Plueckthun (J Mol Biol. 309(3):657-70, 2001). In various embodiments, a manipulated IgG1 antibody is assumed. In various embodiments, the antibody is the IgG1z or IgG1z-SEFL2 antibody described herein.

[0166] Framework region (or FR) residues are variable domain residues other than hypervariable region residues.

[0167] As will be discussed later, antibodies as envisioned herein, including monoclonal, human, humanized, and other antibodies described herein, are typically produced by recombinant methods or other methods that manipulate the genetic code in vitro or in vivo, and therefore do not necessarily reflect specific antibodies found in nature.

[0168] In various embodiments, antigen-binding proteins specifically bind to cell surface proteins so that IL-10 or IL-10 mutant proteins are directed to specific cell types. Such target cell types include myeloid cells, CD8+ T cells, CD4+ T cells, and B cells. Antigen-binding proteins may bind to TREM-1 for the directing of the IL-10 moiety to myeloid cells; to PD1, CD8, LAG3, NKG2D, NKG7, or other CD8+ T cell surface proteins for CD8+ T cells; to CD4+ T cells to CD4 or other CD4+ T cell surface proteins, such as CD30, OX40, 41BB, ICOS; and to B cells to CD20, CD19, or other B cell surface proteins, such as BAFFR.

[0169] In various embodiments, antigen-binding proteins specifically bind to cell surface proteins that play a role in immune checkpoint regulation. Examples of such immune checkpoint modulators include PD-1, CTLA4, CD28, CD80, and CD86.

[0170] In various embodiments, antigen-binding proteins specifically bind to cell surface proteins that are more highly expressed in more specific tissues or organs (e.g., the intestines or lungs) in other organs or tissues. Examples of such tissue- or organ-specific cell surface proteins include MAdCAM1 in the intestines and surfactants or RAGE in the lungs. Fusion proteins of such antigen-binding proteins with IL-10 mutant proteins are useful for treating inflammatory diseases such as IBD, asthma, and chronic obstructive pulmonary disease (COPD).

[0171] In various embodiments, antigen-binding proteins specifically bind to cell surface proteins that are more highly expressed in tumor / cancerous cells than in non-cancerous cells. Examples of such tumor / cancer-specific antigens include BCMA, CD19, CD20, CD22, CD70, CD123, CEA, CDH3, CLDN6, CLL1, CS1, DCAF4L2, FLT3, GABRP, MageB2, MART-1, MSLN, MUC1 (e.g., MUC1-C), MUC12, MUC13, MUC16, mutFGFR3, PRSS21, PSMA, RNF43, STEAP1, STEAP2, TM4SF5, PD-1, CTLA4, EGFR, VEGF, OX40, or FcRL5.

[0172] Anti-TREM-1 antigen-binding protein Previous studies to characterize TREM-1 signaling have used TREM-1 agonist antibodies to mimic receptor ligand activation (Tessarz et al., Immunol Lett 116(2):111-6, 2008; Vandestienne et al., J Clin Invest 131(2):e142468, 2021). Decoy peptides have also been attempted to modulate TREM-1 activity (see, for example, International Patent Publication No. 2014037565).

[0173] This disclosure encompasses the use of amino acid molecules encoding target-specific antibodies. The anti-TREM-1 antigen-binding proteins described herein modulate the interaction between human TREM-1 and its ligand in different ways.

[0174] In one embodiment, an amino acid sequence and / or sequence numbers 41, 61, 81, 101, 121, 141, 161, 181, 202, 222, 242, 262, 282, 302 and 2186 is identical to 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 of the heavy chain variable region described in SEQ ID NOs. 41, 61, 81, 101, 121, 141, 161, 181, 201, 2 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 light chain variable region described in 21, 241, 261, 281, 301, and 2185, wherein the antibody further comprises at least one, two, three, four, five, or all of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3. In one embodiment, the amino acid sequence having a percentage of identity with the light chain variable region may comprise one, two, or three of the light chain CDRs. In other embodiments, the amino acid sequence having an identity percentage with the heavy chain variable region may include one, two, or three of the heavy chain CDRs.

[0175] In another embodiment, all three HCDRs in the heavy chain variable region or CDR of the antibody sequences described above: the heavy chain CDR1 sequence described in SEQ ID NOs: 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276, 296 and 2196; the heavy chain CDR2 sequence described in SEQ ID NOs: 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277, 297 and 2197; and SEQ ID NOs: 38, 58 The following antigen-binding proteins are provided, comprising polypeptides having amino acid sequences that are at least approximately 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 CDR3 sequences described in 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298, and 2198.

[0176] In related embodiments, all three LCDRs in the light chain variable region or CDR of the antibody sequences described above: light chain CDR1 sequences described in SEQ ID NOs: 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290 and 2190; light chain CDR2 sequences described in SEQ ID NOs: 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271, 291 and 2191; and SEQ ID NOs: 32, 5 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 light chain CDR3 sequence described in 2, 72, 92, 132, 152, 172, 192, 212, 232, 252, 272, 292, and 2192.

[0177] The antibodies of this disclosure may have one or more amino acid substitutions in the CDR region of the antibody, such as non-conservative or conservative substitutions. Consensus sequences of the TREM-1 antibody heavy and light chain CDR and / or variable region sequences disclosed herein are also conceivable. For example, a TREM-1 antibody or antigen-binding protein / fusion protein may include the following sequences: In various embodiments, the TREM-1 antibody is X1ASQSX2X3X4NLA (SEQ ID NO: 2199) (where X1 is R or Q, X2 is V or I, X3 is N or S, and X4 is S, H, I, V, or A); QASX1DIX2X3X4LN (SEQ ID NO: 2204) (where X1 is R or Q, X2 is R, S, N, or F, X3 is K or N, and X4 is H, Y, or D); RASQSV It contains an antigen-binding domain that includes a light chain variable region containing an LCDR1 amino acid sequence selected from the group consisting of NSNLA (SEQ ID NO: 2212); QASQDIRKHLN (SEQ ID NO: 2213); RASQDISSNLN (SEQ ID NO: 2214); QASQDIHLN (SEQ ID NO: 2215); RASQGIRKWLA (SEQ ID NO: 2216) or RASQSVNSNLA (SEQ ID NO: 2217) and SGDKLGERVS (SEQ ID NO: 2218).

[0178] In various embodiments, the TREM-1 antibody includes an antigen-binding domain having a light chain variable region containing an LCDR2 amino acid sequence selected from the group consisting of GAX1X2RAT ​​(SEQ ID NO: 2200) (where X1 is S or Y and X2 is T or I); amino acid sequence X1X2X3X4LET (SEQ ID NO: 2206) (where X1 is D, G or H, X2 is A, V or T, X3 is S, A or Y and X4 is T or N); GASTRAT (SEQ ID NO: 2219); DASNLET (SEQ ID NO: 2220); and AASRLQS (SEQ ID NO: 2221).

[0179] In various embodiments, the TREM-1 antibody is QX1X2X3X4X5X6PX7T (SEQ ID NO: 2201) (where X1 is Q, H, or E; X2 is F or Y; X3 is K, Y, or I; X4 is N, T, L, I, or M; X5 is W, F, H, or Y; X6 is absent or P; X7 is W, N, Y, H, or L); QX1YX3X4X5PX6T (SEQ ID NO: 2207) (where X1 is Q, X2 is D, A, or G; X3 is N or K; X4 is L or I; X5 is I or L); it contains an antigen-binding domain having a light chain variable region containing an LCDR3 amino acid sequence selected from the group consisting of QQFKNWPPT (SEQ ID NO: 2222); QHYDNLPIT (SEQ ID NO: 2223); LQAHGFPWT (SEQ ID NO: 2224); QQYDNLPLT (SEQ ID NO: 2225); and QFWPPWT (SEQ ID NO: 2226).

[0180] In various embodiments, the TREM-1 antibody includes an antigen-binding domain having a heavy chain variable region containing an HCDR1 amino acid sequence selected from the group consisting of X1X2X3MX4 (SEQ ID NO: 2202) (where X1 is A, R, T, or S, X2 is Y or N, X3 is A or W, and X4 is S or N); a sequence X1YDIN (SEQ ID NO: 2208) (where X1 is R or S); GYYX1H (SEQ ID NO: 2723) (where X1 is M or I); AYAMS (SEQ ID NO: 2227); RYDIN (SEQ ID NO: 2228); and SYWMS (SEQ ID NO: 2229).

[0181] In various embodiments, the TREM-1 antibody is X1X2X3X4X5X6X7X8X9YYX 10 X 11 X 12 VKG (Sequence ID 2205) (where X1 is T, E, or S; X2 is nonexistent or M, V, or I; X3 is S, R, or K; X4 is G, or Q; X5 is S, D, or H; X6 is G, S, L, or A; X7 is S, G, or R; X8 is T, S, P, or E; X9 is T, I, and X10 is A or V, and X 11 is D or E, and X 12 is S or A); X1X2NPX3X4GX5X6GX7X8X9X 10 FX 11 X 12 (Sequence ID 2209) (where X1 is W or R, X2 is M or L, X3 is N, Q or K, X4 is S, A or R, X5 is N or Q, X6 is S, A or T, X7 is S, Q or Y, X8 is V or T, X9 is Q or K, X 10 is K or N, and X 11 is R or Q, and X 12 It contains an antigen-binding domain that has a heavy chain variable region containing an HCDR2 amino acid sequence selected from the group consisting of G or D); TSGSGSTTYYADSVKG (SEQ ID NO: 2230); WMNPNSGNSSVQKFRG (SEQ ID NO: 2231); NIKQDGSEEYYVDSVKG (SEQ ID NO: 2232); and TSGSGTYYADSVKG (SEQ ID NO: 2669).

[0182] In various embodiments, the TREM-1 antibody is X1X2X3X4X5X6X7FX8YYX9 (Sequence ID 2203) (where X1 is V, E, A or G, X2 is A, F, Y or G, X3 is G, S, Y or W, X4 is S or R, X5 is absent or N, X6 is F, S, Y or absent, X7 is L or F or absent, X8 is D or E, and X9 is Y, H or S);X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 FX 13 X 14 (Sequence ID 2210) (where X1 is G, L, or R; X2 is G, I, or R; X3 is Y, R, I, G, or A; X4 is T, S, Y, or V; X5 is S or Y; X6 is S, A, I, or R; X7 is W, A, or S; X8 is nonexistent or S; X9 is nonexistent or F, W, or Y; X10 is R, S, H, K, or E, and X 11 is W, H, Y, or F, and X 12 is Y, V, A, or S, and X 13 is D or Q, and X 14 The sequence includes an antigen-binding domain having a heavy chain variable region containing an HCDR3 amino acid sequence selected from the group consisting of L, Y, I, or H; VAGSNFLFDY (SEQ ID NO: 2670); GGITSSWRWYFDL (SEQ ID NO: 2671); GGITSSWSRWYFDL (SEQ ID NO: 2672); and DYGDSFDY (SEQ ID NO: 2673).

[0183] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 90 (LCDR1), 91 (LCDR2), 92 (LCDR3), 96 (HCDR1), 97 (HCDR2), and 98 (HCDR3); v) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); vi) Sequence IDs 130 (LCDR1), 131 (LCDR2), 132 (LCDR3), 136 (HCDR1), 137 (HCDR2), and 138 (HCDR3); vii) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); viii) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); ix) Sequence IDs 190 (LCDR1), 191 (LCDR2), 192 (LCDR3), 196 (HCDR1), 197 (HCDR2), and 198 (HCDR3); x) Sequence IDs 210 (LCDR1), 211 (LCDR2), 212 (LCDR3), 216 (HCDR1), 217 (HCDR2), and 218 (HCDR3); xi) Sequence IDs 230 (LCDR1), 231 (LCDR2), 232 (LCDR3), 236 (HCDR1), 237 (HCDR2), and 238 (HCDR3); xii) Sequence IDs 250(LCDR1), 251(LCDR2), 252(LCDR3), 256(HCDR1), 257(HCDR2), and 258(HCDR3); xiii) Sequence IDs 270 (LCDR1), 271 (LCDR2), 272 (LCDR3), 276 (HCDR1), 277 (HCDR2), and 278 (HCDR3); xiv) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3); and xv) Sequence IDs 2190 (LCDR1), 2191 (LCDR2), 2192 (LCDR3), 2196 (HCDR1), 2197 (HCDR2), and 2198 (HCDR3) Includes a set of CDR sequences selected from.

[0184] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); v) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); vi) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); and vii) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3) Includes a set of CDR sequences selected from.

[0185] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein includes a set of CDR sequences selected from the light chain sequence numbers 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); and the heavy chain sequence numbers 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3).

[0186] In the relevant embodiments, the framework residues are modified. The modifiable heavy chain framework region is located within the region designated H-FR1, H-FR2, H-FR3, and H-FR4 surrounding the heavy chain CDR residue, and the modifiable light chain framework region residues are located within the region designated L-FR1, L-FR2, L-FR3, and L-FR4 surrounding the light chain CDR residue. The amino acids within the framework region may be substituted with any suitable amino acids identified, for example, in the human framework or human consensus framework. Although the framework region may be modified, the antigen-binding proteins or antibodies described herein are further conceivable to retain the CDR, LCDR1-3, and / or HCDR-3 of the parent antibody.

[0187] An antibody fragment includes a intact full-length antibody, preferably a part of the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fcab and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, etc., antibodies (e.g., diabodies, tribodies, tetrabodies); minibodies; chelated recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; binding domain immunoglobulin fusion proteins; camelized antibodies; VHH-containing antibodies; and other polypeptides formed from antibody fragments. See, e.g., Holliger&Hudson, 2005 Nat.Biotech. 23:1126-36; Eyer&Hruska, Veterinarni Medicina 57:439-513, 2012.

[0188] The antigen-binding compounds of the present disclosure preferably have a binding affinity for TREM-1 of 10 -8 、10 -9 、10 -10 、10 -11 、10 -12 、10 -13 、10 -14 、10 -15 M or less when measured by surface plasmon resonance or KinexA. The antigen-binding compounds of the present disclosure have a binding affinity for TREM-1 from 10 -9 ~10 -12 、or 10 -10 ~10 -13 、or 10 -10 ~10 -15 M. The SPR assay is performed using standard methods, for example, at 25 °C (e.g., room temperature).

[0189] In various embodiments, the antibody is an IgG1 antibody. In certain embodiments, the antibody is an IgG1z antibody.

[0190] Examples of full-length antibodies that bind to TREM-1 are provided, having the heavy chain amino acid sequences described in SEQ ID NOs: 26, 46, 66, 86, 106, 126, 146, 166, 186, 206, 226, 246, 266, 286, and 2186; and the corresponding light chain amino acid sequences described in SEQ ID NOs: 25, 45, 65, 85, 105, 125, 145, 165, 185, 205, 225, 245, 265, 285, and 2185.

[0191] Antigen-binding proteins containing IL-10 Antigen-binding proteins between IL-10 mutant proteins and antibodies or other binding proteins that target subpopulations of immune cells are further envisioned herein. Exemplary antibodies or antigen-binding proteins include antibodies or binding proteins that target TREM-1 or checkpoint inhibitors, such as PD-1, PD-L1, PD-L2, CTLA4, antibodies or binding proteins that target TNFα, or antibodies or binding proteins that target IL-12 / IL-23.

[0192] Antibodies against checkpoint inhibitors include, but are not limited to, PD-1 antibodies such as pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp.), nivolumab (Opdivo®, Bristol-Myers Squibb), and U.S. Patent Nos. 8,735,553; 8,617,546; 8,008,449; 8,741,295; 8,552,154; 8,354,509; 8,779,105; 7,563,869; 8,287,856; 8,927,697; 8,088,905; and 7,595,048. Antibodies against PD-1 described in the following publications; specifications No. 8,168,179; specifications No. 6,808,710; specifications No. 7,943,743; specifications No. 8,246,955; and specifications No. 8,217,149 and the International Patent Publication Brochure No. 2019 / 140196; anti-CTLA-4 antibodies, such as ipilimumab (YERVOY®) and tremelimumab, and anti-PD-L1 antibodies, such as durvalumab.

[0193] Exemplary sequences of IL-10 mutant protein / anti-TREM-1 antibody antigen-binding protein are SEQ ID NOs: 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 102 1, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 107 1, 1073, 1075, 1077, 1079, 1081, 1083 and 1085 and sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 90 4, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966 ,968,970,972,974,976,978,980,982,984,986,988,990,992,994,996,998,1000,1002,1004,1006,1008,1010,1012,1014,1016,1018,1020,1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072,Further TREM-1 heavy chain IL10 antigen-binding proteins are described in SEQ ID NOs: 1074, 1076, 1078, 1080, 1082, 1084, and 1086. ,2363,2365,2367,2369,2371,2373,2375,2377,2379,2381,2383,2385,2387,2389,2391,2393,2395,2397,2399,2401,2403,2405,2407,2409,2411,2143,2415,2417,2419,2421,2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485, 2 This is described in 487, 2489, 2491, 2493, 2495, 2497, 2498, 2499, 2501, 2503, 2505, 2507, 2509, 2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533, 2535, 2537, 2539 and 2726-2776.

[0194] Examples of monovalent anti-TREM-1 antibody antigen-binding proteins are shown in amino acid sequences 1087-1422 and Table 13B.

[0195] IL-10 mutant proteins and anti-TREM-1 antibodies described in SEQ ID NOs. 303-862, as well as SEQ ID NOs. 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066 TREM-1 modifications described in 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134 and 2136 Variable regions and domains of heteroantibody heavy chains, and sequencing numbers 2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2 These are nucleic acids encoding antigen-binding proteins that contain the TREM-1 variant antibody light chain variable region, as described in 302, 2304, 2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, and 2358.

[0196] An antigen-binding protein comprising an antigen-binding moiety and one or two IL-10 moieties is provided herein, wherein a. the antigen-binding moiety is an antibody or antibody fragment, b. each IL-10 moiety is independently monovalent or bivalent, c. each IL-10 moiety is independently selected from one or more human IL-10 mutant proteins having a sequence that is 90% identical to SEQ ID NO: 2, and d. at least one IL-10 moiety is covalently bound to the antigen-binding moiety. In various embodiments, an antigen-binding protein is provided herein in which at least one IL-10 moiety is fused to the C-terminus of the antigen-binding moiety.

[0197] In various embodiments of antigen-binding proteins, a. the antigen-binding portion is an antibody, and b. the IL-10 portion is fused to each heavy chain of the antibody.

[0198] In various embodiments of the antigen-binding protein, each IL-10 moiety is a monomer. In various embodiments, the antigen-binding protein contains two different mutant protein monomers. In various embodiments, the antigen-binding protein contains two identical mutant protein monomers.

[0199] In various embodiments, with respect to the antigen-binding protein: each IL-10 moiety comprises an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 2, and each IL-10 moiety independently comprises at least one mutation selected from mutations in helix loop AB, helix loop CD, helix loop DE, helix A, helix B, helix C, helix D, helix E and / or helix F.

[0200] In various embodiments, at least one IL-10 moiety includes at least one mutation in helix A. In various embodiments, at least one IL-10 moiety includes at least one mutation in helix F. In various embodiments, at least one IL-10 moiety includes at least one mutation in helix loop AB.

[0201] In various embodiments, IL-10 mutant proteins reduce the suppression of TNF-α production in myeloid cells, decrease the level of CD8+ T cell stimulation, and / or decrease the level of B cell stimulation compared to wt IL-10.

[0202] In various embodiments, IL-10 mutant protein antigen-binding proteins suppress TNF-α production in bone marrow cells. In various embodiments, IL-10 mutant protein antigen-binding proteins suppress TNF-α production in bone marrow cells while reducing CD8+ T cell and B cell activation.

[0203] In various embodiments, the treatment inhibits TNF-α production in monocytes without CD8+ T cell stimulation and / or B cell stimulation in the subject.

[0204] In various embodiments, each IL-10 mutant protein independently has residues N10, H14, F15, P20, M22, L23, R24, R27, D28, K34, T35, Q38, M39, K40, D41, Q42, L43, D44, N45, L46, L47, L48, K49, F56, K57, Y59, L60, Q3, E67, Q70, M77, Q79, N82, Q83, D84 This includes mutations in one or more of the following helices: P85, D86, I87, A89, H90, S93, T100, L103, H109, ​​R110, L112, E115, N116, A127, K130, I136, Y137, K138, S141, E142, D144, I145, E151, M154, M156, K157, N160, and / or the addition of 4 to 8 amino acids between helix D and helix E.

[0205] In various embodiments, each IL-10 mutant protein is at least 95% identical to the amino acid sequence described in SEQ ID NO: 2. In various embodiments, each IL-10 mutant protein independently contains one or more mutations selected from the group consisting of R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, or I87A of SEQ ID NO: 2, which optionally include the addition of an amino acid between helix D and helix E. In various embodiments, each IL-10 mutant protein independently and optionally includes the addition of an amino acid between helix D and helix E, such as N10Q, N10I, N10K, R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, I87A, H14Q, F15Y, M22V, K49T, K49S, F56Y, K57N, Y59T, L60Q, Q63E, Q63L, E The mutant protein contains one or more mutations selected from the group consisting of 67C, Q70E, Q70K, M77R, M77V, Q79R, Q79C, D84R, A89P, H90E, H90Q, S93E, S93Q, T100R, L103E, H109D, R110P, R110Q, L112V, E115K, N116D, N116Q, A127M, K130Q, I136C, Y137C, M154V, M156C, K157N, or N160D. In various embodiments, the amino acid between helix D and helix E is GGGSGG (SEQ ID NO: 2676). Optionally, the IL-10 mutant protein may have a combination of mutations listed in Table 1, Table 16, Table 17, or Table 21.

[0206] In various embodiments, each IL-10 mutant protein is at least 95% identical to the amino acid sequence described in SEQ ID NO: 2. In various embodiments, each IL-10 mutant protein independently contains one or more mutations selected from the group consisting of N10, R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, or I87A of SEQ ID NO: 2, which optionally include the addition of six amino acids between helix D and helix E. In various embodiments, each IL-10 mutant protein independently and optionally includes the addition of six amino acids between helix D and helix E, such as N10Q, N10I, N10K, R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, I87A, H14Q, F15Y, M22V, K49T, K49S, F56Y, K57N, Y59T, L60Q, Q63E, Q It includes one or more mutations selected from the group consisting of 63L, E67C, Q70E, Q70K, M77R, M77V, Q79R, Q79C, D84R, A89P, H90E, H90Q, S93E, S93Q, T100R, L103E, H109D, R110P, R110Q, L112V, E115K, N116D, N116Q, A127M, K130Q, I136C, Y137C, M154V, M156C, K157N, or N160D. In various embodiments, the amino acid between helix D and helix E is GGGSGG (SEQ ID NO: 2676).In various embodiments, the amino acid sequences of the IL-10 mutant protein are as follows: SEQ ID NOs. 3-10 and SEQ ID NOs. 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2 180, 2182, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 241 8, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, This is described in 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2540 and 2777-2791.

[0207] In various embodiments, the antigen-binding protein comprises at least one linker fused to at least one C-terminus of the antigen-binding moiety, and the IL-10 mutant protein moiety is covalently bonded to the C-terminus of each linker. In various embodiments, the linker is 4 to 18 amino acids long. In various embodiments, the linker is 6 amino acids long. In various embodiments, the linker is a trimer, tetramer, pentamer, hexamer, heptamer, or octamer peptide. In various embodiments, the linker contains a GS residue. Further linkers envisioned for use are described below.

[0208] In various embodiments, the antigen-binding protein further comprises a human heavy chain constant region bound to the heavy chain variable region of the antibody portion of the antigen-binding protein. In various embodiments, the antigen-binding protein further comprises a human light chain constant region bound to the light chain variable region of the antibody portion of the antigen-binding protein.

[0209] In various embodiments of the antigen-binding protein, the antigen-binding portion is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, Fab, F(ab’)2, Fab2, monovalent IgG, scFv, scFv-Fc, IgG1 antibody, IgG2 antibody, IgG3 antibody, and IgG4 antibody. In various embodiments, the antigen-binding portion is 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 portion is monovalent IgG. In various embodiments, the heavy chain constant region of the antigen-binding portion is selected from the heavy chain constant regions of IgG, IgM, IgA, IgD, IgE, fragments thereof, combinations thereof, and modifications thereof in which 1 to 10 heavy chain framework amino acids are replaced with corresponding amino acids from another human antibody constant region.

[0210] In various embodiments of the antigen-binding protein, the antigen-binding portion binds to human PD-1 having the amino acid sequence set forth in SEQ ID NO: 22. In various embodiments, the antigen-binding portion binds to human TREM-1 having the amino acid sequence set forth in SEQ ID NO: 20. In various embodiments, the antigen-binding portion binds to human TREM-1 with a binding affinity of at least 10 -8 M.

[0211] In various embodiments, the antigen-binding portion is 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 Binds to the antigen due to its binding affinity for M.

[0212] In various embodiments of antigen-binding proteins, the anti-TREM-1 antigen-binding moiety is a. Light chain variable domain, i. Light chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290 and 2190; ii. Light chain CDR2 containing an amino acid sequence selected from SEQ ID NOs: 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271, 291 and 2191; iii. Light chain CDR3 containing an amino acid sequence selected from SEQ ID NOs: 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292 and 2192. Light chain variable domains including; and b. Heavy chain variable domain, i. Heavy chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276, 296, and 2196; ii. Heavy chain CDR2 containing an amino acid sequence selected from SEQ ID NOs: 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277, 297 and 2197; and iii. Heavy chain CDR3 containing an amino acid sequence selected from SEQ ID NOs: 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298 and 2198. Heavy chain variable domains including Includes.

[0213] In various embodiments of antigen-binding proteins, a. Light chain CDR1 sequences are described in SEQ ID NOs: 30, 50, 70, 110, 150, 170, or 290; b. Light chain CDR2 sequences are described in SEQ ID NOs: 31, 51, 71, 111, 151, 171, or 291; c. Light chain CDR3 sequences are described in SEQ ID NOs: 32, 52, 72, 112, 152, 172, or 292. d. Heavy chain CDR1 sequences are described in SEQ ID NOs: 36, 56, 76, 116, 156, 176, or 296; e. Heavy chain CDR2 sequences are described in SEQ ID NOs: 37, 57, 77, 117, 157, 177, or 297; f. The heavy chain CDR3 sequence is described in SEQ ID NOs: 38, 58, 78, 118, 158, 178, or 298.

[0214] In various embodiments of antigen-binding proteins, a. The light chain CDR1 sequence is described in SEQ ID NO: 50 or 110; b. The light chain CDR2 sequence is described in SEQ ID NO: 51 or 111; c. The light chain CDR3 sequence is described in sequence number 52 or 112; d. The heavy chain CDR1 sequence is described in SEQ ID NO: 56 or 116; e. The heavy chain CDR2 sequence is described in SEQ ID NO: 57 or 117; f. The heavy chain CDR3 sequence is described in SEQ ID NO: 58 or 118.

[0215] In various embodiments of antigen-binding proteins, the antigen-binding protein is i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 90 (LCDR1), 91 (LCDR2), 92 (LCDR3), 96 (HCDR1), 97 (HCDR2), and 98 (HCDR3); v) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); vi) Sequence IDs 130 (LCDR1), 131 (LCDR2), 132 (LCDR3), 136 (HCDR1), 137 (HCDR2), and 138 (HCDR3); vii) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); viii) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); ix) Sequence IDs 190 (LCDR1), 191 (LCDR2), 192 (LCDR3), 196 (HCDR1), 197 (HCDR2), and 198 (HCDR3); x) Sequence IDs 210 (LCDR1), 211 (LCDR2), 212 (LCDR3), 216 (HCDR1), 217 (HCDR2), and 218 (HCDR3); xi) Sequence IDs 230 (LCDR1), 231 (LCDR2), 232 (LCDR3), 236 (HCDR1), 237 (HCDR2), and 238 (HCDR3); xii) Sequence IDs 250(LCDR1), 251(LCDR2), 252(LCDR3), 256(HCDR1), 257(HCDR2), and 258(HCDR3); xiii) Sequence IDs 270 (LCDR1), 271 (LCDR2), 272 (LCDR3), 276 (HCDR1), 277 (HCDR2), and 278 (HCDR3); xiv) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3); and xv) Sequence IDs 2190 (LCDR1), 2191 (LCDR2), 2192 (LCDR3), 2196 (HCDR1), 2197 (HCDR2), and 2198 (HCDR3) Includes a set of CDR sequences selected from.

[0216] In various embodiments of antigen-binding proteins, the anti-TREM-1 antigen-binding moiety is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299, and 2183; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299 and 2183. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186; ii. Sequences encoded by polynucleotide sequences that are at least 80% identical to amino acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 and 2184; and iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2186. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0217] In various embodiments of antigen-binding proteins, the anti-TREM-1 antigen-binding moiety is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 121, 161, 181, and 301; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 39, 59, 79, 119, 159, 179, and 299; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a nucleic acid sequence selected from SEQ ID NOs: 39, 59, 79, 119, 159, 179, and 299. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 42, 62, 82, 122, 162, 182, and 302; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 40, 60, 80, 120, 160, 180, and 300; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 120, 160, 180, and 300. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0218] In various embodiments of antigen-binding proteins, the anti-TREM-1 antigen-binding moiety is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs: 61 and 121; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to a nucleic acid sequence selected from sequence numbers 59 and 119; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs. 59 and 119. A light chain variable domain containing an amino acid sequence selected from the group consisting of; and b. Heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs. 62 and 122; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from SEQ ID NOs. 60 and 120; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs. 60 and 120. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: Includes.

[0219] In various embodiments, the anti-TREM-1 antigen-binding protein or the anti-TREM1 antigen-binding portion of the antigen-binding protein is i) The light chain variable domain described in Sequence ID No. 41 and the heavy chain variable domain described in Sequence ID No. 42; ii) The light chain variable domain described in Sequence ID No. 61 and the heavy chain variable domain described in Sequence ID No. 62; iii) The light chain variable domain described in Sequence ID No. 81 and the heavy chain variable domain described in Sequence ID No. 82; iv) The light chain variable domain described in Sequence ID No. 101 and the heavy chain variable domain described in Sequence ID No. 102; v) The light chain variable domain described in Sequence ID No. 121 and the heavy chain variable domain described in Sequence ID No. 122; vi) The light chain variable domain described in Sequence ID No. 141 and the heavy chain variable domain described in Sequence ID No. 142; vii) The light chain variable domain described in Sequence ID No. 161 and the heavy chain variable domain described in Sequence ID No. 162; viii) The light chain variable domain described in Sequence ID No. 181 and the heavy chain variable domain described in Sequence ID No. 182; ix) The light chain variable domain described in Sequence ID No. 201 and the heavy chain variable domain described in Sequence ID No. 202; x) The light chain variable domain described in Sequence ID No. 221 and the heavy chain variable domain described in Sequence ID No. 222; xi) The light chain variable domain described in Sequence ID No. 241 and the heavy chain variable domain described in Sequence ID No. 242; xii) The light chain variable domain described in Sequence ID No. 261 and the heavy chain variable domain described in Sequence ID No. 262; xiii) The light chain variable domain described in Sequence ID No. 281 and the heavy chain variable domain described in Sequence ID No. 282; xiv) The light chain variable domain described in Sequence ID No. 301 and the heavy chain variable domain described in Sequence ID No. 302; or xv) Light chain variable domain described in Sequence ID No. 2185 and heavy chain variable domain described in Sequence ID No. 2186 Includes.

[0220] In various embodiments of the antigen-binding protein, the amino acid sequence may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs. 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185 and SEQ ID NOs. 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186.

[0221] In various embodiments of the antigen-binding protein, the antigen-binding moiety includes an amino acid sequence that is at least 90% identical to the heavy chain variable region amino acid sequence selected from SEQ ID NOs: 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186.

[0222] In various embodiments of the antigen-binding protein, the antigen-binding portion includes an amino acid sequence that is at least 90% identical to the light chain variable region amino acid sequence selected from SEQ ID NOs: 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185.

[0223] In various embodiments of the antigen-binding protein, the antigen-binding portion includes a heavy chain amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 42, 62, 82, 122, 142, 162, 182, and 302, and a light chain amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 41, 61, 81, 121, 141, 161, 181, and 301.

[0224] In various embodiments of the antigen-binding protein, the antigen-binding portion includes a heavy chain amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs. 62 and 122, and a light chain amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs. 61 and 121.

[0225] In various embodiments, the antigen-binding protein is SEQ ID NOs: 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 97 3, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, Heavy chain amino acid sequences selected from 1079, 1081, 1083 and 1085, and sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966 ,968,970,972,974,976,978,980,982,984,986,988,990,992,994,996,998,1000,1002,1004,1006,1008,1010,1012,1014,1016,1018,1020,1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072,It contains a light chain amino acid sequence selected from 1074, 1076, 1078, 1080, 1082, 1084, and 1086.

[0226] In various embodiments, variants of the TREM-1 antibody heavy chain and / or light chain variable regions are provided herein. TREM-1 antibody heavy chain variable region variant sequences are SEQ ID NOs: 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963, 1965, 1 967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005, 2007, 2011, 2013, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 20 The TREM-1 antibody light chain variable region variant sequences are listed in Tables 10 and 11, and the TREM-1 antibody light chain variable region variant sequences are SEQ ID NOs: 2543, 2544, 2544. 5, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592,2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 22 As described in Tables 85, 2287, 2289, 2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, Tables 10 and 11. The TREM-1-IL-10 antigen-binding protein is thought to contain the TREM-1 antibody variant heavy and / or light chain sequences disclosed herein.

[0227] In various embodiments of antigen-binding proteins, one or more heavy chain framework amino acids of the antigen-binding protein are substituted with corresponding 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 substituted with corresponding amino acids from another human antibody amino acid sequence.

[0228] In various embodiments, the anti-TREM-1 antigen-binding protein further comprises a human light chain constant region bound to the light chain variable region.

[0229] In various embodiments of the antigen-binding protein, the antigen-binding protein comprises two light chains and two heavy chains, each heavy chain comprising an IL-10 moiety attached to the C-terminus of the heavy chain; each heavy chain IL-10 moiety antigen-binding protein is represented by SEQ ID NOs: 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 88 9, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967 ,969,971,973,975,977,979,981,983,985,987,989,991,993,995,997,999,1001,1003,1005,1007,1009,1011,1013,1015,1017,1019,1021,1023,1025,1027,1029,1031,1033,1035,1 It comprises an amino acid sequence that is at least 90% identical to a sequence selected from 037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083 and 1085;And each light chain is sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924 ,926,928,930,932,934,936,938,940,942,944,946,948,950,952,954,956,958,960,962,964,966,968,970,972,974,976,978,980,982,984,986,988,990,9 92, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, and 1086.

[0230] In various embodiments, the antigen-binding protein may be monovalent. In various embodiments, the heavy chain IL-10 partial antigen-binding protein may be SEQ ID NOs: 1087, 1090, 1093, 1096, 1105, 1108, 1111, 1114, 1117, 1123, 1126, 1129, 1132, 1138, 1141, 1147, 1150, 1153, 1156, 1159, 116 2, 1165, 1168, 1171, 1174, 1177, 1180, 1183, 1186, 1189, 1192, 1195, 1198, 1201, 1204, 1207, 1210, 1213, 1216, 1219, 1222, 1225, 1228, 1231, 1237, 1240, 1243, 1246, 125 2, 1255, 1258, 1261, 1264, 1267, 1270, 1273, 1276, 1279, 1285, 1288, 1294, 1297, 1300, 1303, 1309, 1312, 1315, 1318, 1321, 1324, 1333, 1336, 1342, 1345, 1348, 1351, 135 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 4, 1357, 1360, 1363, 1366, 1369, 1372, 1375, 1378, 1381, 1384, 1387, 1390, 1393, 1396, 1399, 1402, 1408, 1411, 1414, 1417, and 1420.In various embodiments, the light chain is sequence numbers 1088, 1091, 1094, 1097, 1106, 1109, 1112, 1115, 1118, 1124, 1127, 1130, 1133, 1139, 1142, 1148, 1151, 1154, 1157, 1160, 1163, 1166, 1169, 1172, 1175, 1178, 1181, 1184, 1187, 1190, 1193, 1196, 1199, 1202, 1205, 1208, 1211, 1214, 1217, 1220, 1223, 1226, 1229, 1232, 1238, 1241, 1244, 1247, 1253, 1256, 1 259, 1262, 1265, 1268, 1271, 1274, 1277, 1280, 1286, 1289, 1295, 1298, 1301, 1304, 1310, 1313, 1316, 1319, 1322, 1325, 1334, 1337, 1343, 1346, 1349, 1352, 1355, 13 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 58, 1361, 1364, 1367, 1370, 1373, 1376, 1379, 1382, 1385, 1388, 1391, 1394, 1397, 1400, 1403, 1409, 1412, 1415, 1418, and 1421.In various embodiments, the monovalent antigen-binding protein is, for example, SEQ ID NOs: 1089, 1092, 1095, 1098, 1107, 1110, 1113, 1116, 1119, 1125, 1128, 1131, 1134, 1140, 1143, 1149, 1152, 1155, 1158, 1161, 116 4, 1167, 1170, 1173, 1176, 1179, 1182, 1185, 1188, 1191, 1194, 1197, 1200, 1203, 1206, 1209, 1212, 1215, 1218, 1221, 1224, 1227, 1230, 1233, 1239, 1242, 1245, 1248 ,1254,1257,1260,1263,1266,1269,1272,1275,1278,1281,1287,1290,1296,1299,1302,1305,1311,1314,1317,1320,1323,1326,1335,1338,1344,1347,1350, It further includes Fc regions as described in 1353, 1356, 1359, 1362, 1365, 1368, 1371, 1374, 1377, 1380, 1383, 1386, 1389, 1392, 1395, 1398, 1401, 1404, 1410, 1413, 1416, 1419, and 1422.

[0231] In various embodiments of the antigen-binding protein, the antigen-binding protein comprises two light chains and two heavy chains, each heavy chain comprising an IL-10 moiety attached to the C-terminus of the heavy chain; each heavy chain IL-10 moiety antigen-binding protein is sequence numbers 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 199 9, 2001, 2003, 2005, 2007, 2011, 2013, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 2 The sequence includes an amino acid sequence selected from 081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131, 2133, 2135, and an amino acid sequence that is at least 90% identical to the sequences listed in Tables 10, 11, 16, and 17;Each light chain corresponds to sequence numbers 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 257 5, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2233, 2235, 2237, 2239, 2 241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 230 The sequence includes an amino acid sequence selected from 9, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, and an amino acid sequence that is at least 90% identical to the sequences listed in Tables 10 and 11. In various embodiments of the antigen-binding protein, the antigen-binding protein comprises two light chains and two heavy chains, each heavy chain comprising an IL-10 moiety bound to the C-terminus of the heavy chain;Each heavy chain IL-10 partial antigen-binding protein is sequence numbers 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 19 A sequence selected from 45, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005 and 2007 is at least 90% identical to A Each light chain contains a mino acid sequence, and each light chain is sequence numbers 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605.

[0232] In various embodiments of the antigen-binding protein, the antigen-binding protein comprises two light chains and two heavy chains, each heavy chain comprising an IL-10 moiety bound to the C-terminus of the heavy chain; each heavy chain IL-10 moiety antigen-binding protein corresponds to Sequence IDs 2011, 2013, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049. ,2051,2053,2055,2057,2059,2061,2063,2065,2067,2069,2071,2073,2075,2077,2079,2081,2083,2085,2087,2089,2091,2093,2095,2097,2099,2101,2103,2105,2107,2109,2111,2113,2115,2117,2119,2121,2123,2125,2127,2129,2 The amino acid sequence contains an amino acid sequence that is at least 90% identical to sequences selected from 131, 2133 and 2135, and each light chain contains an amino acid sequence that is at least 90% identical to sequences 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2 It contains an amino acid sequence that is at least 90% identical to a sequence selected from 291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355 and 2357.

[0233] In various embodiments, the antigen-binding protein comprises a heavy chain amino acid sequence that is at least 90% identical to the sequence described in any one of SEQ ID NOs. 2726-2776. In various embodiments, the antigen-binding protein comprises a heavy chain amino acid sequence described in any one of SEQ ID NOs. 2726-2776. In various embodiments, the antigen-binding protein has a heavy chain IL-10 partial amino acid sequence selected from the group consisting of SEQ ID NOs. 2727-2732.

[0234] In various embodiments, the antigen-binding protein comprises the heavy chain amino acid sequence of SEQ ID NO: 2727 or 2728 and the light chain amino acid sequence described in SEQ ID NO: 976 or 2554, or other 63F8 or 63F8.001 light chains. In various embodiments, the antigen-binding protein comprises the heavy chain amino acid sequence of SEQ ID NO: 2729, 2730, 2731 or 2732 and the light chain amino acid sequence described in SEQ ID NO: 992 or 2555, or other 64D7 or 64D7.001 light chains.

[0235] In various embodiments of antigen-binding proteins, the anti-TREM-1 antigen-binding moiety inhibits the binding of TREM-1 to the TREM-1 ligand.

[0236] In various embodiments, at least one IL-10 moiety is fused to the N-terminus of the antigen-binding moiety. In various embodiments, at least one IL-10 moiety is fused to the N and C-terminuses of the antigen-binding moiety. In various embodiments, the IL-10 moiety is fused to an internal site in the antigen-binding moiety, for example, between the CH1 domain and the hinge region of the heavy chain or at a site described in U.S. Patent No. 8,008,453, which is incorporated herein by reference. In various embodiments, at least one IL-10 moiety is fused to the heavy chain and / or light chain of the antigen-binding moiety. In various embodiments, the heavy chain and / or light chain are modified or manipulated heavy or light chains.

[0237] nucleic acid molecule This disclosure also provides IL-10 mutant proteins, antigen-binding proteins, and isolated nucleic acids encoding antigen-binding proteins containing the antigen-binding moiety and IL-10 moiety described herein, which include, for example, IL-10 mutant protein sequences, 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 of their components and combinations. 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 with the nucleic acids of the present invention. The terms “similarity percentage,” “identity percentage,” and “homology percentage” when referring to specific sequences are used as described in the University of Wisconsin GCG® software program. The nucleic acids of this disclosure also include complementary nucleic acids. In some cases, sequences will be perfectly complementary when aligned (no mismatch). In other cases, there may be up to about 20% mismatch in the sequences. In one embodiment of the present invention, nucleic acids encoding both the heavy chain and the light chain of the antibody of the present disclosure are provided.

[0238] The nucleic acids of this disclosure may be cloned into vectors such as plasmids, cosmids, bacmids, phages, artificial chromosomes (BAC, YAC), or viruses, into which another gene sequence or element (either DNA or RNA) may be inserted to result in replication of the bound sequence or element. In some embodiments, the expression vector contains a constitutively active promoter segment (but not limited to CMV, SV40, elongation factor, or LTR sequence) or an inducible promoter sequence, such as a steroid-inducible pIND vector (Invitrogen), where the expression of the nucleic acid can be regulated. The expression vector of the present invention may further include a regulatory sequence, such as an internal ribosome entry site. The expression vector may be introduced into cells, for example, by transfection.

[0239] Also provided is an expression vector comprising the following operablely linked elements; a transcription promoter; a first nucleic acid molecule encoding the heavy chain of the IL-10 mutant protein, antigen-binding protein, antibody, or antigen-binding fragment or fusion protein of the Disclosure; a second nucleic acid molecule encoding the light chain of the antigen-binding protein, antibody, or antigen-binding fragment of the Disclosure; and a transcription terminator. In another embodiment, the Disclosure provides an expression vector comprising the following operablely linked elements; a first transcription promoter; a first nucleic acid molecule encoding the heavy chain of the IL-10 mutant protein, antigen-binding protein, antibody, or antigen-binding fragment or fusion protein of the Disclosure; a first transcription terminator; a second transcription promoter; a second nucleic acid molecule optionally encoding the light chain of the antigen-binding protein, antibody, or antigen-binding fragment of the Disclosure; and a second transcription terminator.

[0240] The secretion signal peptide sequence may also be optionally encoded by an expression vector operably linked to the target coding sequence, thereby allowing the expressed polypeptide to be secreted by recombinant host cells for easier isolation of the target polypeptide from the cells, if necessary. For example, in one embodiment, the signal peptide sequence may be added to / fused to the amino terminus of any of the IL-10 mutant protein, antigen-binding protein, antibody or its antigen-binding fragment or fusion protein polypeptide sequences described herein.

[0241] Recombinant host cells are also provided that contain such vectors and express antigen-binding proteins containing IL-10 mutant proteins, antigen-binding protein heavy and light chains, or antigen-binding moieties and IL-10 moieties. The recombinant host cells may be prokaryotic cells, such as Escherichia coli (E. coli) cells, or eukaryotic cells, such as mammalian cells or yeast cells. Examples of yeast cells include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris cells. Examples of mammalian cells include VERO, HeLa, Chinese hamster ovary (CHO), W138, baby hamster kidney (BHK), COS-7, MDCK, human fetal kidney cell line 293, healthy dog ​​kidney cell line, healthy cat kidney cell line, monkey kidney cells, African green monkey kidney cells, COS cells, and non-tumor mouse myoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH / 3T3 cells, LMTK31 cells, mouse Sertoli cells, human cervical cancer cells, buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumor cells, TRI cells, MRC 5 cells, and FS4 cells. Recombinant protein-producing cells of this disclosure also include any known insect-expressing cell lines, such as fall armyworm (Spodoptera frugiperda) cells. In one embodiment, the cells are mammalian cells. In a particular embodiment, the mammalian cells are CHO cells.

[0242] Methods for purifying proteins are known in the art and are used herein for the recovery of recombinant proteins from cell culture media. For example, methods for purifying proteins and antibodies are known in the art and may be used for the production of antibodies according to this disclosure. In some embodiments, the method for purifying proteins and antibodies includes filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. The filtration step may include ultrafiltration, optionally ultrafiltration, and dialysis. Filtration is preferably performed at least about 5 to 50 times, more preferably 10 to 30 times, and most preferably 14 to 27 times. Affinity column chromatography may be performed using, for example, PROSEP® affinity chromatography (Millipore, Billerica, Mass.). In various embodiments, the affinity chromatography step includes PROSEP®-vA column chromatography. The eluate may be washed in a solvent washing agent. Cation exchange chromatography may include, for example, SP-Sepharose cation exchange chromatography. Anion exchange chromatography may include, for example, Q-Sepharose fast-flow anion exchange. The anion exchange step is preferably unbound, thereby allowing for the removal of contaminants including DNA and BSA. The antibody product is preferably nanofiltered using, for example, a Pall DV 20 Nanofilter. The antibody product may be concentrated using, for example, ultrafiltration and dialysis. The method may further include a step of size exclusion chromatography to remove aggregates.

[0243] In various embodiments, the nucleotide sequences described in SEQ ID NOs: 11-18 are useful for expressing the IL-10 mutant protein or its fragment or variant as described herein.

[0244] In one embodiment, different nucleic acid molecules encode the heavy chain variable region and the light chain variable region of a target-specific antibody. In another embodiment, the same nucleic acid molecule encodes the heavy chain and light chain variable region of a target-specific antibody. In one embodiment, the nucleic acid encodes either the target-specific antibody of this disclosure or a polypeptide encoded by the nucleic acid described herein.

[0245] In one embodiment, 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 the VH amino acid sequences described in SEQ ID NOs: 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186. The nucleic acid molecules of this disclosure further include nucleic acids that hybridize to nucleic acid sequences encoding the heavy chain variable region amino acid sequences of SEQ ID NOs. 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186 under highly stringent conditions such as those described herein, or nucleic acids having one of the heavy chain variable region nucleic acid sequences of SEQ ID NOs. 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184.

[0246] In one embodiment, 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 the VL amino acid sequences described in SEQ ID NOs: 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185. The nucleic acid molecules of this disclosure further include nucleic acids that hybridize to nucleic acid sequences encoding the light chain variable region amino acid sequences of SEQ ID NOs. 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301 and 2185 under highly stringent conditions such as those described herein, or nucleic acids having one of the light chain variable region nucleic acid sequences of SEQ ID NOs. 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299 and 2183.

[0247] In one embodiment, the nucleic acid molecule of the Disclosure comprises a nucleotide sequence encoding the VL amino acid sequence of an anti-TREM-1 antigen-binding protein or a portion thereof as described herein. In a related embodiment, the VL amino acid sequence is a consensus sequence. In one embodiment, the nucleic acid encodes the amino acid sequence of the light chain CDR of the antibody. In one embodiment, the portion is a contiguous portion comprising LCDR1-CDR3. In a related embodiment, the LCDR1-3 amino acid sequences are a consensus sequence. In one embodiment, the portion comprises at least one, two, or three light chain CDR1, CDR2, or CDR3 regions, optionally with different human or human consensus frameworks, and optionally with one, at most two, or at most three mutations in three collective CDRs.

[0248] In one embodiment, the nucleic acid molecule of the Disclosure comprises a nucleotide sequence encoding the VH amino acid sequence of an anti-TREM-1 antigen-binding protein or a portion thereof as described herein. In a related embodiment, the VH amino acid sequence is a consensus sequence. In one embodiment, the nucleic acid encodes the amino acid sequence of the heavy chain CDR of the antibody. In one embodiment, the portion is a contiguous portion comprising HCDR1-CDR3. In a related embodiment, the HCDR1-3 amino acid sequences are a consensus sequence. In one embodiment, the portion comprises at least one, two, or three heavy chain CDR1, CDR2, or CDR3 regions, optionally with different human or human consensus frameworks, and optionally with one, at most two, or at most three mutations in three collective CDRs.

[0249] The nucleic acid sequences of the heavy and light chains of specific antigen-binding proteins are described in SEQ ID NOs. 303-526 (bivalent) and 527-862 (monovalent). The nucleic acid sequences of the variable region of the heavy chain of the TREM-1 mutant antibody are described in SEQ ID NOs. 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 20 As described in 70, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134 and 2136, TR The nucleotide sequences of the EM-1 mutant antibody light chain variable region are: 2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290, 2 This is described in 292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356 and 2358.

[0250] In exemplary embodiments, the antibodies of the Disclosure include a human kappa (κ) or human lambda (λ) light chain or amino acid sequence derived therefrom, or a human heavy chain or sequence derived therefrom, or both the heavy chain and light chain together in single-chain, dimer, tetramer, or other forms.

[0251] Linker A linker or spacer comprises a peptide or other moiety used to join two molecules by either a covalent or non-covalent bond. The peptide moiety is herein assumed to provide increased spacing of the IL-10 sequence for stability and proper folding in monomeric form within an IL-10 mutant protein polypeptide. The peptide moiety is also herein assumed to be used in antigen-binding proteins to provide a link or spacer between the antigen-binding moiety in the antigen-binding protein and the IL-10 monomer / mutant protein to which it is fused.

[0252] The peptide linker that binds the Fc region to the IL-10 mutant protein may be any of the peptide linkers described herein. In some embodiments, the linker may be 2-40, 3-40, 3-30, or 3-20 amino acid lengths. In various embodiments, the linker may be 3-25, 4-18, 4-20, 5-20, 6-18, or 10-20 amino acid lengths. In various 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 acid lengths. In various embodiments, the linker is 4-18 amino acid lengths. In certain embodiments, the peptide linker that binds the Fc region to the IL-10 mutant protein is at least 4 amino acids, at least 5 amino acids, or at least 6 amino acid lengths. In other embodiments, the peptide linker that binds the Fc region to the carboxyl-terminal Fab fragment is at least 8 amino acids long.

[0253] In one embodiment, the linker may be bound to the C-terminus of the heavy chain of the antigen-binding moiety in the antigen-binding protein. In various embodiments, the linker is a trimer, tetramer, pentamer, hexamer, heptamer, octamer, or repeating trimers, tetramers, pentamers, or hexamers. Exemplary linkers include Gly-Gly-Gly-Gly (SEQ ID NO: 2677), Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 2705), Gly-Gly-Gly-Pro (SEQ ID NO: 2706), Gly-Gly-Gly-Gln (SEQ ID NO: 2707), Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 2708), Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2725), (Gly3Ser) 2( (Sequence code 2709), (Gly4Ser) 2( (Sequence code 2710), (Gly3Ser) 3( (Sequence code 2711), (Gly4Ser)3 (Sequence code 2712), (Gly3Ser) 4( (Sequence code 2713), (Gly4Ser) 4( (Sequence ID 2714), (Gly3Ser)5 (Sequence ID 2715), (Gly4Ser)5 (Sequence ID 2716), (Gly3Ser) 6(Sequence ID 2717), (Gly4Ser)6 (Sequence ID 2718), Gly-Ser-Gly-Ser-Ala-Thr-Gly-Gly-Ser-Gly-Ser-Ser-Ala-Ser-Ser-Gly-Ser-Gly-Ser-Ala-Thr-His-Leu (Sequence ID 2719), Gly-Ser-Gly-Ser-Ala-Thr-Gly-Gly-Ser-Gly-Ser-Val-Ala-Ser-Ser-Gly-Ser-Gly-Ser-Ala-Thr-His-Leu (Sequence ID 272 Examples include 0), Gly-Ser-Gly-Ser-Ala-Thr-Gly-Gly-Ser-Gly-Ser-Ser-Ala-Ser-Ser-Gly-Ser-Gly-Ser-Ala-Thr-His-Leu (Sequence ID 2721), Gly-Ser-Gly-Ser-Ala-Thr-Gly-Gly-Ser-Gly-Ser-Gly-Ala-SeGGGr-Ser-Gly-Ser-Gly-Ser-Ala-Thr-Gly-Ser (Sequence ID 2722), and the linkers described in the examples.

[0254] The linker or spacer peptide moiety is also inserted into the IL-10 mutant protein for stability. For example, a sequence of 4 to 8 amino acids can be added within the DE helix loop of wild-type IL-10 or the IL-10 mutant protein. In various embodiments, the linker is 3, 4, 5, 6, 7, or 8 amino acids long. In various embodiments, the linker is 6 amino acids long. In various embodiments, the linker has the sequence GGGSGG (SEQ ID NO: 2676).

[0255] derivative Suitable detectable molecules may be directly or indirectly conjugated to the IL-10 mutant protein, antigen-binding protein, or fusion protein of this disclosure. Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like. For indirect conjugation of detectable or cytotoxic molecules, the detectable or cytotoxic molecule may be conjugated with a member of a complement / anti-complement pair, where the other member is conjugated to a binding polypeptide or antibody moiety. For these purposes, biotin / streptavidin is an exemplary complement / anti-complement pair.

[0256] The IL-10 mutant proteins, antigen-binding proteins, and antigen-binding proteins comprising an antigen-binding moiety and an IL-10 moiety of the present disclosure also include derivatives modified by covalent bonding of any type of molecule to an antibody, for example, so that the covalent bond does not prevent the antibody from binding to its epitope. Examples of preferred derivatives include, but are not limited to, fucosylated, glycosylated, acetylated, PEGylated, phosphorylated, or amidated derivatives. The IL-10 mutant proteins, antigen-binding proteins, and antigen-binding proteins comprising an antigen-binding moiety and an IL-10 moiety and derivatives thereof of the present disclosure may be derivatized by known protective / blocking groups, proteolytic cleavage, linkage to cell ligands or other proteins, etc. In some embodiments of the present disclosure, at least one heavy chain of the IL-10 mutant protein or antigen-binding protein or antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety is PEGylated. In some embodiments, the PEGylation is by N-linking or by linkage by an amino acid (e.g., lysine) side chain.

[0257] Glycosylation can contribute to the effector function of antibodies, particularly IgG1 antibodies. Therefore, in some embodiments, the IL-10 mutant protein, antigen-binding protein, or antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety of the present disclosure may contain one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. Polypeptide glycosylation is typically N-linked or O-linked. N-linking refers to the binding of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic binding of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-bonded glycosylation refers to the bonding of one sugar, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0258] In certain embodiments, glycosylation of IL-10 mutant proteins, antigen-binding proteins, and antigen-binding proteins containing the antigen-binding moiety and IL-10 moiety described herein is increased by adding one or more glycosylation sites, for example, to the Fc region of the binding protein. Addition of glycosylation sites to antigen-binding proteins can be conveniently achieved by modifying the amino acid sequence to contain one or more of the above-described tripeptide sequences (in the case of N-linked glycosylation sites). Modification can also be made by adding one or more serine or threonine residues to the start sequence or by substitution with one or more serine or threonine residues (in the case of O-linked glycosylation sites). For ease of implementation, the antigen-binding protein amino acid sequence can be modified at the DNA level, particularly by mutating the DNA encoding the target polypeptide at pre-selected bases, such that codons are generated and translated to desired amino acids.

[0259] This disclosure also includes the production of IL-10 mutant proteins, antigen-binding proteins, and antigen-binding proteins containing an antigen-binding moiety and an IL-10 moiety, having a modified carbohydrate structure that results in modified effector activity, including antigen-binding proteins that are fucosylated or have reduced fucosylation, exhibiting improved ADCC activity. Various methods for reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity has been shown to be mediated by the binding of an antibody molecule to the FcγRIII receptor, which depends on the carbohydrate structure of N-linked glycosylation at the N297 residue of the CH2 domain. Non-fucosylated antibodies bind to this receptor with increased affinity than naturally occurring fucosylated antibodies and more efficiently induce FcγRIII-mediated effector function. For example, recombinant production of non-fucosylated antibodies in CHO cells with knocked-out α-1,6-fucosyltransferase enzyme results in antibodies with 100-fold increased ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). Similar effects can be achieved, for example, by reducing the activity of α-1,6-fucosyltransferase enzyme or other enzymes in the fucosylation pathway by siRNA or antisense RNA treatment, by manipulating cell lines to knock out the enzyme, or by culturing with selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell lines, such as Lec13 or the rat hybridoma YB2 / 0 cell line, naturally produce antibodies with lower fucosylation levels (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003).For example, it has been shown that increasing the level of bisected carbohydrates in cells overexpressing the GnTIII enzyme by recombinant antibodies can increase ADCC activity (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0260] In other embodiments, glycosylation of IL-10 mutant proteins, antigen-binding proteins, and antigen-binding proteins comprising the antigen-binding moiety and IL-10 moiety described herein is reduced or eliminated, for example, by removing one or more glycosylation sites from the Fc region of the binding protein. Amino acid substitutions that eliminate or modify N-linked glycosylation sites may reduce or eliminate N-linked glycosylation of antigen-binding proteins. In certain embodiments, the bispecific antigen-binding proteins described herein include mutations at position N297 (EU numbering), such as N297Q, N297A, or N297G. In a particular embodiment, the bispecific antigen-binding protein of the present invention includes an Fc region from a human IgG1 antibody having the N297G mutation. To improve the stability of molecules containing the N297 mutation, the Fc region of the molecule may be further manipulated. For example, in one embodiment, one or more amino acids in the Fc region are substituted with cysteine ​​to promote disulfide bonding in the dimeric state. Therefore, residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (EU numbering) in the IgG1 Fc region may be substituted with cysteine. In one embodiment, certain pairs of residues are substituted with cysteine ​​so that they preferentially form disulfide bonds with each other, thereby inhibiting or preventing disulfide bond scrambling. In certain embodiments, pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In certain embodiments, the bispecific antigen-binding proteins described herein include an Fc region from a human IgG1 antibody having mutations at R292C and V302C. In such embodiments, the Fc region may also include the N297G mutation.

[0261] Modifications of the IL-10 mutant protein, antigen-binding protein, and antigen-binding protein containing the antigen-binding moiety and IL-10 moiety of the present disclosure to increase the blood half-life may also be desirable, for example, by incorporating or adding a salvage receptor-binding epitope (e.g., by mutation in an appropriate region, or by incorporating the epitope into a peptide tag that is subsequently fused to the IL-10 mutant protein, antigen-binding protein, and antigen-binding protein containing the antigen-binding moiety and IL-10 moiety at any terminal or midway, for example by DNA or peptide synthesis; see, for example, International Publication No. 96 / 32478) or by adding a molecule such as PEG or other water-soluble polymers containing polysaccharide polymers. Preferably, the salvage receptor-binding epitope consists of one or more amino acid residues from one or two loops of the Fc region, constituting a region moved to a similar position in the antigen-binding protein. In one embodiment, three or more residues from one or two loops of the Fc region are moved. In one embodiment, the epitope is taken from the CH2 domain of the Fc region (e.g., the IgG Fc region) and transferred to the CH1, CH3, or VH region, or two or more such regions, of the IL-10 mutant protein, antigen-binding protein, and antigen-binding protein containing the antigen-binding moiety and the IL-10 moiety. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region, VL region, or both of the antigen-binding protein. For a description of Fc variants and their interactions with salvage receptors, see International Publication Brochures 97 / 34631 and 96 / 32478.

[0262] The IL-10 mutant proteins, antigen-binding proteins, and antigen-binding proteins comprising antigen-binding moieties and IL-10 moieties of the present disclosure include variants having one or more amino acid substitutions, deletions, additions, or substitutions that preserve their biological properties. Those skilled in the art can generate variants having one or more amino acid substitutions, deletions, additions, or substitutions. These variants may include, in particular, (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 contain substituents, and (d) variants in which the polypeptide is fused with another peptide or polypeptide, e.g., a fusion partner, a protein tag, or another chemical moiety that can impart useful properties to the polypeptide, e.g., an epitope for an antibody, a polyhistidine sequence, a biotin moiety, etc. The antibodies and bispecific antibodies of the present invention may include variants in which an amino acid residue from one species is substituted with a corresponding residue from another species at either a conserved or non-conserved position. 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 skilled in the art.

[0263] Treatment methods IL-10 potently inhibits inflammatory cytokines, including TNFα and IL-23 production from bone marrow cells. Both TNFα and IL-23 are effective targets for the treatment of inflammatory bowel disease. IL-10 also suppresses antigen-presenting cells, including inhibiting the induction of MHCII, CD86, and ICAM1. Although IL-10 has a higher EC50, it activates CD8+ T and B cells, which may contribute to the dose-limiting toxicity of IL-10 in clinical trials. rhIL-10 has a low PK (t1 / 2 = 2.5-4 hours) and failed to achieve cell type selectivity in clinical settings. High doses of rhIL-10 stimulate CD8+ T and B cells, while low doses show insufficient tissue and bone marrow cell coverage. Myeloid cell-specific IL-10R1 knockout mice developed colitis similar to that of IL-10 knockout mice, suggesting that IL-10 inhibition in myeloid cells is sufficient for the treatment of inflammatory bowel disease (IBD) (Zigmond et al., Immunity 40(5):720-33, 2014). Therefore, it is hypothesized herein that by targeting IL-10 to myeloid cells or another target region using an anti-TREM-1 mAb / IL10 mutant protein bispecific method, sufficient anti-inflammatory activity in myeloid cells can be achieved without stimulating activity in CD8+ cells and B cells.

[0264] Human patients with homozygous loss-of-function mutations in IL-10, IL-10R1, or IL-10R2 developed severe infantile ileoblastic disease (IBD) (Kotlarz et al., Gastroenterology, 143:347-355, 2012; Glocker, NEJM, 61(21):2033-45, 2009). IL-10 is one of the genetic mutations associated with IBD (GWAS locus, rs3024505 RAF 0.16 or 1.46, p-value 10). -42 Jostins et al., Nature 491:119-124, 2012, deCODE rs3024505 IBD or 1.13, p-value 0.024).

[0265] The trigger receptor expressed in bone marrow cells (TREM1, TREM-1) is an Ig family member expressed in neutrophils, monocytes, and macrophages. TREM-1 inhibition may be desirable for IBD treatment because TREM-1 KO mice are viable and protected from DSS colitis and T-cell transfer colitis (Weber, PLoS Pathog, 10(1):1003900, 2014). Treatment with TREM-1-Fc fusion improved survival and reduced TNFα induction after LPS inoculation in mice (Bouchon, Nature, 410:1103-7, 2001). TREM-1 has increased expression in inflammatory IBD tissue (Schenk et al., J Clin Invest. 117:3097-3106, 2007), and CX3CR1 cre IL10R1 fl / fl TREM-1 exhibits increased expression in colonic macrophages in mice (Zimond, Immunity, 40(5):720-33, 2014), binds to DAP12, and induces Syk phosphorylation upon activation. Due to its high expression on the surface of bone marrow cells, TREM-1 is hypothesized here to play a desirable targeting partner for IL-10 antigen-binding proteins.

[0266] PGLYRP1 (peptidoglycan-recognizing protein 1) has recently been reported as a ligand for TREM-1 (Read, J.Immunol. 194:1417-1421, 2015), and other potential ligands remain undetermined. Anti-TREM-1 antibodies have been reported to reduce the secretion of inflammatory cytokines from lamina propria cells isolated from IBD patients stimulated by the TREM-1 agonist PGLYRP-1 / peptidoglycan (Brynjolfsson et al., Inflamm Bowel Dis 22(8):1803-11, 2016).

[0267] Reading the efficacy of treatment in IBD involves evaluating the IL-10 IBD risk variant rs3024505 in the regulation of IL-10 expression, assessing heterogeneity of IL-10 levels in IBD patient serum and tissue, and evaluating the potential correlation with disease severity and the response to treatment for the IBD risk variant.

[0268] Further measures to determine efficacy include analyzing the reduced IL-10-inducible expression profile in myeloid cells and IBD tissue, the heterogeneity of myeloid-derived cytokines (e.g., TNFα, IL-23) that contributed to IL-10 suppression in IBD samples, and determining the heterogeneity and number of monocytes and macrophages in IBD inflammatory tissue. Therapy as described herein with IL-10 mutant proteins or antigen-binding proteins is thought to maintain suppression of myeloid cell activity and eliminate IL-10 activation of CD8+ T cells and B cells. Therapy is thought to reduce levels of inflammatory cytokines such as TNF-α and IL-23 in the subject.

[0269] Treatment with an anti-TREM-1 antigen-binding protein or an antigen-binding protein comprising the antigen-binding moiety and IL-10 moiety of the present disclosure, including an anti-TREM-1 antigen-binding protein, is thought to reduce TREM-1 expression in inflammatory cells.

[0270] Crohn's disease involves abnormal inflammation of any part of the digestive tract from mouth to anus, but in most patients, the abnormal inflammation is localized to the ileocolon, small intestine, and colorocorectal region. Typically, the inflammation is discontinuous. Common symptoms include abdominal pain, loss of appetite, weight loss, fever, diarrhea, right lower abdominal distension and / or tenderness, constipation, vomiting, and perianal discomfort and discharge. Other possible symptoms include, in particular, peripheral arthritis, developmental delay, episcleritis, aphthous stomatitis, erythema nodosum, pyoderma gangrenosum, kidney stones, urinary dilution disorders and alkalinization, malabsorption, and gallstones. See, for example, Strober et al., Medical Immunology, 10th Edition, Section III, Ch.35 (2001); Merck Manual of Diagnosis and Therapy, 17th Edition, Section 3, Ch.31 (1999). Macrophages isolated from Crohn's disease patients result in increased levels of IL-12, IFNγ, TNFα, and other inflammatory cytokines.

[0271] Ulcerative colitis differs from Crohn's disease in several ways. First, ulcerative colitis is generally limited to the colon, while Crohn's disease can occur throughout the entire gastrointestinal tract. Second, unlike Crohn's disease, where inflammation can penetrate the entire intestinal wall or other parts of the gastrointestinal tract, ulcerative colitis primarily involves inflammation of only the superficial layer of the intestine. Finally, ulcerative colitis typically involves a continuous area of ​​inflammation, rather than the discontinuous sites of inflammation characteristic of Crohn's disease. Similar to Crohn's disease, ulcerative colitis is mainly found in urban areas. Also, due to the familial clustering of cases, genetic factors are likely to play a role in ulcerative colitis. Autoantibodies are observed more frequently in ulcerative colitis patients than in Crohn's disease patients. Autoantibodies are often directed towards colonic epithelial cell components. Antineutrophil cytoplasmic antibodies with specificity for catalase, α-enolase, and lactoferrin are the most commonly seen. In some cases, such antibodies cross-react with colonic microorganisms.

[0272] In clinical trials, Crohn's disease activity is often scored using the Crohn's Disease Activity Index (CDAI). The CDAI provides a disease activity score based on eight factors, including (1) the number of times liquid or loose stools occur per day, (2) the patient's assessment of the amount of abdominal pain per day, (3) the patient's assessment of overall health, (4) the patient's reporting of other symptoms, including arthritis, iritis, uveitis, erythema nodosum, pyoderma gangrenosum, aphthous stomatitis, anal fissure, fistula or abscess, other fistulas or fever, (5) the patient's reporting of taking romorothil or other sedatives for diarrhea, (6) abdominal mass, (7) hematocrit level, and (8) body weight. See, for example, Best et al. (1976), Gastroenterol. 70:439-444, the relevant portion of which is incorporated herein by reference.

[0273] The symptoms of ulcerative colitis are variable. They may include diarrhea, tenesmus, abdominal cramps, bloody mucus in the stool, fever, and rectal bleeding. Toxic megacolon, a potentially life-threatening condition in which the colon is dilated beyond approximately 6 centimeters and may lose its muscle tone and / or perforation, may also occur. Other conditions that may accompany ulcerative colitis include peripheral arthritis, ankylosing spondylitis, sacroiliitis, anterior uveitis, erythema nodosum, pyoderma gangrenosum, episcleritis, autoimmune hepatitis, primary sclerosing cholangitis, cirrhosis, and growth retardation and developmental delay in children.

[0274] In one embodiment, patients suffering from inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, may be treated before, after, or concurrently with existing therapies for IBD with IL-10 mutant protein, anti-TREM-1 antigen-binding protein, or other antigen-binding proteins or IL-10-containing antigen-binding proteins disclosed herein. Existing treatments for IBD include, for example, sulfasalazine, 5-aminosalicylic acid and its derivatives (such as orsalazine, valsalazid, and mesalamine), anti-TNF antibodies (including infliximab, adalimumab, golimumab, and certolizumab pegol), oral or parenteral corticosteroids (including prednisone, methylprednisolone, budesonide, or hydrocortisone), adrenocorticotropic hormone, antibiotics (including metronidazole, ciprofloxacin, or rifaximin), azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, tacrolimus, and thalidomide.

[0275] In one embodiment, the Disclosure provides a method for inhibiting one or more inflammatory cytokines, such as TNFα, in a mammal requiring such treatment, comprising administering a therapeutically effective amount of the IL-10 mutant protein, antigen-binding protein or antibody, or a fusion protein described herein, to a subject requiring such treatment. In a preferred embodiment, the subject is a mammal. In one embodiment, the subject is a human. The method may be used to treat diseases characterized by increased expression or activity of TNFα. The IL-10 mutant protein, antigen-binding protein or antibody, or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein, may be administered together with another pharmaceutical product, in the same formulation, or separately.

[0276] Rheumatoid arthritis (RA) is a chronic disease characterized by systemic symptoms as well as symptoms particularly related to the joints. Symptoms generally include synovitis resulting in painful and swollen joints, abnormal laboratory findings such as elevated levels of rheumatoid factor, anti-citrulline-modified protein (anti-CCP) antibodies and C-reactive protein (CRP), and elevated erythrocyte sedimentation rate (ESR). Less common symptoms include various extra-articular symptoms, such as those affecting tendons, ligaments, blood vessels, heart, and lungs. Disease activity can often be measured using a variety of indicators. See, for example, Anderson et al. (2012), Arthritis Care & Res. 64(5):640-647 (the portion describing such indicators is incorporated herein by reference). Elements of such scoring indicators include the number of tender joints, the number of swollen joints, functional assessments, and various laboratory findings such as CRP and ESR.

[0277] In one embodiment, a patient suffering from RA may be treated before, after, or concurrently with drug treatment in the current use of RA with an IL-10 mutant protein, an anti-TREM-1 antigen-binding protein or other antigen-binding protein or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as disclosed herein. Therapies currently used for rheumatoid arthritis (RA) include, in particular, nonsteroidal anti-inflammatory drugs (NSAIDs) (such as aspirin and cyclooxygenase-2 (COX-2) inhibitors), disease-modifying anti-inflammatory drugs (DMARDs such as methotrexate, leflunomide, and sulfasalazine), antimalarial drugs (such as hydroxychloroquine), cyclophosphamide, D-nishiramine, azathioprine, gold salts, tumor necrosis factor inhibitors (such as etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol), CD20 inhibitors such as rituximab, IL-1 antagonists such as anakinra, IL-6 inhibitors such as tocilizumab, Janus kinase inhibitors (such as tofacitinib), abatacept, and corticosteroids.

[0278] In either a separate composition or a fusion protein, a combination of an IL-10 mutant protein, an IL-10 mutant protein, and an antigen-binding protein is further conceivable to increase the therapeutic index (TI) of IL-10 as an anti-inflammatory agent. In various embodiments, the TI of an IL-10 mutant protein is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more when administered alone or in combination with the antigen-binding proteins of this disclosure.

[0279] In various embodiments, IL-10 mutant proteins reduce the suppression of TNF-α production in myeloid cells, decrease the level of CD8+ T cell stimulation, and / or decrease the level of B cell stimulation compared to wt IL-10. In various embodiments, IL-10 mutant protein antigen-binding proteins suppress TNF-α production in myeloid cells. In various embodiments, IL-10 mutant protein antigen-binding proteins suppress TNF-α production in myeloid cells while reducing CD8+ T cell and B cell activation.

[0280] In certain embodiments, TNF-α inhibition is measured using isolated PBMCs. PBMCs are isolated from the blood of a subject (human, mouse, rat, cynomolgus monkey, etc.), stimulated in vitro with LPS, and TNFα levels before and after stimulation in the presence of different test molecules are determined, for example, using ELISA ALPHALISA or the MSD Vplex TNFα detection kit.

[0281] In certain embodiments, the measurement of CD8+ T cell and / or B cell activation is performed using a whole blood assay. Whole blood is isolated from the subject and stimulated in vitro in the presence of different test molecules, and the level of CD8+ T cell stimulation or B cell activation is determined, for example, by detecting the pSTAT3 level in the sample using a FACS assay.

[0282] Dosage and Administration The method of this disclosure includes the step of administering a pharmaceutical composition comprising an IL-10 mutant protein, an antigen-binding protein, or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein. In certain embodiments, the pharmaceutical composition is a sterile composition.

[0283] The amount of therapeutic composition in a given dose may vary depending on the size of the individual receiving the treatment and the characteristics of the disease being treated.

[0284] This disclosure provides compositions comprising an IL-10 mutant protein, antigen-binding protein, or antibody as described herein, or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising an IL-10 mutant protein, antigen-binding protein, or antibody as described herein, or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein, may be formulated according to known methods for preparing pharmaceutically useful compositions, thereby combining a therapeutic antibody in a mixture with a pharmaceutically acceptable carrier. A composition is described as containing a “pharmaceutically acceptable carrier” if its administration is tolerable by a recipient patient. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. See, for example, Getman), ed., Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company (1995).

[0285] For pharmaceutical applications, the polypeptides of this disclosure are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration may be performed by bolus infusion, controlled release, for example, using a minipump or other suitable technique, or by infusion over a typical period of one to several hours. Generally, the pharmaceutical formulation will contain the IL-10 mutant protein, antigen-binding protein or antibody of this disclosure, or an antigen-binding protein containing the anti-TREM-1 and IL-10 moieties described herein, in combination with a pharmaceutically acceptable carrier such as saline, buffered saline, or 5% dextrose in water. The formulation may contain one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc. When using such combination therapy, the IL-10 mutant protein, antigen-binding protein or antibody of this disclosure, or an antigen-binding protein containing the anti-TREM-1 and IL-10 moieties described herein, may be combined in a single formulation or administered in separate formulations. Methods of formulation are well known in the art and are disclosed, for example, in Gennaro, ed., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa. (1990), which is incorporated herein by reference. The therapeutic dose is generally in the range of 0.1 to 100 mg / kg patient body weight per day, preferably 0.5 to 20 mg / kg per day, and the exact dose is determined by a clinician in accordance with recognized standards, taking into account the nature and severity of the condition being treated, the characteristics of the patient, etc. Dose determination is within the scope of the skill of the art. More generally, the antibody will be administered over a period of one week or less, often over a period of one to three days. Generally, the dose of antibody administered will vary depending on factors such as the patient's age, weight, height, sex, systemic condition, and medical history. Typically, it is desirable to provide the recipient with an antibody dose in the range of approximately 1 pg / kg to 10 mg / kg (amount of drug / patient body weight), but lower or higher doses may be administered depending on the situation.

[0286] Administration of the IL-10 mutant protein, antigen-binding protein, or antibody of this disclosure, or antigen-binding proteins containing the antigen-binding moiety and IL-10 moiety described herein, to a subject may be performed by intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, local catheter perfusion, or direct intrafocal injection. In various embodiments, administration is intravenous or subcutaneous. When administering the IL-10 mutant protein, antigen-binding protein, or antigen-binding protein containing the antigen-binding moiety and IL-10 moiety by injection, administration may be performed by continuous infusion or by single or multiple bolus administration.

[0287] Further routes of administration include oral, mucosal, pulmonary, and transdermal. Oral delivery is preferred for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid-based systems (see, for example, DiBase et al., “Oral Delivery of Microencapsulated Proteins”, in Sanders et al., eds., Protein Delivery: Physical Systems, pp. 255-288, Plenum Press (1997)). The feasibility of intranasal delivery is exemplified by methods such as insulin administration (see, for example, Hinchcliffe et al., Adv. Drug Deliv. Rev., 35:199 (1999)). Dry or liquid particles containing the antibody of the present invention can be prepared using a dry powder disperser, a liquid aerosol generator, or a sprayer and inhaled (e.g., Pettit et al., TIBTECH, 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev., 35:235 (1999)). This method is exemplified by the AERX® diabetes management system, a portable electronic inhaler that delivers aerosolized insulin to the lungs. Studies have shown that proteins as large as 48,000 kDa were delivered across the skin at therapeutic concentrations using low-frequency ultrasound, demonstrating the feasibility of transdermal administration (Mitragotri et al., Science, 269:850 (1995)).

[0288] For therapeutic purposes, a composition comprising the IL-10 mutant protein, antigen-binding protein, or antibody of this disclosure, or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein, and a pharmaceutically acceptable carrier, is administered to a patient in a therapeutically effective dose. A combination of the IL-10 mutant protein, antigen-binding protein, or antibody of this disclosure, or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein, and a pharmaceutically acceptable carrier is described as being administered in a "therapeutably effective dose" if the amount administered is physiologically significant. A drug is physiologically significant if its presence results in a detectable physiological change in the recipient patient. For example, a drug used to treat inflammation is physiologically significant if its presence reduces the inflammatory response. Effective treatment can be evaluated in various ways. In one embodiment, effective treatment is determined by the reduction of inflammation. In other embodiments, effective treatment is characterized by the inhibition of inflammation. In yet another embodiment, an effective therapy is measured by improvements in the patient's health, including signs such as weight gain, physical recovery, reduced pain, good growth, and subjective indicators of better health from the patient.

[0289] The amount of IL-10 mutant protein in a given dose may vary depending on the size of the individual receiving the treatment and the characteristics of the disease being treated. The dose of IL-10 mutant protein administered is in the range of approximately 0.05 mg / kg to 1 mg / kg or approximately 0.05 to 0.5 mg / kg. In various embodiments, when fused with the TREM-1 antibody described herein, the dose of IL-10 mutant protein is approximately 0.05 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.75 mg / kg or 1.0 mg / kg, or, if indicated as the weight administered, approximately 1.0 μg to 50 μg or approximately 1.0 μg, 3.0 μg, 5.0 μg, 7.5 μg, or 10 μg.

[0290] For example, antigen-binding proteins containing an anti-TREM-1 antibody sequence, or antigen-binding proteins containing both an antigen-binding moiety and an IL-10 moiety, are thought to be administered in doses of 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg or higher. IL-10 mutant proteins or antigen-binding proteins can be formulated at concentrations of 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, 200 mg / ml, or 250 mg / ml. In various embodiments, for example, an antigen-binding protein or fusion protein containing an anti-TREM-1 antibody sequence is administered in doses of approximately 0.05 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1.0 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, or 5 mg / kg.

[0291] The compositions described herein are administered once a week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every six months.

[0292] Pharmaceutical compositions comprising the IL-10 mutant protein, antigen-binding protein, or antibody described herein, or an antigen-binding protein comprising the antigen-binding moiety and IL-10 moiety described herein, may be supplied in liquid, aerosol, or solid form. Liquid forms are exemplified by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-release formulations. The latter form is exemplified by mini osmotic pumps and implants (Bremer et al., Pharm. Biotechnol., 10:239 (1997); Ranade, “Implants in Drug Delivery”, in Ranade et al., eds., Drug Delivery Systems, pp.95-123, CRC Press (1995); Bremer et al., “Protein Delivery with Infusion Pumps”, in Sanders et al., eds., Protein Delivery: Physical Systems, pp.239-254, Plenum Press (1997); Yewey et al., “Delivery of Proteins from a Controlled Release Injectable Implant”, in Sanders et al., eds., Protein Delivery: Physical Systems, pp.93-117, Plenum Press (1997).

[0293] The formulation may also contain two or more active compounds, preferably those with complementary activities that do not adversely affect each other, if required for the specific indication being treated. Alternatively, or in addition, the composition may include agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0294] In one embodiment, the IL-10 mutant protein, antigen-binding protein, or antibody of this disclosure, or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein, is administered in combination therapy, i.e., in combination with other agents, such as therapeutic agents useful for treating conditions or disorders, such as autoimmune disorders and inflammatory diseases. The term “combined” in this context means that the agents are administered simultaneously or sequentially, substantially simultaneously. When administered sequentially, at the start of administration of the second compound, the first of the two compounds is still detectable at an effective concentration at the site of treatment.

[0295] The therapeutic agents disclosed herein may be administered simultaneously in the same formulation. It is further conceivable that the agents may be administered in separate formulations and simultaneously, where simultaneously means that the agents are administered within 30 minutes of each other. It is further conceivable that a second agent may be administered simultaneously.

[0296] In another embodiment, the IL-10 mutant protein is administered before the administration of the antigen-binding protein composition. Pre-administration refers to the administration of the drug within the period from one week before treatment with the other drug to 30 minutes before the administration of the other drug. It is further conceivable that the drug is administered after the administration of the other composition or drug. Subsequent administration means administration from 30 minutes after antibody treatment to one week after antibody administration, for example, at 30 minutes, 1 hour, 2 hours, 4 hours, 1 day, and 2 days. A second one is further conceivable.

[0297] For example, a combination therapy may include one or more IL-10 mutant proteins, antigen-binding proteins or antibodies of the Disclosure, or antigen-binding proteins comprising an antigen-binding moiety and an IL-10 moiety as described herein, which are co-compounded and / or co-administered with one or more further therapeutic agents, such as one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory drugs, metabolic inhibitors, enzyme inhibitors and / or cytotoxic agents or cell growth inhibitors.

[0298] Therapeutic agents used in combination with the IL-10 mutant protein, antigen-binding protein or antibody of this disclosure or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein include agents that inhibit various stages of an inflammatory response. In one embodiment, the IL-10 mutant protein, antigen-binding protein or antibody of this disclosure or an antigen-binding protein comprising an antigen-binding moiety and an IL-10 moiety as described herein may be co-compounded and / or co-administered with one or more further agents such as other cytokines or growth factor antagonists (e.g., soluble receptors, peptide inhibitors, small molecules, ligand fusions); or antibodies or their antigen-binding fragments that bind to other targets (e.g., antibodies that bind to other cytokines or growth factors, their receptors or other cell surface molecules); and anti-inflammatory cytokines or their agonists. Non-limiting examples of agents that may be used in combination with the antibodies described herein include, but are not limited to, one or more interleukin (IL) or receptor antagonists, e.g., antagonists of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-17A-F, IL-18, IL-20, IL-21, IL-22, IL-23, IL-25, IL-31, IL-32, IL-33; cytokines or growth factors or receptor antagonists, e.g., LT, EMAP-II, GM-CSF, FGF, and PDGF. The antibodies of the present invention can also be combined with inhibitors of antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, and CD20 (for example, the CD20 inhibitor rituximab (RITUXAN®), CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands (including CD154 (gp39 or CD40L) or LFA-1 / ICAM-1 and VLA-4 / VCAM-1) (Yusuf-Makagiansar et al., Med.Res.Rev., 22:146-167 (2002)).Examples of antagonists that can be used in combination include the antagonists of IL-1, IL-6, IL-12, TNFα, IL-15, IL-18, IL-20, IL-22, IL-23, and IL-31.

[0299] In other embodiments, one or more IL-10 mutant proteins, antigen-binding proteins, or antigen-binding proteins comprising the antigen-binding moiety and IL-10 moiety of the Disclosure may be co-compounded and / or co-administered with one or more anti-inflammatory agents, immunosuppressants, or metabolic or enzyme inhibitors. Non-limiting examples of agents or inhibitors that may be used in combination with the antibodies described herein include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), e.g., ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such as prednisolone; cytokine-suppressing anti-inflammatory drugs (CSAIDs); nucleotide biosynthesis inhibitors, e.g., purine biosynthesis inhibitors; folic acid antagonists (e.g., methotrexate). Examples include sart (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-glutamic acid); and one or more inhibitors of pyrimidine biosynthesis, such as dihydroorotate dehydrogenase (DHODH) inhibitors. Preferred therapeutic agents for use in combination with one or more antibodies of the present invention, such as bispecific antibodies, include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folic acid antagonists (e.g., methotrexate).

[0300] Further inhibitors include corticosteroids (oral, inhaled, and topical injectable); immunosuppressants, e.g., cyclosporine, tacrolimus (FK-506); and mTOR inhibitors, e.g., sirolimus (rapamycin-RAPAMUNE® or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); agents that interfere with signaling by inflammatory cytokines such as IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib and their variants; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesterase type IV inhibitor); phospholipase inhibitors, e.g., inhibitors of cytoplasmic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone analogs); and blood vessels. Examples include inhibitors of endothelial growth factor or growth factor receptors, such as VEGF inhibitors and / or VEGF-R inhibitors; and one or more inhibitors of angiogenesis. Preferred therapeutic agents for use in combination with the IL-10 mutant protein, antigen-binding protein or antibody of this disclosure or antigen-binding proteins containing anti-TREM-1 and IL-10 moieties as described herein are immunosuppressants, such as cyclosporine and tacrolimus (FK-506); mTOR inhibitors, such as sirolimus (rapamycin) or rapamycin derivatives, such as soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2 inhibitors, such as celecoxib and its variants; and phospholipase inhibitors, such as cytoplasmic phospholipase 2 (cPLA2) inhibitors, such as trifluoromethyl ketone analogs.

[0301] kit This disclosure also envisions a kit comprising one or more containers containing the IL-10 mutant protein, antigen-binding protein, or antibody of this disclosure, or an antigen-binding protein comprising the antigen-binding moiety and IL-10 moiety described herein, in a pharmaceutically acceptable carrier or composition. The IL-10 mutant protein, antigen-binding protein, or antibody of this disclosure, or an antigen-binding protein comprising the antigen-binding moiety and IL-10 moiety described herein, may be provided in the form of an injectable solution for single or multiple doses, either as a unit dose or as a sterile powder to be reconstituted before injection. Alternatively, such a kit may include a dry powder disperser, a liquid aerosol generator, or a sprayer for administering the therapeutic agent. Such a kit may further include instructions and written information regarding indications and use of the pharmaceutical composition.

[0302] Optionally, syringes, e.g., disposable or pre-filled syringes, sterile sealed containers, e.g., vials, bottles, containers and / or kits or packages, containing any of the above-mentioned IL-10 mutant protein, antigen-binding protein, or antigen-binding protein or composition comprising the anti-TREM-1 moiety and the IL-10 moiety, along with suitable instructions for use, are also envisioned.

[0303] In further embodiments, the present invention provides (a) a composition comprising an IL-10 mutant protein, antigen-binding protein or antibody of the present disclosure or an antigen-binding protein comprising an anti-TREM-1 moiety and an IL-10 moiety as described herein; (b) a container comprising the composition; and (c) a product or unit dose form comprising a label affixed to the container or a package insert contained in the container referring to the use of the antibody in the treatment of an immune-related disease.

[0304] In another embodiment, the composition or kit comprises a further active ingredient, which may be, for example, a further antibody or anti-inflammatory agent, a cytotoxic agent or other agent as described herein. Preferably, the composition is sterile. [Examples]

[0305] Example 1: Generation of IL-10 mutant protein This specification hypothesizes that IL-10 mutant proteins, which exhibit different binding to IL-10R1 / R2 and different immunostimulatory activity compared to wild-type IL-10, may be useful as immunotherapies.

[0306] A stable IL-10 monomer, IL-10M1, was created by inserting a 6-amino acid linker (GGGSGG) (SEQ ID NO: 2676) between loops D and E of IL-10 (Josephson et al., J. Biol. Chem, 275:13552-13557, 2000). IL-10M1 was modeled to the structure of hIL-10 / IL-10R1. The 24 residues of hIL-10 are contained within the binding interface, which consists of two interaction surfaces: site Ia - helix F and the AB loops of IL-10 and L2-L4 of IL-10R1, as well as site Ib - N-terminus of helix A and the C-terminus of helix F of IL-10 and L5-L6 of IL-10R1.

[0307] An IL-10 mutagenesis algorithm was constructed based on the structural differences between viral IL-10 (83% homology) and cynomolgus monkey (cyno) IL-10. In fusion proteins, for example when fused to an antibody, IL-10M1 was used as a scaffold for mutant protein design to facilitate proper folding of IL-10, allowing the last two α-helices to fold into the N-terminal globular domain and form functional monomers. A combination of structural and sequence analysis described later was used to facilitate focusing on potential residues for mutation. To narrow the size of the mutant protein panel, ΔΔG binding energy calculations were used to determine the mutations that would have the most destabilizing impact on the receptor interface.

[0308] The algorithm maximizes mutational diversity at site Ib by focusing on residues with side chains facing the interface, using the sequences, mutations, and known structures of IL-10 and IL-10 / IL-10R1. Single point mutations were screened, and Trp, Phe, Pro (with some exceptions), or Cys residues were not mutated. Sequence diversity was maximized by selecting at least one mutation at each residue on the binding surface.

[0309] Further methods include selecting conservative mutations in the CD loop, such as mutating each residue in the AB loop to Gly or Pro to favor the disordered three-dimensional structure, including cleavage into the N-terminal and C-terminal flexible loops, and narrowing the mutation panel using energy calculations (i.e., ΔΔG binding).

[0310] A total of 235 mutations were generated and screened. Mutations in the following residues were examined for binding to IL-10 R1: P20, L23, R24, R27, D28, K34, T35, Q38, M39, K40, D41, Q42, L43, D44, N45, L46, L47, L48, K49, N82, Q83, D84, P85, D86, I87, K138, S141, E142, D144, I145, or E151. N-terminal and C-terminal cleavage were also included in the panel if they altered IL-10 activity. Mutant numbering is based on Sequence ID No. 2, which is wild-type human IL-10 lacking the 18-amino acid signal peptide.

[0311] Mutations generated and selected for further analysis include: AB loop, D41G, L46K; site Ia, Q38E (helix A), Q38R (helix A), K34D (helix A), K138L (helix F), K138D (helix F); site Ib, R27L (helix A). Each of these mutant proteins also contained the GGGSGG (SEQ ID NO: 2676) linker between helices D and E of IL-10. Exemplary sequences are shown in Table 1.

[0312] [Table 1]

[0313] In addition to mutation analysis, linkers of different sizes were generated to be inserted between the D and E helices. Using Rosetta Remodel (Huang et al., PLoS One. 2011;6(8):e24109), the protein structures of IL-10 with different amino acid insertions in the helical structure, including trimers, tetramers, pentamers, and hexamers, were modeled. The GS linker received high marks in the model. The modeled linker sequences are listed in Table 2.

[0314] [Table 2]

[0315] Table 3 shows the inhibition of LPS-induced TNF in 293 cells by selected IL-10 mutant proteins and IL-10 mutant protein fusion with anti-TREM-1 antibody 1B12. The heavy chain of the fusion molecule has the identified sequence; the respective light chain sequences are those of antibody 1B12 (SEQ ID NO: 2185).

[0316] [Table 3]

[0317] The purpose of IL-10 mutant proteins is to maintain / restore IL-10 inhibitory activity in monocytes and macrophages, and to reduce IL-10 stimulation in CD8+ T cells and B cells.

[0318] Example 2 - Anti-TREM-1 antibody It was hypothesized that fusing IL-10 to a target region on the cell surface would be beneficial in extending its half-life and activity. A high-affinity anti-TREM-1 monoclonal antibody was generated, and its ability to target IL-10 to immune cells and extend its half-life was evaluated.

[0319] Fully human antibodies against human TREM-1 were generated by immunizing XENOMOUSE® transgenic mice. See, for example, U.S. Patent Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; and 7,064,244.

[0320] Mice were immunized with human and / or cynomolgus monkey TREM1 protein, TREM1 expression vectors, and / or TREM1-expressing CHO cells. For genetic immunization, mice were immunized 16 times over 8 weeks using the HELIOS® Gene Gun system according to the manufacturer's instructions (BioRad, Hercules, California). Briefly, expression vectors encoding either human TREM-1 and DAP12 or cynomolgus monkey TREM-1 and DAP12 were pooled, 2 μg of total DNA was coated onto 1.6 μm gold beads (BioRad, Hercules, California), and delivered to the epidermis of the shaved abdominal area of ​​mice. For soluble protein immunization, mice were immunized with human or cynomolgus monkey recombinant TREM-1 protein representing the N-terminal extracellular domain. Animals were immunized 14 to 17 times over 10 to 12 weeks using subcutaneous injections delivered at two sites along the dorsal midline of the mouse, located at the base of the tail and in the subscapular region, with recombinant protein adjuvanted with either Alum and CpG-ODN or the Sigma Adjuvant System. The initial soluble protein immunization involved delivery of 10 μg, with subsequent booster doses of 5 μg. For cellular immunization, mice were immunized with 2 to 4 million CHO-S cells transiently expressing either human TREM-1 or cynomolgus monkey TREM-1, adjuvanted with Alum and CpG-ODN. Animals were immunized a total of 13 times over 10 weeks, alternating between intraperitoneal and subcutaneous injections at the base of the tail, either once or twice weekly. Animals were bled, and plasma was collected at various time points during the immunization study, ranging from 4 to 10 weeks, to assess TREM1-specific titer. TREM1-specific plasma titers were monitored using transiently transfected 293T cells by hepatocyte FACS analysis on an ACCURI® flow cytometer (BD Biosciences). Animals with the highest antigen-specific plasma titers for human and cynomolgus monkey TREM1 were euthanized and used for hybridoma generation (Kohler and Milstein, 1975).

[0321] Hybridoma generation: Animals exhibiting suitable antigen-specific serum titers were identified, and spleen and / or inflow region lymph nodes from selected mice were pooled from each sample. Splenocytes and lymphocytes were dissociated from lymphoid tissue by pulverization in suitable culture medium or by using a GENTLEMACS® Dissociator (Miltenyi Biotec) semi-automated tissue dissociation device. IgG-expressing B cells were isolated, grown using standard methods, and fused with suitable cell fusion partners. Hybridoma supernatants were tested for binding to human TREM-1 transiently expressed on HEK293 cells using CELLINSIGHT®. Briefly, HEK293 cells were transiently co-transfected with human TREM-1 and DAP12 or a mammalian expression construct encoding a mock vector and DAP12 alone in a 1:1 ratio using 293Fectin (Invitrogen) according to the manufacturer's protocol. The following day, 15,000 cells / well of transfected HEK293 cells were combined with an equal volume of the hybridoma medium test sample used and 15 μg / mL of final concentration Hoechst 33342 (Pierce) nuclear stain in a 384-well FMAT plate (Corning) at a total volume of 30 μL / well. After incubation at room temperature for 1 hour, the supernatant was aspirated using an AQUAMAX® plate washer, and the wells were washed for two cycles with 50 μL / well of FACS buffer (PBS (Hyclone), 2% FBS (Sigma) in each cycle in AquaMax). The cells were stained with 5 μg / mL of Alexa Fluor 488 goat anti-human IgG Fc (Jackson ImmunoResearch) secondary antibody, shaken in a Big Bear plate shaker, and incubated at room temperature for 20 minutes. The supernatant was aspirated using an AQUAMAX® plate washer, the wells were washed again with 50 μL / well of FACS buffer for two cycles, and 30 μL of FACS buffer was added to each well using a multidrop instrument.The plates were placed in a Big Bear Plate shaker to evenly distribute the cells into the wells, and then read using the Cell Health Profiling Bio-App on the CELLINSIGHT® CX7 platform.

[0322] The TREM-1 specific antibodies identified in the primary screening were evaluated for their cross-reactivity with cynomolgus monkey TREM-1 and their specificity to TREM-1 rather than to DAP12. TREM-1 hybridoma supernatants were tested for binding to transiently expressed human or cynomolgus monkey TREM-1 on HEK293 cells using FACS (samples 1-6) or CELLINSIGHT™ (samples 8-9). For TREM1 antibodies from samples 1-6, HEK293 cells were transiently co-transfected with 293Fectin in a 1:1 ratio with human TREM-1 and DAP12, cynomolgus monkey TREM-1 and DAP12, or a mock vector and a mammalian expression construct encoding human DAP12. The following day, transfected HEK293 cells were transferred to 96-well FACS plates at 50,000 cells / well and incubated with normalized hybridoma supernatant at a final concentration of 2.5 μg / mL at 4°C for 1 hour. Next, the cells were pelleted by centrifugation, the supernatant was removed by flicking, and the wells were washed twice with 200 μL / well FACS buffer. 5 μg / mL of ALEXA FLUOR® 647 goat anti-human IgG Fc (Jackson ImmunoResearch) secondary detection antibody and 2.5 μg / mL of 7-aminoactinomycin-D (Sigma) viability stain were incubated with the cells for 15 minutes at 4°C. The cells were pelleted by centrifugation, the supernatant was removed by flicking, and the wells were washed again with 200 μL / well FACS buffer. TREM-1 hybridoma supernatants exhibiting specific binding to human TREM-1 or cynomolgus monkey TREM-1 were detected by FACS using a BD ACCURI® C6 flow cytometer equipped with an Intellicyt autosampler. Data were reported as geometric mean (GM) multipliers exceeding binding to unrelated control antibodies. Binding results for specific anti-TREM-1 antibodies are shown in Table 4.

[0323] [Table 4]

[0324] High-quality TREM1 antibodies exhibiting TREM1-specific binding and human / cynomolgus monkey cross-reactivity were evaluated for their ability to prevent ligand PGLYRP1 from binding to human TREM1 / DAP12 transiently expressed on HEK293 cells. Briefly, HEK293 cells were transiently co-transfected with 293Fectin in a 1:1 ratio with human TREM1 and DAP12 or a mock vector and a mammalian expression construct encoding human DAP12. The following day, the transfected HEK293 cells were transferred to 96-well FACS plates at 50,000 cells / well and incubated with normalized hybridoma supernatant at a final concentration of 2.5 μg / mL at 4°C for 1 hour. Human PGLYRP1-His (R&D Systems) was combined with PGN-ECndss (peptidoglycan, InvivoGen) and incubated at room temperature for 15 minutes. Then, PGLYRP1 at a final concentration of 7.5 μg / mL and PGN at 30 μg / mL were added to the wells. The plate was then shaken and incubated at 4°C for 15 minutes. The cells were then pelleted by centrifugation, the supernatant was removed by gently flicking, and the wells were washed with 200 μL of FACS buffer. 5 μg / mL of ALEXA FLUOR® 647 human anti-His secondary detection antibody and 2.5 μg / mL of 7-aminoactinomycin-D (Sigma) viability stain were added to the cells, shaken, and incubated at 4°C for 15 minutes. The cells were washed with FACS buffer, pelleted by centrifugation, the supernatant was removed by gently flicking, and the cells were washed again with FACS buffer. Next, cells were run on a BD ACCURI® C6 flow cytometer equipped with an Intellicyt HYPERCYT® autosampler. A total of 518 TREM-1 antibodies exhibited the desired specific PGLYRP1 blocking activity. Receptor-ligand inhibition for selected TREM-1 monoclonal antibodies is summarized in Table 5.

[0325] [Table 5]

[0326] TREM-1 Antibody Relative Affinity Grading by Restriction Antigen Assay: TREM-1 hybridoma supernatants were affinity graded in a panel based on their binding kinetics to soluble TREM-1 in restriction antigen assays using LUMAVIDIN® beads (Luminex) in FACS. Briefly, in-house biotinylated human TREM-1-His antigen (b-huTREM-1-His) was serially diluted in FACS buffer and combined with equivolumes of LUMAVIDIN® beads (uniquely barcoded beads, different for each antigen concentration) to obtain a 5-point 2x serial dilution series starting at a final b-huTREM-1-His antigen concentration of 30 ng / mL. The antigen-bead mixtures were plate-cultured across 3 wells in a 96-well FACS plate and then incubated at room temperature for 30 minutes with protection from light. Next, the beads were pelletized by centrifugation, the supernatant was removed by gently flicking, and the wells were washed twice with 200 μL / well FACS buffer. Then, different beads were resuspended, pooled, and diluted in STABILGUARD® Immunoassay Stabilizer (SurModics) to block nonspecific binding. The normalized TREM-1 hybridoma supernatant was combined with an equal volume of the bead mixture in a FACS plate at a final concentration of 5 μg / mL of test antibody per 0.5 μL of beads / well. The plate was then shaken and incubated overnight at room temperature for approximately 18 hours. Next, the beads were pelletized by centrifugation, the supernatant was removed by gently flicking, and the wells were washed twice with 200 μL / well FACS buffer. ALEXA FLUOR® 488 goat anti-human IgG Fc secondary detection antibody (Jackson ImmunoResearch) was added to a plate at 5 μg / mL, shaken, and incubated at room temperature for 15 minutes while protected from light. The beads were washed with FACS buffer, pelletized by centrifugation, the supernatant was removed by gently flicking, and washed again with FACS buffer.Next, the beads were resuspended and analyzed in a BD ACCURI® C6 flow cytometer equipped with an Intellicyt HYPERCYT® autosampler.

[0327] TREM-1 hybridoma samples showing a signal at least twice as high as the control IgG antibody sample were considered to exhibit a TREM-1 specific binding profile. The antibody binding signal correlates with antibody affinity; the degree of antibody binding to the target antigen TREM-1 correlates with the measured fluorescence intensity, thus allowing for relative comparisons of affinity across the panel. TREM-1 antibodies that showed better binding than benchmark antibody 1B2 in the restriction antigen screen were advanced to light chain screening and human / cynomolgus monkey affinity gap analysis. Table 6 shows antibody binding data for selected TREM-1 antibodies using representative antigen coating concentrations that fell within the linear range of instrumental signal detection.

[0328] [Table 6]

[0329] TREM1 Relative Epitope Binning / Profiling: TREM1 hybridoma supernatants were evaluated by an epitope binning assay (modified antibody-antibody competition assay) using LUMAVIDIN® beads (Luminex) in FACS to determine various relative intrinsic epitope bins in the panel. Briefly, a series of 15 different uniquely barcoded LUMAVIDIN® beads were each combined with an equal volume of in-house biotinylated human TREM-1-His antigen diluted in FACS buffer at a final concentration of 100 ng / mL. The antigen-bead mixtures were plate-cultured across three wells in a 96-well FACS plate and then incubated at room temperature for 30 minutes with protection from light. The beads were then pelletized by centrifugation, the supernatant was removed by flicking, and the wells were washed twice with 200 μL / well FACS buffer. Fifteen different TREM-1 antibodies exhibiting diverse VDJ rearrangements and good quantification, which showed good binding in restriction antigen assays, were selected as reference antibodies for pre-coating the beads. These fifteen antibodies were prepared in FACS buffer at a saturated concentration of 5 μg / mL and incubated with fifteen different LUMAVIDIN® beads at room temperature for 1 hour, protected from light. The beads were pelletized by centrifugation, the supernatant was removed by gently flicking, and the wells were washed three times with 200 μL / well FACS buffer. The different beads were then resuspended, pooled, and diluted in STABILGUARD® Immunoassay Stabilizer (SurModics) to block nonspecific binding. The normalized TREM-1 hybridoma supernatant (test antibody) was combined with an equal volume of the bead mixture in a FACS plate at a final concentration of 5 μg / mL of test antibody per 0.5 μL of beads / well. Next, the plate was shaken and incubated at room temperature for 1 hour while protected from light. The beads were then pelletized by centrifugation, the supernatant was removed by gently flicking, and the wells were washed twice with 200 μL / well of FACS buffer.ALEXA FLUOR® 488 goat anti-human IgG Fc secondary detection antibody (Jackson ImmunoResearch) was added to a plate at 5 μg / mL, shaken, and incubated at room temperature for 15 minutes with protection from light. The beads were washed with FACS buffer, pelletized by centrifugation, the supernatant was removed by light flicking, and washed again with FACS buffer. The beads were then resuspended and run in a BD ACCURI® C6 flow cytometer equipped with an Intellicyt HYPERCYT® autosampler.

[0330] Test antibodies that compete for similar epitopes in the TREM-1 antigen as reference antibodies are prevented from binding, while non-competitive antibodies can bind and generate an additional signal together with the reference antibody. The fully bound antibody is then detected by the secondary antibody. To determine the antibody competition / binding profile of individual test antibodies, the reference-only antibody binding signal was subtracted from the reference-plus test antibody signal for each competition / binding reaction (i.e., across the entire reference antibody set). A summary of relative pitope binning for selected TREM-1 antibodies is shown in Table 7 below.

[0331] [Table 7]

[0332] Determination of the human / cynomolgus monkey affinity gap for TREM1 antibodies: TREM-1 antibodies with unique CDR3 sequences that showed better binding than benchmark antibodies in restriction antigen screening were analyzed for their affinity to human and cynomolgus monkey TREM-1. Binding affinity K D (M), binding rate constant k a (M -1 s -1- ) and the dissociation rate constant k d (s -1The concentration was determined using an OCTET® HTX instrument (Fortebio) in a panel of 114 TREM-1 antibodies. Briefly, TREM-1 hybridoma supernatant normalized to 10 μg / mL in DMEM null medium was prepared by diluting 1:10 in OCTET® assay buffer (10 mM Tris, 0.1% Triton X-100, 150 mM NaCl, 1 mM CaCl2, 0.1 mg / mL BSA, pH 7.6) until a final test concentration of 1 μg / mL was reached. Amine-reactive second-generation AR2G biosensors (Molecular Devices) were pre-incubated in 200 μL of nanopore water at room temperature for at least 10 minutes before use. Next, the AR2G biosensor was activated for 5 minutes in a solution of 20 mM EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (ForteBio) pre-mixed with 10 mM NHS (N-hydroxysulfosuccinimide) (ForteBio) in nanopore water. An in-house generated mouse anti-human Fc monoclonal antibody was bound to the AR2G biosensor at 10 μg / mL in 10 mM sodium acetate buffer at pH 5 for 5 minutes, quenched with 1 M ethanolamine at pH 8.5 for 5 minutes, and then used to capture the antibody from the solution. The TREM-1 test antibody was loaded into the biosensor for 5 minutes, and baseline measurements were taken over 1 minute. Next, recombinant soluble human TREM-1-His protein was bound to the antibody-loaded biosensor in a 3-fold dilution series covering 6 points from 450 nM to 1.85 nM or 150 nM to 0.62 nM. The binding of recombinant human TREM-1 to an antibody-filled sensor was measured for 5 minutes, followed by measurement of dissociation in OCTET® buffer for 10 minutes. Next, the biosensor was regenerated with 10 mM glycine at pH 1.7, and the same TREM-1 antibody was refilled in the same sensor for 5 minutes. The binding and dissociation of cynomolgus monkey TREM-1-His protein were then measured using the same method. The data were referenced using a 0 nM analyte reference sensor.Dynamic analysis was performed using the Geneda Screener software with a 1:1 Langmuir model along with mass transfer. TREM-1 antibodies that met the design objectives and showed a less than 10-fold affinity difference between human and cynomolgus monkey TREM-1 were identified, and their binding affinities are shown in Table 8.

[0333] [Table 8]

[0334] Antibodies were selected based on their ability to bind to human or cynomolgus monkey TREM-1, their lack of binding to TREM2, and their ability to block the binding of PGLYRP1 to TREM-1. Fourteen antibodies were selected for further testing, as listed in Table 9.

[0335] [Table 9]

[0336] Example 3 - Reformatting of anti-TREM-1 antibody Lead anti-TREM-1 antibodies from the XenoMouse® campaign were converted to the IgG1z subtype antibody format by fusing the VL domain of the κ light chain to the CK domain, the VL domain of the λ light chain to the CL domain, and the VH domain to the CH1-CH2-CH3 (221-447) sequence. The CH2 domain of this antibody isotype was engineered for reduced effector function by incorporating the N297G mutation and for improved thermal stability by engineered disulfide bonds (R292C, V302C); this antibody isotype is denoted as IgG1z SEFL2. Lead anti-TREM-1 antibodies were further engineered to remove "hotspots" or residues that were computationally predicted or empirically determined to adversely affect molecular expression, purification, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and / or immunogenicity. Various amino acid mutations in these hotspots were designed based on preservation, covariance, chemical similarity, predictions from structural modeling, and prior knowledge from other antibody engineering campaigns. We designed a small panel of rationally engineered antibodies containing both single mutations and combinations of mutations.

[0337] Recombinant expression constructs for a rationally designed panel of hotspot-manipulated mutants were generated using Golden Gate cloning to assemble: 1) synthetic DNA fragments containing antibody-variable domains, 2) pre-cloned “partial vectors” (R292C, N297G, V302C) containing the required constant domains (i.e., CK or CL, CH1-CH2-CH3 (118-447)), and 3) mammalian expression vector scaffolds. The heavy chain (HC) was assembled into the vector scaffold using a puromycin-selective cassette, and the light chain (LC) was assembled into the vector scaffold using a hygromycin-selective cassette. The HC and LC expression vectors were co-transfected in a 1:1 ratio in CHO-K1 cells with Lipofectamine LTX (Gibco), and a stable pool was generated by passage every 2-3 days in the presence of 10 ug / mL puromycin and 500 ug / mL hygromycin until cell viability was >90% (Vi-CELL BLU, Beckman Coulter). A stable pool was seeded in production medium at a rate of 2e6 viable cells per mL of culture and incubated at 36°C with 5% CO2 for 6 days. The cell supernatant was collected by centrifugation, and the antibodies were purified by magnetic bead affinity chromatography using AmMag® Protein A electromagnetic beads (GenScript) or MAG Sepharose® PrismA (Cytiva). The identity of each mutant was confirmed by intact mass spectrometry. For each mutant, the expression titer in conditioned medium was measured using a Protein A sensor with ForteBio OCTET® (Pall Life Sciences). The percentage of high molecular weight (%HMW) material present after Protein A affinity chromatography was measured by analytical size exclusion chromatography, and the % target protein purity was measured by non-reducing microcapillary electrophoresis (MCE NR) using LabChip GXII (Perkin Elmer). Data for mutants prepared by this process are shown in Table 10.

[0338] Table 10

[0339] Table 11

[0340] Table 12

[0341] Table 13

[0342] Table 14

[0343] A subset of three anti-TREM-1 lead antibodies (30H2(19330), 49A2(19333), and 46H7(19332)) from the XENOMOUSE® campaign were also manipulated by yeast display for improved manufacturability with retained binding to TREM-1. For each antibody, all possible adjacent pairs of residues in all six CDRs were simultaneously mutated to all possible amino acids using degenerate NNK codons to generate libraries. The libraries were presented on the surface of a yeast derivative of BJ5464, where the Fd domain was fused to the N-terminus of the α-aggregin and the LC was not fused to the yeast surface. The efficiency of the display was measured by binding to an ALEXA FLUOR® 647 conjugate anti-Fab antibody. The libraries were sorted using fluorescence-activated cell sorting (FACS) for high binding to biotin-conjugate recombinant TREM-1 ECD) with streptavidin PE as secondary fluorescence. Variable domains present in the sorted binding / display double-positive pool and display-positive pool were amplified using primers specific to the framework 1 (FW1) and HC and LC FW4 domains, and subjected to NGS analysis in Illumina MiSeq for 2 × 300 bp runs. After processing the data by general frequency analysis, mutations were selected, where the ratio of positive binding amino acid frequencies was divided by the positive display amino acid frequencies, and then normalized relative to the parent sequence ratio. Sequences with an enrichment value greater than or equal to the parent sequence were considered beneficial or acceptable diversity and, after affinity maturation, were used for further rational antibody manipulation.

[0344] The top display variable domain was converted to the IgG1z SEFL2 isotype, and cloned using Golden Gate cloning to assemble 1) a synthetic DNA fragment containing the antibody variable domain, 2) a pre-cloned "partial vector" (R292C, N297G, V302C) containing the required constant domains (i.e., CK or CL, CH1-CH2-CH3 (118-447)), and 3) a mammalian expression vector skeleton. The heavy chain (HC) was assembled into the vector skeleton using a puromycin-selective cassette, and the light chain (LC) was assembled into the vector skeleton using a hygromycin-selective cassette. HC and LC expression vectors were co-transfected in a 1:1 ratio in CHO-K1 cells using Lipofectamine LTX (Gibco). A stable pool was generated by subculturing every 2-3 days in the presence of 10 ug / mL puromycin and 500 ug / mL hygromycin until cell viability was >90% (Vi-CELL BLU, Beckman Coulter). The stable pool was seeded into production medium at a rate of 2e6 viable cells per mL of culture and incubated at 36°C with 5% CO2 for 6 days. Antibodies were purified by magnetic bead affinity chromatography using AMMAG® Protein A electromagnetic beads (GenScript). The identity of each molecule was confirmed by intact mass spectrometry. The percentage of high molecular weight (%HMW) material present after protein A affinity chromatography was measured by analytical size exclusion chromatography, and the % target protein purity was measured by non-reducing microcapillary electrophoresis (MCE NR) using LabChip GXII (Perkin Elmer). Data for mutants prepared by yeast display hotspot manipulation are shown in Table 11.

[0345] [Table 15]

[0346] [Table 16]

[0347] [Table 17]

[0348] [Table 18]

[0349] The divalent and monovalent anti-TREM-1 mAbs in HEK293 cells expressing TREM-1 / DAP12 were compared by analyzing the phosphorylation levels of intracellular Syk kinase using p-Syk ALPHALISA® (Perkin-Elmer). Briefly, HEK293 cells stably expressing human TREM1 and DAP12 were cultured at 37°C / 5% CO2 in DMEM / F12 Ham medium (Corning) supplemented with 10% dialyzed FBS (Gibco), 2 mM GlutaMAX (Gibco), 2 mM L-glutamine (Sigma), 1% penicillin / streptomycin (Gibco), and 0.1 mg / mL zeocin (Gibco). One day prior to the experiment, the cells were detached using trypsin-EDTA and centrifuged at 400 × g for 5 minutes. The cell pellet was resuspended in complete medium before a second centrifugation at 400 × g for 5 minutes. After centrifugation, the cells were divided into 1 × 10⁶ cells. 6The cells were resuspended in complete medium at a concentration of 100 μL / well or 50,000 cells / well and seeded onto CELLBIND® 96-well transparent flat-bottom polystyrene plates (Corning) at a final volume of 100 μL / well or 50,000 cells / well. The seeded plates were incubated overnight at 37°C / 5% CO2 for 18–24 hours. On the day of the experiment, 70 μL of medium was taken from each well. The TREM1 antibody was diluted to 3 times the maximum final concentration by a 4-fold serial dilution in assay medium (DMEM / F12HAM medium supplemented with 10% heat-inactivated FBS). The crosslinking reagent Protein G (Sigma) was prepared to 3 times the final concentration using assay medium. The titrated TREM1 antibody was mixed 1:1 with either Protein G or assay medium, and 60 μL of each TREM1 antibody + / - crosslinking reagent was added to each well containing cells. These plates were incubated at room temperature for 1 hour, after which all medium was removed from the wells. The cells were then lysed using 25 μL / well of lysis buffer (M-Per mammalian protein extractant and 1×Halt protease / phosphatase inhibitor). The cells were incubated on ice with lysis buffer for 1 hour, and then 5 μL of the cell lysate was mixed with AlphaLISA® acceptor cocktail (1 nM anti-pSyk, rabbit IgG (anti-phosphoSyk (Tyr525 / 526) (clone C87C1)) (Cell Signaling Technology), 1 nM biotin-anti-Syk, mouse IgG (clone 4D10) (BD Biosciences), 10 μg / mL anti-rabbit IgG) in 1×AlphaLISA® Immunoassay Buffer (PerkinElmer)). AlphaLISA® acceptor beads (PerkinElmer) and 1× Halt inhibitor were transferred to each well of a 384-well white plate (PerkinElmer). The plate was further incubated on ice for 2 hours, then 5 μL of AlphaLISA® donor cocktail (streptavidin-α-donor beads (PerkinElmer) in 1× immunoassay buffer) was added to a final concentration of 40 μg / mL, and incubated in the dark at room temperature for 1 hour.After incubation, the phospho-Syk (pSyk) signal was detected by FRET (fluorescence resonance energy transfer) using an ENVISION® plate reader (PerkinElmer). The results were calculated by the ratio of sample pSyk signal to basal pSyk signal, where potency and maximum signal were compared among the changes for each antibody. Treatment with bivalent anti-TREM-1 mAb alone induced a weak signal in TREM-1 / DAP12 expressing cells. No pSyk signal was observed with monovalent anti-TREM-1 mAb, while bivalent and monovalent anti-TREM-1 mAbs crosslinked with protein G resulted in pSyk induction.

[0350] Antibody Sequence Cladding and Alignment: The VH and VL domains of the input antibody sequence were extracted and aligned to a structure-based IgG numbering system based on Honegger and Pluckthun (J Mol Biol. 309(3):657-70, 2001). Distance matrices were generated from composite multi-sequence alignments using an uncorrected model, where the distance between two sequences is the percentage of mismatch in both the VH and VL domains. Finally, using the distance matrices, a tree was constructed using the UPGMA (Unweighted Joining with Arithmetic Mean) method (Sokal and Michener, University of Kansas Science Bulletin. 38:1409-1438, 1958), and related sequences were grouped based on a branch traversal limit of 0.2. The alignments were manually refined based on amino acid chemical similarity to generate consensus CDR sequences for each cladding group, resulting in the sequences shown in Table 12.

[0351] [Table 19]

[0352] [Table 20]

[0353] Example 4: Generation of IL-10 mutant protein antigen-binding protein The generation and selection of therapeutic fusions of bivalent and monovalent anti-TREM-1 mAb / IL-10 mutant proteins, the PK / PD of the fusions, and the effects of mouse bivalent and monovalent anti-TREM-1 mAb / IL-10 mutant proteins on colitis models will be evaluated.

[0354] IL-10 wild-type, IL-10M1, or IL-10 mutant proteins were fused to the C-terminus of the Fc domain of the anti-TREM-1 antibody. In almost all fusions, the final K residue was removed from the antibody's Fc region (referred to as "desK"). In one example, the linker between the anti-TREM-1 antibody and the IL-10 mutant was GGGGS(G4S)(SEQ ID NO: 2725), and the internal IL-10 linker was GGGSGG(SEQ ID NO: 2676)(G3SG2).

[0355] Preparation of symmetric fusion of anti-TREM-1 Ab fragment with IL-10 mutant protein: Antibody-cytokine fusions were constructed by fused IL-10M1 or an engineered variant of IL-10M1 to the C-terminus of the heavy chain of the anti-TREM-1 IgG1z SEFL2 antibody after removing the final Lys residue (K447-) and adding a 5-amino acid linker with the antibody-cytokine sequence G4S (SEQ ID NO: 2725). Antibody-cytokine recombinant expression constructs were generated using Golden Gate cloning to assemble 1) a synthetic DNA fragment containing the antibody variable domain, 2) a synthetic DNA fragment containing the linker plus the designed cytokine, 3) a pre-cloned "partial vector" (R292C, N297G, V302C) containing the required constant domains (i.e., CK or CL and CH1-CH2-CH3 (118-446)), and 4) a mammalian expression vector scaffold. The fused heavy chain (HC) was assembled into a vector skeleton using a puromycin-selective cassette, and the light chain (LC) was assembled into a vector skeleton using a hygromycin-selective cassette. The fused HC and LC expression vectors were co-transfected in a 1:1 ratio in CHO-K1 cells using LIPOFECTAMINE® LTX (Gibco), and a stable pool was generated by subculturing every 2-3 days in the presence of 10 μg / mL puromycin and 500 ug / mL hygromycin until cell viability was >90% (Vi-CELL BLU, Beckman Coulter). The stable pool was seeded into production medium at a rate of 2e6 viable cells per mL of culture and incubated at 36°C with 5% CO2 for 6 days. The cell supernatant was collected by centrifugation, and the antibodies were purified using an automated, 3-column, tandem chromatography process.

[0356] Preparation of asymmetric fusions of IL-10 mutant proteins with anti-TREM-1 Ab fragments: Asymmetric Fab-HeteroFc-cytokine fusions (aka "monovalent" antibody-cytokine fusions) were prepared by fusing IL-10M1 or a manipulated variant of IL-10M1 to both the C-terminus of the antibody heavy chain and the C-terminus of the huFc(221-446) domain. Both were further manipulated to preferentially heterodimerize by electrostatic steering driven by a reverse-charged mutation ("charge-pair mutation") introduced into the CH3 domain of each chain. Therefore, the Fab-HeteroFc-cytokine fusion was further manipulated to have 1) antibody LC, 2) charge pair mutations E356K and D399K, IL-10M1 or a variant of IL-10M1, the final Lys residue (K447-) was removed, a 5-amino acid linker with the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2725) was added between the antibody and cytokine, and then fused to the C-terminus, IgG1z. The IL-10M1 or IL-10M1 mutant is a three-chain protein containing the huFc(221-446) domain of the IgG1z SEFL2 isotype, which is fused to the C-terminus after further manipulation to have the SEFL2 isotype antibody HC and 3) charge pair mutations K392D, K409D, and K434D (underlined in the following sequences), the final Lys residue (K447-), a 5-amino acid linker with the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2725) (not underlined, bold) is added between the Fc and the cytokine (e.g., IL-10 mutant protein, double underlined): [ka]

[0357] Cell supernatant was collected by centrifugation, and antibody fusions were purified using an automated, 3-column, tandem chromatography process. The antibody fusions were affinity-captured, and immediately afterward, the buffer was replaced by gel filtration using SEPHADEX® G25 resin (Cytiva), followed by further purification by size exclusion using SUPERDEX® 200 Increase resin (Cytiva). The identity of each mutant was confirmed by intact mass spectrometry. For each mutant, the expression titer in conditioned medium was measured using a Protein A sensor with ForteBio OCTET® (Pall Life Sciences). The percentage of high molecular weight (%HMW) material present after size exclusion chromatography (SEC) was measured by analytical SEC, and the % target protein purity was measured by non-reducing microcapillary electrophoresis using LABCHIP® GXII (Perkin Elmer). Aggregation tendency and monomer stability were evaluated by stressing the samples at 40°C for 1 and 2 weeks. Next, stressed and T0 samples were analyzed by analytical SEC to quantify the increase in aggregation (%HMW) and monomer loss at T0 compared to post-stress. The results for selected molecules are shown in Tables 13A and 13B. Bispecific functional activity was measured by inhibiting TNFα production in LPS-stimulated PBMCs.

[0358] [Table 21]

[0359] [Table 22]

[0360] [Table 23]

[0361] [Table 24]

[0362] [Table 25]

[0363] [Table 26]

[0364] [Table 27]

[0365] The first, second, and third melting transitions (Tm1, Tm2, ​​and Tm3, respectively) were measured by differential scanning calorimetry (DSC) using MicroCal(Malvern) in representative subsets of molecules, as shown in Table 14.

[0366] [Table 28]

[0367] Proof-of-Concept (PoC) antibody-cytokine molecules were generated with either a bivalent or monovalent cytokine fusion. The C-terminal IL-10M1 or mutant protein Q38E was fused to a representative anti-TREM1 antibody, 1B12. For all of these PoC molecule morphologies, the cytokine was ligated to the antibody by removing the terminal Lys at the C-terminus of the antibody and adding the Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2725) sequence (as described above). Furthermore, for the PoC molecule morphology with a monovalent cytokine, the Fc portion of the antibody was manipulated to have the same charge-pair mutation described above. The monovalent cytokine was fused to either the positive (E356K and D399K) or negative (K392D, K409D, and K434D) arm of the antibody's heavy chain.

[0368] Fab antibody-cytokine and PoC antibody-cytokine recombinant expression constructs were generated using Golden Gate cloning to assemble the following: 1) a synthetic DNA fragment containing an antibody variable domain, 2) a synthetic DNA fragment containing a linker-plus designed cytokine, 3) a pre-cloned "partial vector" containing the required constant domain (i.e., CK or CL, CH1-CH2-CH3 (118-446) (R292C, N297G, V302C, E356K, D399K), CH2-CH3 (221-446) (R292C, N297G, V302C, K392D, K409D, K434D)), and 4) a mammalian expression vector skeleton. The fused HC and fused Fc domains were assembled into the vector skeleton using a puromycin-selective cassette, and the LC was assembled into the vector skeleton using a hygromycin-selective cassette. Fused HC, fused Fc, and LC expression vectors were co-transfected in a 1:1:1 ratio in CHO-K1 cells using LIPOFECTAMINE® LTX (Gibco). A stable pool was generated by subculturing every 2-3 days in the presence of 10 ug / mL puromycin and 500 ug / mL hygromycin until cell viability was >90% (Vi-CELL BLU®, Beckman Coulter). The stable pool was seeded into production medium at a rate of 2e6 viable cells per mL of culture and incubated at 36°C with 5% CO2 for 6 days.

[0369] Cell supernatant was collected by centrifugation, and antibody fusions were purified using an automated, 3-column, tandem chromatography process. Fab-HeteroFc-cytokine fusions were affinity-captured using CAPTURESELECT® CH1 XL (ThermoFisher), and immediately afterward, the buffer was replaced by gel filtration using SEPHADEX® G25 resin (Cytiva), followed by further purification by size exclusion using SUPERDEX® 200 Increase resin (Cytiva). The identity of each mutant was confirmed by intact mass spectrometry. For each mutant, the expression titer in conditioned medium was measured using a Protein A sensor with ForteBio OCTET® (Pall Life Sciences). The percentage of high molecular weight (%HMW) material present after Protein A affinity chromatography was measured by analytical size exclusion chromatography (SEC), and the % target protein purity was measured by non-reducing microcapillary electrophoresis using LABCHIP® GXII (Perkin Elmer). Aggregation tendency and monomer stability were evaluated by stressing samples at 40°C for 1 and 2 weeks. Next, stressed and T0 samples were analyzed by analytical SEC to quantify the increase in aggregation (%HMW) and monomer loss after stress compared to T0. Bispecific functional activity was measured by inhibiting TNFα production in LPS-stimulated PBMCs, and the results for selected molecules are shown in Table 15.

[0370] [Table 29]

[0371] Monovalent and bivalent antibodies prepared as described above and fused to the IL-10 mutant protein Q38E were tested for monocyte activation. Briefly, monocytes were isolated from frozen PBMCs using the Miltenyi Monocyte Isolation Kit II. Purified monocytes were placed in a 96-round-well TC-treated plate at a rate of 10 per well. 5Cells were cultured on plates in complete medium. The indicated test proteins were serially diluted 1:5 from 11000 pM to 0.005 pM. LPS was added to the cells at a final concentration of 10 ng / mL. The plates were incubated overnight at 37°C in 5.0% CO2. TNFα in the cell supernatant was measured using the MSD Vplex Human TNFα Detection Kit. Figure 2 shows that bivalent anti-TREM-1 is more potent than monovalent anti-TREM-1, and that bivalent IL-10 mutant protein is more potent than monovalent IL-10 mutant protein at its C-terminus.

[0372] The effects of IL-10, IL-10 mutant proteins, and IL-10 / anti-TREM-1 antigen-binding proteins on immune cell activity were evaluated using a whole blood assay. Fresh heparinized human whole blood obtained from healthy, untreated donors was divided into 96-well 2.0 mL deep-well plates. The blood-containing plates were equilibrated at 37°C for at least 30 minutes before adding the IL10 reagents. The indicated test reagents were serially diluted and added to the whole blood. The plates were incubated at 37°C for 30 minutes. The plates were then placed on ice, and staining antibodies against CD3 (clone UCHT-1) and CD4 (clone RPA-T4) were added for a 10-minute incubation. The cells were then treated with 1.4 mL of BD fix / lysis buffer and incubated at 37°C for 20 minutes. The cells were washed with 2% FCS / PBS until the lysed RBCs were clear. To permeabilize the cells, 200 μL of ice-cold BD Perm buffer III was added to each well and mixed thoroughly. After five washes, CD20 (clone H1), CD14 (clone RM052), and pSTAT3 pY705 (clone 4 / P-STAT3) were added to the cells. Covered with aluminum foil to protect from light, the cells were incubated in a plate shaker at 200-300 RPM for 1 hour at ambient temperature. Unbound antibodies were immediately removed by washing twice with 2% FCS / PBS. The cells were resuspended in 200 μL of 2% FCS / PBS wash buffer and subjected to FACS analysis using a BD LSRII flow cytometer. IL-10 alone was found to be CD8+.+ While it induced T cell and B cell proliferation, it did not induce the increased CD8+ T cell and B cell activation induced by IL-10 mutant protein or anti-TREM-1 / IL-10 mutant protein, demonstrating the proof-of-concept of cell-type-specific targeting effects of anti-TREM-1 mAb / IL-10 mutant protein (Figure 3).

[0373] The inhibition of LPS-induced TNFα production from human monocytes was evaluated. Monocytes were isolated from frozen PBMCs using the Miltenyi Monocyte Isolation Kit II. Purified monocytes were placed in a 96-round-well TC-treated plate at a rate of 10 per well. 5 Cells were cultured on plates in complete medium. Shown test proteins containing various IL-10 mutant proteins were serially diluted 1:5 from 11000 pM to 0.005 pM. LPS was added to the cells at a final concentration of 10 ng / mL. The plates were incubated overnight at 37°C in 5.0% CO2. TNFα in the cell supernatant was measured using the MSD Vplex Human TNFα Detection Kit. Figures 4A–4H demonstrate that antigen-binding proteins containing different IL-10 mutant proteins were more potent in reducing TNFα in monocytes compared to cultures containing IL-10 mutant proteins alone, suggesting that TREM-1 binding significantly enhanced the suppression of IL-10 mutant proteins in monocyte activation.

[0374] To evaluate the inhibition of LPS-induced human TNFα production in human monocytes, monocytes were isolated from frozen PBMCs using the Miltenyi Monocyte Isolation Kit II. Purified monocytes were then placed in a 96-round-well TC-treated plate at a rate of 10 per well. 5Cells were cultured on plates in complete medium. The indicated test proteins were serially diluted 1:5 from 11000 pM to 0.005 pM. LPS was added to the cells at a final concentration of 10 ng / mL. The plates were incubated overnight at 37°C in 5.0% CO2. TNFα in the cell supernatant was measured using the MSD Vplex Human TNFα Detection Kit. To evaluate the inhibition of LPS-induced mouse TNFα production, mouse bone marrow was isolated from the femur of C57Bl / 6 mice. Cells were placed in 96-well TC-treated plates, 10 cells per well. 5 Individual cells were cultured on plates in complete culture medium. The indicated test reagents were serially diluted 1:5 from 11000 pM to 0.005 pM. LPS was added to the cells at a final concentration of 10 ng / mL. The plates were incubated overnight at 37°C in 5.0% CO2. Mouse TNFα in the supernatant was measured using the MSD Vplex mouse TNFα detection kit.

[0375] Figure 5 shows a significant improvement in the efficacy of the IL-10 mutant protein in inhibiting TNFα in human monocytes upon TREM-1 binding (Figure 5A). The improvement in TNF inhibition in monocytes is dependent on TREM-1 binding, which is because it has little efficacy against mouse monocytes due to a lack of cross-reactivity with mouse TREM-1 (Figure 5B).

[0376] These results demonstrate that IL-10 mutant proteins and antigen-binding proteins containing immune cell targeting moieties, such as anti-TREM-1 antibodies, can modulate immune cells differently compared to wild-type IL-10. The antigen-binding proteins significantly inhibit TNF-α production from bone marrow cells. The antigen-binding proteins do not stimulate CD8+ T cells and B cells. IL-10 mutant protein antigen-binding proteins can suppress inflammation without stimulating immune cells and therefore may provide therapeutic effects in treating inflammatory bowel disease.

[0377] Example 5 - Stability operation of IL-10M1 To manipulate the yeast display stability of IL-10M1 (SEQ ID NO: 2211) using an unbiased method, an error-prone PCR-based library of IL-10M1 was generated under low mutagenic conditions using the GENEMORPH® II kit and presented on yeast (IL10M1-EP Library). The library was presented on the surface of a yeast derivative of BJ5464, where IL-10M1 was fused to the N-terminus of an α-aggregin. Display efficiency was measured by binding to an ALEXAFLUOR® 647 conjugate anti-HA antibody. The library was sorted using fluorescence-activated cell sorting (FACS) with high binding to biotin-conjugate recombinant IL-10R-Fc using streptavidin PE as secondary fluorescence.

[0378] To identify mutants that functioned better than wild-type IL-10M1 under stress conditions, various stresses were applied to this error-prone yeast library. As a stress for protein expression, the IL-10M1-EP library in yeast cells was induced to present IL-10M1 mutants at 20 or 30°C. To identify mutants exhibiting higher tolerance to this stress, mutants that bound well to IL-10R-Fc and were well presented on the yeast surface (assessed by HA epitope tagging) were sorted by FACS. IL-10M1 sequences from these increased binding pools were amplified along with the initial IL-10M1 EP Library pool using primers outside the IL-10M1 coding sequence. The amplicons were then processed using the Nextera Library Preparation Kit and subjected to NGS analysis in Illumina MiSeq for 2 × 300 bp runs. Mutant sequences that were significantly increased or decreased in the binder pool after 30C-induced stress were selected for recombinant generation, and their stability was evaluated to assess the predictive power of the method (Table 15).

[0379] To identify mutants that can fold better and are less exposed to proteolysis, restricted proteolysis experiments were performed. First, the IL-10M1 error-prone library was induced at 20°C and sorted by FACS to remove non-IL10R binders. This yielded the IL-10M1 EP binder library. Next, the IL-10M1 EP binder library was induced at 20°C and subjected to restricted amounts of trypsin, chymotrypsin, and thermolysin. To identify mutants exhibiting higher resistance to these proteases, mutants bound to IL-10R-Fc and presented better on the yeast surface than the parent IL-10M1 molecule were sorted by FACS and analyzed by NGS as described above. Mutant sequences that were significantly increased or decreased in the protease resistance pool (e.g., increased protein stress, increased temperature stress, increased temperature and protease stress, decreased protease stress, or increased temperature stress) were selected for recombinant generation and their stability was evaluated to assess the predictive power of the method (Table 16).

[0380] [Table 30]

[0381] [Table 31]

[0382] [Table 32]

[0383] Rosetta Stability Manipulation: Stability manipulation was also investigated by computer design. Structural modeling of the IL10M1 mutant protein was performed using RosettaScripts and the Talaris2014 scoring function. Using the "fastrelax" protocol, five cycles of backbone minimization and rotational isomer optimization were performed to local energy minimums for the monomer IL-10M1 model derived from the PDB 1Y6K crystal structure. After relaxation, residues 21-161 were individually screened and computer-mutated to each of the 20 standard amino acids. This was followed by 50 Monte Carlo-based pseudo-annealing steps for the peptide backbone and surrounding residues. All selected designs were also remodeled and scored in the background of L46K decay mutation to confirm that the predicted behavior was unaffected. Table 17 shows the sequence numbers of the IL10 mutant protein fusion with the heavy chain of antibody 61B12; the light chain in each is the same as in antibody 61B12 (sequence number 105, however, lacking the signal sequence).

[0384] [Table 33]

[0385] [Table 34]

[0386] To select a design with improved stability and surface properties, the final models were ranked relative to each other using Talaris2014 scores and hydrophobic solvent-exposed surface area (hSASA). Fifteen high-scoring designs with lower energy and reduced hSASA were ultimately selected for experimental evaluation (Table 18).

[0387] [Table 35]

[0388] An alternative rational design approach investigated the introduction of disulfides to stabilize the forced monomer structure of IL10M1. In this method, residues 21–123 in the template model were screened for their proximity to residues 124–165. Residue pairs were computer-mutated to cysteine, and disulfides were formed if the Cβ atoms were within 6.5 Å of each other. These designs were graded relative to each other using the Talaris2014 score and unweighted disulfide latent conditions. Therefore, five bridge designs with low energy were selected for experimental evaluation (Table 19).

[0389] [Table 36]

[0390] Production and testing as bispecific fusion: Mutant protein variants designed for increased stability were cloned as bivalent C-terminal fusions to anti-TREM1 antibody 61B2.001, recombinantly expressed in CHO cells, and purified as described in Example 3. The identity of each variant was confirmed by intact mass spectrometry, and each was concentrated to 10 ± 1 mg / mL using Slide-A-Lyzer® G2 Dialysis Cassettes, 20K MWCO, 3 mL (ThermoFisher cat.#87735). Expression titers in conditioned medium were measured using a Protein A sensor with ForteBio OCTET® (Pall Life Sciences). The percentage of high molecular weight (%HMW) material present was measured by analytical size exclusion chromatography (SEC), and the % target protein purity was measured by non-reducing microcapillary electrophoresis using LABCHIP® GXII (Perkin Elmer). Aggregation tendency and monomer stability were evaluated by stressing the samples at 40°C for two weeks. Next, the stressed and T0 samples were analyzed by analytical SEC to quantify the increase in aggregation (%HMW) and monomer loss after stress compared to T0. Bispecific functional activity was measured by inhibition of TNFα production in LPS-stimulated PBMCs in a subset of molecules; the results for selected molecules are shown in Table 20.

[0391] [Table 37]

[0392] [Table 38]

[0393] [Table 39]

[0394] [Table 40]

[0395] These results indicate that a subset of stable mutations successfully reduced the degree of aggregation of anti-TREM1 antibody-IL10M1 fusion / antigen-binding protein variants during purification and after 2 weeks of stress at 40°C at moderately high concentrations (10 mg / mL).

[0396] The best stability mutations were combined with mutations designed to correct a deamidation hotspot at the first position of the GGSGG linker (hereinafter referred to as linker [G1]) between the first and second domains of IL10M1 at positions N10, N116, and / or 1. Novel IL10M1 variants were embedded at three different positions: N-terminus, C-terminus, or between Fab and Fc, and fused bivalently to the anti-TREM1 antibodies 63F8.001 and 64D7.001 heavy chains.

[0397] [Table 41]

[0398] The C-terminal IgG-cytokine fusion was expressed in CHO cells, purified, concentrated to 10 mg / mL, and assayed as described in Example 4.

[0399] N-terminal cytokine-IgG fusions were constructed by fusing IL-10M1 or a modified variant of IL-10M1, followed by a 10-amino acid linker with the sequence GGGSGGGS (SEQ ID NO: 2676), to the N-terminus of the heavy chain of anti-TREM-1 IgG1z SEFL2. Antibody-cytokine recombinant expression constructs were generated using Golden Gate cloning to assemble 1) a synthetic DNA fragment containing the antibody variable domain, 2) a synthetic DNA fragment containing the designed cytokine plus linker, 3) a pre-cloned "partial vector" containing the required constant domain, and 4) a mammalian expression vector skeleton. The fused heavy chain (HC) was assembled into the vector skeleton using a puromycin-selective cassette, and the light chain (LC) was assembled into the vector skeleton using a hygromycin-selective cassette. The constructs were expressed in CHO cells, purified, concentrated to 10 mg / mL, and assayed as described in Example 4.

[0400] The embedded Fab-cytokine-Fc molecule was constructed by fusing IL-10M1 or a manipulated variant of IL10M1, along with a lead linker and subsequent linker of sequence GGGG (SEQ ID NO: 2677), into the hinge region of the anti-TREM-1 IgG1z SEFL2 antibody between amino acids C282 and D283 (i.e., EPSKC-GGGG-IL10M1-GGGG-DKTHC (SEQ ID NO: 2724)). The Fab-cytokine-Fc recombinant expression construct was generated using Golden Gate cloning to assemble 1) a synthetic DNA fragment containing the antibody variable domain, 2) a synthetic DNA fragment containing the designed cytokine plus linker, 3) a pre-cloned “partial vector” containing the required constant domain, and 4) a mammalian expression vector skeleton. The fused heavy chain (HC) was assembled into the vector skeleton using a puromycin-selective cassette, and the light chain (LC) was assembled into the vector skeleton using a hygromycin-selective cassette. The construct was expressed in CHO cells, purified, concentrated to 10 mg / mL, and assayed as described in Example 4.

[0401] [Table 42]

[0402] [Table 43]

[0403] The heavy chains in Table 22 were paired with their respective light chain partners, for example, for clone 63F8.001, with SEQ ID NO: 976 or SEQ ID NO: 2554 (but lacking the signal sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 2674)), and for 64D7.001, with SEQ ID NO: 992 or SEQ ID NO: 2555 (but lacking the signal sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 2674)). After fixing the deamidation sites and subjecting them to moderately high concentrations (10 mg / mL) and 10 days of stress at 40°C, several combinations of IL-10M1 mutations were identified that retained low aggregation.

[0404] [Table 44]

[0405] The affinity of selected fusion proteins to human TREM1 and cynomolgus monkey TREM1 was measured by BIACORE® analysis (BIACORE® T200, Sensor Chip: SCM5). BIACORE® conditions were: Sample: IL10 / TREM1 titration in the range of 0.244 nM to 500 nM; Binding: 3 minutes at 50 μl / min; Dissociation: 15 minutes for IL 10 and 30 minutes for TREM1 at 50 μl / min; Regeneration: 30 μl / min (twice) over 30 seconds, containing 10 mM glycine, pH 1.5.

[0406] Affinity was measured for huIL10R α protein #274 from cell-stable transfected Sf 21 cells and huIL10R α protein #9100 expressed in human HEK293 cells. The results are shown in Table ...

Claims

1. A human interleukin-10 (IL-10) mutant protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence described in Sequence ID No. 2, wherein the human interleukin-10 (IL-10) mutant protein has at least one mutation selected from mutations in helix loop AB, helix loop CD, helix loop DE, helix A, helix B, helix C, helix D, helix E and / or helix F.

2. The aforementioned mutations optionally include the addition of six amino acids between helix D and helix E, as shown in Sequence ID No. 2: R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, I87A, H14Q, F15Y, M22V, K49T, K49S, F56Y, K57N, Y59T, L60Q, Q63E, Q63L, E67C, Q70E, Q70K, M7 The IL-10 mutant protein according to claim 2, which is one or more of 7R, M77V, Q79R, Q79C, D84R, A89P, H90E, H90Q, S93E, S93Q, T100R, L103E, H109D, R110P, R110Q, L112V, E115K, N116D, A127M, K130Q, I136C, Y137C, M154V, M156C, K157N, or N160D.

3. Sequence numbers 3-10 or sequence numbers 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2360, 2362, 2364, 2366, 2368, 2370, 2 372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2 436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2500, 2 An IL-10 mutant protein according to claim 1 or 2, having an amino acid sequence described in any one of 502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, or 2777 to 2791.

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the IL-10 mutant protein according to any one of claims 1 to 3.

5. An isolated antigen-binding protein, a. An antibody or antibody fragment; b. Binding to human TREM-1 having the amino acid sequence described in Sequence ID No. 20; c. Light chain variable domain, i. Light chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290 and 2190; ii. Light chain CDR2 containing an amino acid sequence selected from SEQ ID NOs: 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271, 291 and 2191; iii. Light chain CDR3 containing an amino acid sequence selected from SEQ ID NOs: 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292 and 2192 Includes a light chain variable domain that includes; and d. A heavy chain variable domain, i. Heavy chain CDR1 containing an amino acid sequence selected from SEQ ID NOs: 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276, 296 and 2196; ii. Heavy chain CDR2 containing an amino acid sequence selected from SEQ ID NOs: 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277, 297 and 2197; and iii. Heavy chain CDR3 containing an amino acid sequence selected from SEQ ID NOs: 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298 and 2198 An isolated antigen-binding protein containing a heavy chain variable domain.

6. i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 90 (LCDR1), 91 (LCDR2), 92 (LCDR3), 96 (HCDR1), 97 (HCDR2), and 98 (HCDR3); v) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); vi) Sequence IDs 130 (LCDR1), 131 (LCDR2), 132 (LCDR3), 136 (HCDR1), 137 (HCDR2), and 138 (HCDR3); vii) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); viiii) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); ix) Sequence IDs 190 (LCDR1), 191 (LCDR2), 192 (LCDR3), 196 (HCDR1), 197 (HCDR2), and 198 (HCDR3); x) Sequence IDs 210 (LCDR1), 211 (LCDR2), 212 (LCDR3), 216 (HCDR1), 217 (HCDR2), and 218 (HCDR3); xi) Sequence IDs 230 (LCDR1), 231 (LCDR2), 232 (LCDR3), 236 (HCDR1), 237 (HCDR2), and 238 (HCDR3); xi) Sequence IDs 250 (LCDR1), 251 (LCDR2), 252 (LCDR3), 256 (HCDR1), 257 (HCDR2), and 258 (HCDR3); xiiii) Sequence IDs 270 (LCDR1), 271 (LCDR2), 272 (LCDR3), 276 (HCDR1), 277 (HCDR2), and 278 (HCDR3); xiv) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3); or xv) Sequence IDs 2190 (LCDR1), 2191 (LCDR2), 2192 (LCDR3), 2196 (HCDR1), 2197 (HCDR2), and 2198 (HCDR3) The antigen-binding protein according to claim 5, comprising a set of CDR amino acid sequences selected from.

7. a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301 and 2185; or iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299 and 2183. A light chain variable domain comprising an amino acid sequence selected from the group consisting of; and b. A heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186; ii. A sequence encoded by a polynucleotide sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302 and 2186; or iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: The antigen-binding protein according to claim 5 or 6, comprising:

8. i) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 41 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 42; ii) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 61 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 62; iii) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 81 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 82; iv) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 101 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 102; v) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 121 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 122; vi) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 141 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 142; vii) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 161 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 162; viiii) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 181 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 182; ix) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 201 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 202; x) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 221 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 222; xi) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 241 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 242; xi) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 261 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 262; xiiii) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 281 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 282; xiv) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 301 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 302; or xv) Amino acid sequences that are at least 90% identical to the light chain variable domain described in SEQ ID NO: 2185 and amino acid sequences that are at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 2186 An antigen-binding protein according to any one of claims 5 to 7, comprising:

9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain according to any one of claims 5 to 8, a light chain according to any one of claims 5 to 8, or both.

10. An antigen-binding protein comprising an antigen-binding moiety and one or two IL-10 moieties, a. The antigen-binding portion is an antibody or antibody fragment, b. Each IL-10 portion is independently monovalent or divalent. c. Each IL-10 moiety was independently selected from one or more human IL-10 mutant proteins having a sequence that is 90% identical to SEQ ID NO:

2. d. An antigen-binding protein in which at least one IL-10 moiety is covalently bound to the antigen-binding moiety.

11. (a) A polypeptide sequence having formula A-L-M or M-L-A, i) A is the immunoglobulin heavy chain of the IgG antibody that binds to the TREM-1 protein as described in SEQ ID NO: 20, ii) L is a linker peptide containing 4 to 20 amino acids, and iii) M is a polypeptide sequence which is a mutant protein of IL-10 having at least 90% sequence identity with wt IL-10 described in Sequence ID No. 2; and (b) Immunoglobulin light chain of IgG antibody that binds to the TREM-1 protein as described in Sequence ID No. 20 An antigen-binding protein containing, The immunoglobulin heavy chain of (a) and the immunoglobulin light chain of (b) form an IgG antibody moiety that binds to TREM-1, and the protein comprises one or two molecules of the polypeptide of (a) and one or two molecules of the light chain of (b), and optionally, the single polypeptide of (a) comprises an M moiety, wherein the antigen-binding protein.

12. a. Each IL-10 portion contains an amino acid sequence that is at least 90% identical to the amino acid sequence described in Sequence ID No. 2, and b. The antigen-binding protein according to claim 10 or 11, wherein each IL-10 moiety independently comprises at least one mutation selected from mutations in helix loop AB, helix loop CD, helix loop DE, helix A, helix B, helix C, helix D, helix E and / or helix F.

13. Each IL-10 section operates independently. i) Residues of Sequence ID No. 2: N10, H14, F15, P20, M22, L23, R24, R27, D28, K34, T35, Q38, M39, K40, D41, Q42, L43, D44, N45, L46, L47, L48, K49, F56, K57, Y59, L60, Q63, E67, Q70, M77, Q79, N82, Q83, D84, P85, D86, I87, A89 Mutations in one or more of H90, S93, T100, L103, H109, ​​R110, L112, E115, N116, A127, K130, I136, Y137, K138, S141, E142, D144, I145, E151, M154, M156, K157, or N160, or the addition of 4 to 8 amino acids between helix D and helix E; and / or ii) The following sequences of Sequence ID No. 2 optionally include the addition of six amino acids between helix D and helix E: N10Q, N10I, N10K, R27L, K34D, D41G, L46K, Q38E, Q38R, Q38D, K138L, K138D, I87A, H14Q, F15Y, M22V, K49T, K49S, F56Y, K57N, Y59T, L60Q, Q63E, Q63L, E67C, Q70E, One or more mutations selected from the group consisting of Q70K, M77R, M77V, Q79R, Q79C, D84R, A89P, H90E, H90Q, S93E, S93Q, T100R, L103E, H109D, R110P, R110Q, L112V, E115K, N116D, N110Q, A127M, K130Q, I136C, Y137C, M154V, M156C, K157N, or N160D. An antigen-binding protein according to any one of claims 10 to 12, comprising:

14. The IL-10 portion is sequence numbers 3 to 10 or sequence numbers 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2360, 2362, 2364, 2366, 236 8, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 24 34, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2500, 2 An antigen-binding protein according to any one of claims 10 to 13, having an amino acid sequence described in any one of 502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, or 2777 to 2791.

15. Sequence numbers 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 9 29, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997 An antigen-binding protein according to any one of claims 10 to 14, having a heavy chain amino acid sequence described in any one of 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, and 1085.

16. Sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 9 30, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998 ,1000,1002,1004,1006,1008,1010,1012,1014,1016,1018,1020,1022,1024,1026,1028,1030,1032,1034,1036,1038,1040,1042,1044,1046,1048,1050,1052,10 An antigen-binding protein according to any one of claims 10 to 15, having a light chain amino acid sequence described in any one of 54, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, and 1086.

17. Sequence numbers 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 9 23, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 98 5, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, Any of 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, and 1085 The heavy chain amino acid sequence described in one document and sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976 ,978,980,982,984,986,988,990,992,994,996,998,1000,1002,1004,1006,1008,1010,1012,1014,1016,1018,1020,1022,1024,1026,1028,1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080,An antigen-binding protein according to any one of claims 10 to 16, having the corresponding light chain amino acid sequences described in 1082, 1084, and 1086.

18. Sequence numbers 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 195 An antigen-binding protein according to any one of claims 10 to 14, having a heavy chain amino acid sequence described in any one of 5, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005, and 2007.

19. Sequence numbers 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579 An antigen-binding protein according to any one of claims 10 to 14 or 18, having a light chain amino acid sequence described in any one of 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605.

20. Sequence numbers 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1 The heavy chain amino acid sequence described in any one of 953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005 and 2007, and sequence numbers 2543, 2544, 2545 ,2546,2547,2548,2549,2550,2551,2552,2553,2554,2555,2556,2557,2558,2559,2560,2561,2562,2563,2564,2565,2566,2567,2568,2569,2570,2571,2572,2573,2574,2575,2576,2577,2578,2579,2580,2581, An antigen-binding protein according to any one of claims 10 to 14 or 18 or 19, having the corresponding light chain amino acid sequence described in 2582, 2583, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605.

21. Sequence numbers 2011, 2013, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 208 An antigen-binding protein according to any one of claims 10 to 14, having a heavy chain amino acid sequence described in any one of 3, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131, 2133, and 2135.

22. Sequence numbers 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, An antigen-binding protein according to any one of claims 10 to 14 or 21, having a light chain amino acid sequence described in any one of 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, and 2357.

23. Sequence numbers 2011, 2013, 2015, 2017, 2019, 2021, 2026, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 20 The heavy chain amino acid sequence described in any one of 81, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131, 2133 and 2135, and sequence numbers 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2 An antigen-binding protein according to any one of claims 10 to 14 or 21 or 22, having the corresponding light chain amino acid sequences described in 311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355 and 2357.

24. The aforementioned antigen-binding portion is a. Light chain variable domain, i. A light chain CDR1 sequence containing an amino acid sequence selected from SEQ ID NOs: 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290, and 2190; ii. A light chain CDR2 sequence containing an amino acid sequence selected from SEQ ID NOs: 31, 51, 71, 91, 111, 131, 151, 171, 191, 211, 231, 251, 271, 291 and 2190; iii. Light chain CDR3 sequence containing an amino acid sequence selected from SEQ ID NOs: 32, 52, 72, 92, 112, 132, 152, 172, 192, 212, 232, 252, 272, 292 and 2192 Light chain variable domains including; and b. A heavy chain variable domain, i. A heavy chain CDR1 sequence containing an amino acid sequence selected from SEQ ID NOs: 36, 56, 76, 96, 116, 136, 156, 176, 196, 216, 236, 256, 276, 296, and 2196; ii. Heavy chain CDR2 sequences containing amino acid sequences selected from SEQ ID NOs: 37, 57, 77, 97, 117, 137, 157, 177, 197, 217, 237, 257, 277, 297 and 2197; and iii. Heavy chain CDR3 sequence containing an amino acid sequence selected from SEQ ID NOs: 38, 58, 78, 98, 118, 138, 158, 178, 198, 218, 238, 258, 278, 298 and 2198 Heavy chain variable domains including The antigen-binding protein according to any one of claims 10 to 23, comprising an anti-TREM-1 antigen-binding moiety.

25. The aforementioned antigen-binding portion is i) Sequence IDs 30 (LCDR1), 31 (LCDR2), 32 (LCDR3), 36 (HCDR1), 37 (HCDR2), and 38 (HCDR3); ii) Sequence IDs 50 (LCDR1), 51 (LCDR2), 52 (LCDR3), 56 (HCDR1), 57 (HCDR2), and 58 (HCDR3); iii) Sequence IDs 70 (LCDR1), 71 (LCDR2), 72 (LCDR3), 76 (HCDR1), 77 (HCDR2), and 78 (HCDR3); iv) Sequence IDs 90 (LCDR1), 91 (LCDR2), 92 (LCDR3), 96 (HCDR1), 97 (HCDR2), and 98 (HCDR3); v) Sequence IDs 110 (LCDR1), 111 (LCDR2), 112 (LCDR3), 116 (HCDR1), 117 (HCDR2), and 118 (HCDR3); vi) Sequence IDs 130 (LCDR1), 131 (LCDR2), 132 (LCDR3), 136 (HCDR1), 137 (HCDR2), and 138 (HCDR3); vii) Sequence IDs 150 (LCDR1), 151 (LCDR2), 152 (LCDR3), 156 (HCDR1), 157 (HCDR2), and 158 (HCDR3); viiii) Sequence IDs 170 (LCDR1), 171 (LCDR2), 172 (LCDR3), 176 (HCDR1), 177 (HCDR2), and 178 (HCDR3); ix) Sequence IDs 190 (LCDR1), 191 (LCDR2), 192 (LCDR3), 196 (HCDR1), 197 (HCDR2), and 198 (HCDR3); x) Sequence IDs 210 (LCDR1), 211 (LCDR2), 212 (LCDR3), 216 (HCDR1), 217 (HCDR2), and 218 (HCDR3); xi) Sequence IDs 230 (LCDR1), 231 (LCDR2), 232 (LCDR3), 236 (HCDR1), 237 (HCDR2), and 238 (HCDR3); xi) Sequence IDs 250 (LCDR1), 251 (LCDR2), 252 (LCDR3), 256 (HCDR1), 257 (HCDR2), and 258 (HCDR3); xiiii) Sequence IDs 270 (LCDR1), 271 (LCDR2), 272 (LCDR3), 276 (HCDR1), 277 (HCDR2), and 278 (HCDR3); xiv) Sequence IDs 290 (LCDR1), 291 (LCDR2), 292 (LCDR3), 296 (HCDR1), 297 (HCDR2), and 298 (HCDR3); or xv) Sequence IDs 2190 (LCDR1), 2191 (LCDR2), 2192 (LCDR3), 2196 (HCDR1), 2197 (HCDR2), and 2198 (HCDR3) The antigen-binding protein according to any one of claims 10 to 24, wherein the anti-TREM-1 antigen-binding moiety comprises a set of CDR amino acid sequences selected from the above.

26. The anti-TREM-1 antigen binding portion is a. Light chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301, and 2185; ii. Sequences encoded by polynucleotide sequences that are at least 80% identical to amino acid sequences selected from sequence numbers 41, 61, 81, 101, 121, 141, 161, 181, 201, 221, 241, 261, 281, 301 and 2185; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 39, 59, 79, 99, 119, 139, 159, 179, 199, 219, 239, 259, 279, 299 and 2183. A light chain variable domain comprising an amino acid sequence selected from the group consisting of; and b. A heavy chain variable domain, i. A sequence that is at least 80% identical to an amino acid sequence selected from sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186; ii. Sequences encoded by polynucleotide sequences that are at least 80% identical to amino acid sequences selected from sequence numbers 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, and 2186; iii. A sequence encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of nucleic acid sequences selected from SEQ ID NOs: 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, and 2184. Heavy chain variable domain containing an amino acid sequence selected from the group consisting of the following: An antigen-binding protein according to any one of claims 10 to 25, comprising:

27. The aforementioned antigen-binding portion is i) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 41 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 42; ii) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 61 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 62; iii) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 81 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 82; iv) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 101 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 102; v) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 121 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 122; vi) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 141 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 142; vii) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 161 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 162; viiii) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 181 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 182; ix) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 201 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 202; x) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 221 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 222; xi) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 241 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 242; xi) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 261 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 262; xiiii) An amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 281 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 282; xiv) an amino acid sequence that is at least 90% identical to the light chain variable domain described in SEQ ID NO: 301 and an amino acid sequence that is at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 302; or xv) Amino acid sequences that are at least 90% identical to the light chain variable domain described in SEQ ID NO: 2185 and amino acid sequences that are at least 90% identical to the heavy chain variable domain described in SEQ ID NO: 2186 The antigen-binding protein according to any one of claims 10 to 26, wherein the anti-TREM-1 antigen-binding moiety includes the anti-TREM-1 antigen-binding moiety.

28. a. Consists of two light chains and two heavy chains, b. Each heavy chain includes an IL-10 portion attached to the C-terminus of the heavy chain; c. Each heavy chain IL-10 partial antigen-binding protein is sequence numbers 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 98 7, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 10 It comprises an amino acid sequence that is at least 90% identical to a sequence selected from 45, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083 and 1085; and d. Each light chain IL-10 portion is sequence numbers 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 92 6, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 9 98, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054 An antigen-binding protein according to any one of claims 24 to 27, comprising an amino acid sequence that is at least 90% identical to a sequence selected from 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084 and 1086.

29. Sequence numbers 2137, 2139, 2141, 2143, 2145, 2147, 2149, 2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2359, 2361, 2363, 2365, 2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2143, 2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441 ,2443,2445,2447,2449,2451,2453,2455,2457,2459,2461,2463,2465,2467,2469,2471,2473,2475,2477,2479,2481,2483,2485,2487,2489,2491,2493,2495,2497,2498,2499,2501,2503,250 An antigen-binding protein according to any one of claims 10 to 14 or 24 to 27, having a heavy chain IL-10 partial amino acid sequence selected from the group consisting of 5, 2507, 2509, 2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533, 2535, 2537, 2539 and 2726 to 2776.

30. An antigen-binding protein according to any one of claims 10 to 14 or 24 to 28, having a heavy chain IL-10 partial amino acid sequence selected from the group consisting of SEQ ID NOs: 2727 to 2732.

31. The antigen-binding protein according to claim 30, comprising the heavy chain amino acid sequence of SEQ ID NO: 2727 or 2728 and the light chain amino acid sequence of SEQ ID NO: 976 or 2554.

32. The antigen-binding protein according to claim 30, comprising the heavy chain amino acid sequence of SEQ ID NO: 2729, 2730, 2731, or 2732 and the light chain amino acid sequence of SEQ ID NO: 992 or SEQ ID NO: 2555.

33. An antigen-binding protein comprising an antigen-binding moiety and one or two IL-10 moieties, a. The antigen-binding portion is an antibody or antibody fragment; b. Each IL-10 portion is independently monovalent or divalent; c. Each IL-10 moiety was independently selected from one or more human IL-10 mutant proteins having a sequence that is 90% identical to SEQ ID NO: 2; d. At least one IL-10 moiety is covalently bound to the antigen-binding moiety, and / or e. An antigen-binding protein wherein the antigen-binding portion competes with the antigen-binding portion described in any one of claims 24 to 27 for binding to the human TREM-1 protein.

34. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain region of an antigen-binding protein according to any one of claims 10 to 33, the light chain region of an antigen-binding protein according to any one of claims 10 to 33, or both.

35. A method for treating inflammatory diseases in individuals who require it, i) The IL-10 mutant protein according to any one of claims 1 to 3; ii) The anti-TREM-1 antigen-binding protein according to any one of claims 5 to 8; or iii) Antigen-binding protein according to any one of claims 10 to 33 A method including administering [a substance].

36. The method according to claim 35, wherein the inflammatory disease is selected from the group consisting of inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, rheumatoid arthritis, psoriasis, psoriatic arthritis, and cytokine release syndrome (CRS).

37. A composition for use in treating inflammatory diseases, i) The IL-10 mutant protein according to any one of claims 1 to 3; ii) The anti-TREM-1 antigen-binding protein according to any one of claims 5 to 8; or iii) Antigen-binding protein according to any one of claims 10 to 33 A composition containing the following:

38. Use of a composition comprising the IL-10 mutant protein according to any one of claims 1 to 3 in the preparation of a drug for treating an inflammatory disease.

39. The use of a composition in the preparation of a drug for treating an inflammatory disease, wherein the composition is i) The IL-10 mutant protein according to any one of claims 1 to 3; ii) The anti-TREM-1 antigen-binding protein according to any one of claims 5 to 8; or iii) Antigen-binding protein according to any one of claims 10 to 33 Includes, use.

40. Use of a composition comprising an anti-TREM-1 antibody or antigen-binding fragment according to any one of claims 5 to 8 or 10 to 33 in combination with an anti-IL-10 mutant protein according to any one of claims 1 to 3 in the preparation of a drug for treating an inflammatory disease.

41. An isolated antigen-binding protein, a. An antibody or antibody fragment; b. Binding to human TREM-1 having the amino acid sequence described in Sequence ID No. 20; c. Light chain variable domain, i. Amino acid sequence X １ ASQSX ２ X ３ X ４ NLA (Sequence ID 2199) (where X １ is R or Q, and X ２ is V or I, and X ３ is N or S, X ４ Light chain CDR1 containing S, H, I, V, or A; ii. Amino acid sequence GAX １ X ２ RAT (SEQ ID NO: 2200) (where X １ is S or Y, and X ２ is T or I) containing light chain CDR2; and iii. Amino acid sequence QX １ X ２ X ３ X ４ X ５ X ６ PX ７ T (Sequence No. 2201) (where X １ is Q, H, or E, and X ２ is F or Y, and X ３ is K, Y, or I, and X ４ is N, T, L, I or M; X ５ is W, F, H, or Y, and X ６ is non-existent or P; X ７ (is W, N, Y, H, or L) light chain CDR3 Includes a light chain variable domain that includes; and d. A heavy chain variable domain, i. Amino acid sequence X １ X ２ X ３ MX ４ (Sequence No. 2202) (where X １ is A, R, T, or S, and X ２ is Y or N, and X ３ is A or W, and X ４ Heavy chain CDR1 containing S or N; ii. 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 (Sequence No. 2205) (where X １ is T, E, or S, X ２ is either nonexistent, or M, V, or I, X ３ is S, R, or K, and X ４ is G or Q, and X ５ is S, D, or H, and X ６ is G, S, L, or A, X ７ is S, G, or R, and X ８ is T, S, P or E, and X ９ is T or I, X １０ is A or V, and X １１ is D or E, X １２ Heavy chain CDR2 containing S or A; and iii. Amino acid sequence X １ X ２ X ３ X ４ X ５ X ６ X ７ FX ８ YYX ９ (Sequence No. 2203) (where X １ is V, E, A or G, and X ２ is A, F, Y, or G, and X ３ is G, S, Y, or W, and X ４ is S or R, X ５ is either nonexistent or N, X ６ is F, S, Y or non-existent, X ７ is L or F or non-existent, X ８ is D or E, X ９ (This is a heavy chain CDR3 containing Y, H, or S) An isolated antigen-binding protein containing a heavy chain variable domain.

42. a. Light chain variable domain, i. Light chain CDR1 containing the amino acid sequence RASQSVNSNLA (SEQ ID NO: 2212); ii. Light chain CDR2 containing the amino acid sequence GASTRAT (SEQ ID NO: 2219); iii. Light chain CDR3 containing amino acid sequence QQFKNWPPT (SEQ ID NO: 2222) Light chain variable domains including; and b. A heavy chain variable domain, i. Heavy chain CDR1 containing the amino acid sequence AYAMS (SEQ ID NO: 2227); ii. Heavy chain CDR2 containing the amino acid sequence TSGSGSTTYYADSVKG (SEQ ID NO: 2230); and iii. Heavy chain CDR3 containing amino acid sequence VAGSNFLFDY (SEQ ID NO: 2670) Heavy chain variable domains including The isolated antigen-binding protein according to claim 41, comprising

43. An isolated antigen-binding protein, a. An antibody or antibody fragment; b. Binding to human TREM-1 having the amino acid sequence described in Sequence ID No. 20; c. Light chain variable domain, i. Amino acid sequence QASX １ DIX ２ X ３ X ４ LN (Sequence ID 2204) (where X １ is R or Q, and X ２ is R, S, N, or F, and X ３ is K or N, and X ４ Light chain CDR1 containing H, Y, or D; ii. Amino acid sequence X １ X ２ X ３ X ４ LET (SEQ ID NO: 2206) (where X １ is D, G or H, X ２ is A, V or T, X ３ is S, A or Y, X ４ is T or N) and contains a light chain CDR2; iii. Amino acid sequence QX １ YX ３ X ４ X ５ PX ６ T (SEQ ID NO: 2207) (where X １ is Q or H, X ２ is D, A or G, X ３ is N or K; X ４ is L or I, X ５ is I or L) and comprises a light chain CDR3 Includes a light chain variable domain that includes; and d. A heavy chain variable domain, i. Amino acid sequence X １ YDIN (Sequence ID 2208) (where X １ Heavy chain CDR1 containing R or S; ii. 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 (Sequence No. 2209) (where X １ is W or R, X ２ is M or L, X ３ is N, Q, or K, and X ４ is S, A, or R, and X ５ is N or Q, and X ６ is S, A, or T, and X ７ is S, Q, or Y, and X ８ is V or T, X ９ is Q or K, and X １０ is K or N, and X １１ is R or Q, and X １２ Heavy chain CDR2 containing G or D; and iii. 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 (Sequence number 2210) (where X １ is G, L, or R, and X ２ is G, I, or R, and X ３ is Y, R, I, G or A, and X ４ is T, S, Y, or V, and X ５ is S or Y, and X ６ is S, A, I or R, and X ７ is W, A, or S, and X ８ is either non-existent or S, X ９ is either nonexistent, or F, W, or Y, and X １０ is R, S, H, K or E, and X １１ is W, H, Y, or F, and X １２ is Y, V, A or S, and X １３ is D or Q, and X １４ Heavy chain CDR3 (which is L, Y, I, or H) An isolated antigen-binding protein containing a heavy chain variable domain.

44. a. Light chain variable domain, i. Light chain CDR1 containing the amino acid sequence QASQDIRKHLN (SEQ ID NO: 2213); ii. Light chain CDR2 containing the amino acid sequence DASNLET (SEQ ID NO: 2220); and iii. Light chain CDR3 containing amino acid sequence QHYDNLPIT (SEQ ID NO: 2223) Light chain variable domains including; and b. A heavy chain variable domain, i. Heavy chain CDR1 containing the amino acid sequence RYDIN (SEQ ID NO: 2228); ii. Heavy chain CDR2 containing the amino acid sequence WMNPNSGNSSVQKFRG (SEQ ID NO: 2231); and iii. Heavy chain CDR3 containing the amino acid sequence GGYTSSWRWYFDL (SEQ ID NO: 2671) or GGYTSSWSRWYFDL (SEQ ID NO: 2672) Heavy chain variable domains including The antigen-binding protein according to claim 43, comprising:

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