SLC15A4 inhibitory antibody and application thereof

Abstract

The present disclosure relates to an SLC15A4 inhibitory antibody and an application thereof. Specifically, the present disclosure provides an SLC15A4 inhibitory antibody or an antigen binding fragment thereof, the antibody or the antigen binding fragment thereof being capable of inhibiting binding of SLC15A4 to TASL. The disclosure also provides a nucleic acid molecule encoding the SLC15A4 inhibitory antibody or the antigen binding fragment thereof, a chimeric antigen receptor containing the SLC15A4 inhibitory antibody or the antigen binding fragment thereof, an antibody drug conjugate or a composition, and applications thereof.

Description

Technical Field

[0001] This disclosure relates to the biomedical field, specifically to SLC15A4 inhibitory antibodies and their applications. Background Technology

[0002] SLC15A4, also known as PHT1, is a member of the SLC15 family of solute carriers and is mainly expressed in antigen-presenting cells, such as plasmacytic dendritic cells (pDCs) and B cells. SLC15A4 is an endosomally resident proton-coupled amino acid transporter that primarily mediates the transport of L-histidine and oligopeptides.

[0003] SLC15A4 is crucial for the activation of Toll-like receptors (TLRs) 7 / 8 / 9, determining the levels of inflammation-related cytokines such as type I interferon-1 (IFN-I), tumor necrosis factor-α (TNFα), and interleukin-6 (IL-6) produced by TLR7 / 8 activation. Recent studies have found that mTOR is essential for TLR7 / 8 signaling activation and the activation of downstream IRF5 and IRF7, and SLC15A4 can mediate cytokine production by regulating mTOR activity. More importantly, SLC15A4 must form a complex with the TLR adaptor protein TASL (TLR adaptor interacting with SLC15A4 on the lysosome), thereby recruiting TASL to the lysosome and activating IRF5 via the conserved pLxIS motif at the C-terminus of TASL, thus activating the expression of inflammatory factors and the production of type I interferon-1. SLC15A4 and TASL are both indispensable for the activation of TLR7 / 8 / 9. Knockout of either SLC15A4 or TASL will prevent TLR7 / 8 / 9 from being activated to produce inflammatory factors and type I interferon.

[0004] TLR7 / 8 / 9, expressed on the lysosomal membrane, are key pattern recognition receptors for the body to recognize abnormal nucleic acid signals and play an important regulatory role in response to pathogen infection and bodily damage. However, overactivation of TLR7 / 8 / 9 can lead to uncontrolled and excessive secretion of type I interferon and pro-inflammatory cytokines, resulting in autoimmune diseases such as systemic lupus erythematosus.

[0005] Systemic lupus erythematosus (SLE) is a typical autoimmune disease. Studies have found that excessive activation of pDCs to produce type I interferon and excessive activation of B cells to produce antibodies against autologous dsDNA and anti-ribonucleoprotein (snRNAP) are the main causes of SLE. Increasing evidence suggests that Slc15A4 is associated with various inflammatory diseases, including SLE, colitis, inflammatory bowel disease (IBD), psoriasis, and type II diabetes. Slc15a4 loss-of-function mouse models ('feeble') or Slc15a4 gene knockout mouse models (Slc15a4...) are used to further investigate these factors. - / - Mice showed a significant reduction in the severity of SLE, IBD, and psoriasis. Furthermore, genome-wide association studies (GWAS) have shown that SLC15A4 is also closely associated with inflammatory diseases such as SLE in the human population.

[0006] In addition to playing a crucial role in TLR signaling, SLC15A4 is also involved in other innate immune pathways, including activation of NOD1 / 2 signaling and inflammasome activation. These studies support the possibility that specific inhibition of SLC15A4 could be a potential therapeutic strategy for inflammatory diseases.

[0007] Despite extensive research confirming SLC15A4 as a key inflammatory regulatory molecule, providing a solid foundation for the development of related immunomodulators, there are currently no clinically available treatments targeting SLC15A4. In recent years, only two studies have reported several small-molecule inhibitors of SLC15A4. However, no large-molecule drugs with specificity and safety have been reported. This may be due to the high conservation of SLC15A4 across species, making the development of large-molecule drugs still challenging. Summary of the Invention

[0008] To address one of the aforementioned technical problems in the prior art, this disclosure provides an inhibitory antibody or antigen-binding fragment of SLC15A4 that can effectively bind to SLC15A4 and inhibit the formation of a complex between SLC15A4 and TASL, thereby inhibiting the activation of the downstream TLR signaling pathway, and thus can be used for the prevention and treatment of autoimmune diseases and / or inflammatory diseases.

[0009] According to one aspect of this disclosure, an antibody or antigen-binding fragment thereof that binds to SLC15A4 is provided, said antibody or antigen-binding fragment thereof inhibiting the binding of SLC15A4 to TASL.

[0010] In some embodiments, the antibody or its antigen-binding fragment binds to all or part of the CTD domain corresponding to human SLC15A4, thereby stabilizing SLC15A4 in an outwardly open conformation.

[0011] In some embodiments, the antibody or its antigen-binding fragment binds to a region corresponding to amino acids 348–532 of human SLC15A4.

[0012] In some embodiments, the antibody or its antigen-binding fragment may bind to a region corresponding to amino acids 348–366, 429–453, and / or 511–532 of human SLC15A4.

[0013] In some embodiments, the antibody or its antigen-binding fragment may bind to one or more amino acid residues corresponding to R350, T360, K431, E432, T434, N436, T438, N441, H445, N526 and F524 of human SLC15A4.

[0014] In some embodiments, the human SLC15A4 may have an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with it.

[0015] In some embodiments, the antibody or its antigen-binding fragment may include: (1) a heavy chain variable region complementarity-determining region (HCDR): HCDR1 having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 11 and 32-41, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region; HCDR2 having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any one of SEQ ID NO: 11 and 32-41, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region; and HCDR3 having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in SEQ ID NO: 11 and 32-41; and HCDR3 having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in SEQ ID NO: 11 and 32-41. The amino acid sequence of HCDR3 contained in the heavy chain variable region shown in any one of SEQ ID NO:11 and 32-41, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or, (2) Light chain variable region complementarity-determining region (LCDR): LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region shown in any one of SEQ ID NO:12 and 42-47, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region; LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region shown in any one of SEQ ID NO:12 and 42-47, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region; and, LCDR3, having the amino acid sequence of LCDR2 contained in the light chain variable region shown in any one of SEQ ID NO:12 and 42-47, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region; and, LCDR3, having the amino acid sequence of LCDR2 contained in the light chain variable region shown in SEQ ID NO:12 and 42-47; The amino acid sequence of LCDR3 contained in the light chain variable region shown in any one of NO:12 and 42-47, or an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

[0016] In some embodiments, the aforementioned HCDR1-3 and / or LCDR1-3 can be defined by any numbering system commonly used by those skilled in the art. In some embodiments, the three HCDRs contained in the heavy chain variable region and / or the three LCDRs contained in the light chain variable region are defined by rules of Kabat, Chothia, IMGT, Martin, Contact, Honegger, Gelfand, or combinations thereof.

[0017] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:11, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0018] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:32, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0019] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:33, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:42, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0020] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:34, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:43, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0021] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:35, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0022] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:36, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:44, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0023] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:37, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:45, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0024] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:38, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:46, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0025] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:39, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0026] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:40, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:47, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0027] In some embodiments, the antibody or its antigen-binding fragment may include: an amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:41, or HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions; and / or, an amino acid sequence having LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions.

[0028] In some embodiments, the antibody or its antigen-binding fragment may include: HCDR1 having an amino acid sequence as shown in GX1SFTX2Y; HCDR2 having an amino acid sequence as shown in X3HPX4DSE; and HCDR3 having an amino acid sequence as shown in CAX5WGX6X7X8X9X. 10 DX 11 DX 12 FDX 13 The amino acid sequence shown in W; LCDR1, which has the following characteristics: KASQNVGX 14 NVX 15The amino acid sequences shown are: LCDR2, which has the amino acid sequence shown as SASYRYS (SEQ ID NO: 17); and LCDR3, which has the amino acid sequence shown as CQQYNX. 16 X 17 The amino acid sequence shown in PYTF, where X1 to X 17 Without particular restriction, the amino acid residues may be absent and / or selected from any natural amino acid residues, such as glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), phenylalanine (F), aspartic acid (D), asparagine (N), glutamine (Q), threonine (T), aspartic acid (N), glutamic acid (E), lysine (K), arginine (R), and histidine (H).

[0029] In some embodiments, X1 may be selected from Y, F, W, or H. In a preferred embodiment, X1 may be selected from Y or F.

[0030] In some embodiments, X2 may be selected from R, N, K, H, G, Q, S, T, Y, or C. In a preferred embodiment, X2 may be selected from R or N.

[0031] In some embodiments, X3 may be absent. In some embodiments, X3 may be selected from A, V, L, I, F, P, M, or W. In a preferred embodiment, X3 may be I.

[0032] In some embodiments, X4 may be selected from G, N, Q, S, T, Y, or C. In a preferred embodiment, X4 may be selected from S or T.

[0033] In some embodiments, X5 may be selected from K, R, H, I, A, V, L, I, F, P, M, or W. In a preferred embodiment, X5 may be selected from R or I.

[0034] In some embodiments, X6 may be selected from G, N, Q, S, T, Y, C, A, V, L, I, F, P, M, or W. In a preferred embodiment, X6 may be selected from A or S.

[0035] In some embodiments, X7 may be selected from Y, F, W, or H. In a preferred embodiment, X7 may be selected from Y or F.

[0036] In some embodiments, X8 may be selected from G, N, Q, S, T, Y, C, Y, F, W, or H. In a preferred embodiment, X8 may be selected from Y, F, or N.

[0037] In some embodiments, X9 may be selected from K, R, H, A, V, L, I, F, P, M, or W. In a preferred embodiment, X9 may be selected from K, R, or I.

[0038] In some implementations, X 10 It can be selected from Y, F, W, H, G, N, Q, S, T, Y, or C. In a preferred embodiment, X 10 It can be selected from Y or N.

[0039] In some implementations, X 11 It can be selected from Y, F, W, H, G, N, Q, S, T, Y, or C. In a preferred embodiment, X 11 You can choose from W or R.

[0040] In some implementations, X 12 It can be selected from Y, F, W, H, G, N, Q, S, T, Y, C, D, or E. In a preferred embodiment, X 12 It can be selected from Y, F, S or D.

[0041] In some implementations, X 13 It can be selected from G, N, Q, S, T, Y, or C. In a preferred embodiment, X 13 It can be selected from S or Y.

[0042] In some implementations, X 14 It can be selected from G, N, Q, S, T, Y, C, R, K, or H. In a preferred embodiment, X 14 It can be selected from T, R or K.

[0043] In some implementations, X 15 It can be selected from A, V, L, I, F, P, M, W, D, or E. In a preferred embodiment, X 15 You can choose from V or D.

[0044] In some implementations, X 16 It can be selected from G, N, Q, S, T, Y, or C. In a preferred embodiment, X 16 It can be selected from N or S.

[0045] In some implementations, X 17 It can be selected from A, V, L, I, F, P, M, W, Y, F, or H. In a preferred embodiment, X 17 You can choose from F or Y.

[0046] In some embodiments, the HCDR1 of the antibody or its antigen-binding fragment may have an amino acid sequence as shown in SEQ ID NO:13, 48 or 49, or an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the antibody.

[0047] In some embodiments, the HCDR2 of the antibody or its antigen-binding fragment may have an amino acid sequence as shown in SEQ ID NO:14, 50 or 51, or an amino acid sequence with one or more amino acid substitutions, deletions or additions.

[0048] In some embodiments, the HCDR3 of the antibody or its antigen-binding fragment may have an amino acid sequence as shown in any one of SEQ ID NO:15, 52-61, or an amino acid sequence with one or more amino acid substitutions, deletions or additions.

[0049] In some embodiments, the LCDR1 of the antibody or its antigen-binding fragment may have an amino acid sequence as shown in SEQ ID NO:16, 62 or 63, or an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the antibody.

[0050] In some embodiments, the antibody or its antigen-binding fragment LCDR2 may have an amino acid sequence as shown in SEQ ID NO:17, or an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the antibody.

[0051] In some embodiments, the LCDR3 of the antibody or its antigen-binding fragment may have an amino acid sequence as shown in SEQ ID NO:18, 64 or 65, or an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the antibody.

[0052] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 14, and 15, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0053] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 50, and 52, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0054] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:48, 50, and 53, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:62, 17, and 64, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0055] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:48, 50, and 54, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:63, 17, and 65, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0056] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 50, and 55, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0057] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 50, and 56, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0058] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 50, and 57, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0059] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:49, 50, and 58, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0060] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 51, and 59, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0061] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 50, and 60, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0062] In some embodiments, the antibody or its antigen-binding fragment may include: having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 13, 50, and 61, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions; and / or having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, 17, and 18, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions.

[0063] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment may have an amino acid sequence as shown in any one of SEQ ID NO: 11 and 32-41, or an amino acid sequence having at least 85% sequence identity with the other.

[0064] In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment may have an amino acid sequence as shown in any one of SEQ ID NO: 12 and 42-47, or an amino acid sequence having at least 85% sequence identity with the other.

[0065] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:11, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it.

[0066] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:32, or an amino acid sequence having at least 85% sequence identity with the latter; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with the latter.

[0067] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:33, or an amino acid sequence having at least 85% sequence identity with the latter; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:42, or an amino acid sequence having at least 85% sequence identity with the latter.

[0068] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:34, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:43, or an amino acid sequence having at least 85% sequence identity with it.

[0069] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:35, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it.

[0070] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:36, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:44, or an amino acid sequence having at least 85% sequence identity with it.

[0071] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:37, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:45, or an amino acid sequence having at least 85% sequence identity with it.

[0072] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:38, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:46, or an amino acid sequence having at least 85% sequence identity with it.

[0073] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:39, or an amino acid sequence having at least 85% sequence identity with the latter; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with the latter.

[0074] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:40, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:47, or an amino acid sequence having at least 85% sequence identity with it.

[0075] In some embodiments, the antibody or its antigen-binding fragment may include: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:41, or an amino acid sequence having at least 85% sequence identity with it; and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it.

[0076] In some embodiments, the antibody may be, but is not limited to, IgA, IgD, IgE, IgG, or IgM.

[0077] In some embodiments, the antibody may be of the IgG type, such as IgG1, IgG2, IgG3 or IgG4.

[0078] In some embodiments, the antigen-binding fragment may be one known in the art. In specific embodiments, the antigen-binding fragment may include scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, dsFv.

[0079] In some embodiments, the antibody may be a biantibody, a bispecific antibody, or a multispecific antibody.

[0080] In some embodiments, the antibody or its antigen-binding fragment may further include a heavy chain constant region and / or a light chain constant region.

[0081] In some embodiments, the heavy chain constant region may be selected from the IgG1, IgG2, IgG3 or IgG4 heavy chain constant regions.

[0082] In some embodiments, the heavy chain constant region may be the IgG2a heavy chain constant region.

[0083] In some embodiments, the antibody or antigen-binding fragment includes, but is not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0084] The antibody or antigen-binding fragment thereof disclosed herein can inhibit the binding of SLC15A4 to TASL. In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein binds to the C-terminal domain (CTD) of SLC15A4. In specific embodiments, the antibody or antigen-binding fragment thereof disclosed herein binds to loops 7-8, 9-10, and / or loops 11-12 of SLC15A4. In specific embodiments, the antibody or antigen-binding fragment thereof disclosed herein can form hydrogen bonds or hydrophobic interactions with any one or more sites of R350, T360, K431, E432, T434, N436, T438, N441, H445, F524, and N526 of SLC15A4.

[0085] The antibody or its antigen-binding fragment disclosed herein can bind to SLC15A4, stabilizing SLC15A4 in an outward (inner lysosome lumen side) conformation, thereby preventing SLC15A4 from forming a complex with TASL.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein does not bind to the SLC15A4 / TASL complex. In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein can inhibit the expression of type I interferon and inflammatory cytokines, inhibit the activation of TLR7 / 8 / 9 signaling pathways, inhibit the expression of type I interferon and inflammatory cytokines on the THP1 cell line, and / or inhibit the expression of type I interferon on peripheral blood mononuclear cells. In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein can effectively inhibit the activation of IFN-α signaling in human PBMCs induced by TLR7 / 8 agonists (e.g., R848) or TLR9 agonists (e.g., CpGA).

[0087] According to another aspect of this disclosure, a nucleic acid molecule is provided that encodes the antibody or an antigen-binding fragment thereof.

[0088] According to another aspect of this disclosure, an expression vector is provided that contains the nucleic acid molecule.

[0089] In some embodiments, the expression vector includes prokaryotic expression vectors and eukaryotic expression vectors.

[0090] In some embodiments, the eukaryotic expression vector includes yeast expression vectors, mammalian expression vectors, insect expression vectors, etc.

[0091] According to another aspect of this disclosure, a host cell is provided comprising the nucleic acid molecule and / or the expression vector. In some embodiments, the host cell is a conventional host cell in the art, provided that the expression vector stably expresses the carried nucleic acid molecule as the antibody or its antigen-binding fragment described in this invention.

[0092] In some embodiments, the host cell is selected from prokaryotic cells and eukaryotic cells.

[0093] In some embodiments, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0094] In some embodiments, the eukaryotic cells include yeast cells, mammalian cells, insect cells, etc.

[0095] In some embodiments, the mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats.

[0096] In some embodiments, the mammalian cells include CHO cells, 293 cells, 293T cells, Vero cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells.

[0097] According to another aspect of this disclosure, a chimeric antigen receptor is provided, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises an antibody or an antigen-binding fragment thereof as described in this disclosure.

[0098] According to another aspect of this disclosure, a modified immune cell is provided that comprises the chimeric antigen receptor described in this disclosure.

[0099] According to another aspect of this disclosure, an antibody-drug conjugate is provided, comprising: an antibody or an antigen-binding fragment thereof as described in this disclosure; and a drug covalently linked to the antibody or the antigen-binding fragment thereof.

[0100] In some embodiments, the antibodies or antigen-binding fragments described herein may be covalently linked to therapeutic agents, such as cytotoxins, drugs (e.g., immunosuppressants), or radiotoxins, to obtain the antibody-drug conjugates.

[0101] In some embodiments, the antibodies or antigen-binding fragments described in this disclosure may also be conjugated with radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates.

[0102] According to another aspect of this disclosure, a detection kit is provided, the kit comprising the antibody or an antigen-binding fragment thereof. In some embodiments, the kit can be used to detect the presence and / or amount of SLC15A4 in a sample.

[0103] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising the antibody or antigen-binding fragment thereof described in this disclosure, and a pharmaceutically acceptable carrier.

[0104] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventional in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents, etc.

[0105] In some embodiments, the pharmaceutical composition may further comprise other small molecule drugs, antibodies, or peptides. In some embodiments, the antigen or antigen-binding fragment of this disclosure may be administered sequentially or simultaneously with the other small molecule drugs, antibodies, or peptides. The other small molecule drugs, antibodies, or peptides may be selected as needed.

[0106] In some embodiments, the other small molecule drugs, antibodies, or peptides include, but are not limited to, anti-IFNα antibodies, anti-IFN-γ receptor antibodies, soluble IFN-γ receptors, anti-TNF antibodies, anti-TNF receptor antibodies and / or soluble TNF receptors, Flt3 ligand antagonists, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (e.g., prednisone, hydrocortisone), immunosuppressants (e.g., cyclophosphamide, azathioprine, and methotrexate), antimalarial drugs (e.g., hydroxychloroquine), biological agents that inhibit the production of dsDNA antibodies (e.g., LJP394), drugs containing mesalazine (including sulfasalazine and drugs containing 5-aminosalicylic acid (5-ASA), olsalazine, and balsalazine), non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (e.g., prednisone, hydrocortisone), and TNF inhibitors (including adilimumab). Inasip Infliximab (Remicade), immunosuppressants (such as 6-mercaptopurine, azathioprine, and cyclosporine A), antibiotics, COX-2 inhibitors, analgesics, corticosteroids (such as prednisone and hydrocortisone), immunosuppressants (such as methotrexate), B-cell depleting agents (such as Rituxan) TM B-cell agonists (e.g., LymphoStat-B) TM ), anti-TNF-α agents (e.g., EMBREL) TM , and REMICADE TM ), glucocorticoids, methotrexate, synthetic vitamin A, cyclosporine, and T-cell inhibitors (e.g., Raptiva) TM ).

[0107] In some embodiments, the route of administration of the pharmaceutical composition may be parenteral, injection, oral, or topical. The pharmaceutical composition may be formulated into a form suitable for administration, such as a solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, or in the form of powder, tablet, capsule, granules, injection, or infusion.

[0108] According to another aspect of this disclosure, a method for diagnosing, treating, and / or preventing SLC15A4-related disease is provided, the method comprising administering to a subject in need an antibody of the present disclosure or an antigen-binding fragment thereof, a nucleic acid molecule of the present disclosure, an expression vector of the present disclosure, a host cell of the present disclosure, a chimeric antigen receptor of the present disclosure, an immune cell of the present disclosure, and / or an antibody-drug conjugate.

[0109] According to another aspect of this disclosure, the use of the antibodies of this disclosure or antigen-binding fragments thereof, the nucleic acid molecules of this disclosure, the expression vectors of this disclosure, the host cells of this disclosure, the chimeric antigen receptors of this disclosure, the immune cells of this disclosure, and / or antibody-drug conjugates in the diagnosis, treatment, and / or prevention of SLC15A4-related diseases is provided.

[0110] According to another aspect of this disclosure, the use of the antibody or antigen-binding fragment thereof of this disclosure in the preparation of reagents for diagnosing SLC15A4-related diseases is provided.

[0111] According to another aspect of this disclosure, the use of the antibodies or antigen-binding fragments thereof, the nucleic acid molecules thereof, the expression vectors thereof, the host cells thereof, the chimeric antigen receptors thereof, the immune cells thereof, and / or antibody-drug conjugates thereof in the preparation of medicaments for treating and / or preventing SLC15A4-related diseases is provided. In some embodiments, the SLC15A4-related diseases may include inflammatory diseases, which may be caused by elevated expression of type I interferon or inflammatory cytokines.

[0112] In some implementations, the SLC15A4-related diseases may include tumors, systemic lupus erythematosus, colitis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, and celiac disease), type II diabetes, multiple sclerosis, psoriasis, autoimmune thyroid disease, rheumatoid arthritis, lupus, and glomerulonephritis.

[0113] In some embodiments, the autoimmune thyroid disease may include, but is not limited to, autoimmune primary hypothyroidism, Graves' disease, Hashimoto's thyroiditis, and destructive thyroiditis with hypothyroidism. Attached Figure Description

[0114] Figure 1 This demonstrates the SLC15A4 monoclonal antibody screening strategy. Figure 1 A shows the antibody immunization and screening strategy; Figure 1 B shows the chromatography and SDS-PAGE results of the SLC15A4 (32-558) protein sample used for immunization.

[0115] Figure 2 The SLC15A4 monoclonal antibody XY_107 binds to the SLC15A4 membrane surface and mediates endocytosis. Figure 2 A shows the SLC15A4 bound to the membrane surface by XY_107 and the membrane-localized SLC15A4 mutant; Figure 2 BD shows that XY_107 binds to SLC15A4 on the surface of the THP1 membrane and mediates endocytosis.

[0116] Figure 3 The SCL15A4 monoclonal antibody XY_107Fab fragment (Fab107) was shown to inhibit TLR7 / 8 / 9 pathway activation. Figure 3 A and 3C show IL6 expression; Figure 3 B and 3D images show the expression of TNFα; Figure 3 E shows the relative expression of IFNB1; Figure 3 F and Figure 3 G shows that Fab107 effectively inhibits the activation of IFN-α signaling in human PBMCs induced by R848 or CpGA.

[0117] Figure 4 The SLC15A4 monoclonal antibody XY_107Fab fragment (Fab107) showed that it inhibited R848-induced THP1 activation and differentiation. Figure 4 A shows that Fab107 inhibits R848-induced THP1 activation and adhesion; Figure 4 B and Figure 4 C showed that Fab107 inhibited R848-induced upregulation of CD14 and HLA-DR expression on the THP1 membrane surface.

[0118] Figure 5 The SLC15A4 monoclonal antibody XY_107 was shown to bind to SLC15A4-dependent LL7-8, LL8-9, and LL11-12. Figure 5 A shows the construction strategy for the SLC15A4 mutant; Figure 5B shows the ability of XY_107 to bind to the SLC15A4 mutant.

[0119] Figure 6 The XY_107Fab fragment (Fab107) of the SLC15A4 monoclonal antibody showed that it effectively bound the SLC15A4 monomer. Figure 6 A and Figure 6 C), cannot effectively bind to the SLC15A4-TASL complex ( Figure 6 B and Figure 6 C).

[0120] Figure 7 This shows the amino acid sequence comparison results of the heavy chain variable region of the SLC15A4 monoclonal antibody XY_107 and antibodies derived from the same germline.

[0121] Figure 8 This shows the amino acid sequence alignment results of the light chain variable region of the SLC15A4 monoclonal antibody XY_107 and antibodies derived from the same germline.

[0122] Figure 9 The SLC15A4 monoclonal antibody XY_107Fab fragment (Fab107) binds to the SLC15A4 CTD, stabilizing SLC15A4 and maintaining its conformation facing the endosomal side (outer side of the cell membrane). Figure 9 A-9B shows Fab107 combined with SLC15A4CTD; Figure 9 C-9E shows the interaction sites between Fab107 and SLC15A4; Figure 9 F shows that Fab107 cannot bind effectively after mutation at the key site of SLC15A4; Figure 9 G shows that Fab107 conflicts with SLC15A4, which opens to the cytoplasmic side. Detailed Implementation

[0123] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0124] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0125] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0126] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0127] The terms “SLC15A4” and “SLC15A4 antigen” are used interchangeably to include variants, isotypes, species homologs of human SLC15A4, and analogs that share at least one common epitope with SLC15A4. Therefore, the antibodies of the present invention may cross-react with SLC15A4 from species other than humans in certain circumstances, or with other proteins structurally related to human SLC15A4 (e.g., human SLC15A4 homologs). In other circumstances, the antibodies may be completely specific to mouse SLC15A4 and do not exhibit species or other types of cross-reactivity.

[0128] The term "antibody" in this disclosure encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0129] The term "monoclonal antibody" in this disclosure refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that, apart from possibly trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies comprised in the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0130] The terms “antibody or antigen-binding fragment thereof” and “antibody” are used interchangeably herein to refer to antibodies that are substantially similar in structure to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, consisting of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant domains) from the N-terminus to the C-terminus. Each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant domain) from the N-terminus to the C-terminus. The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: κ light chain and λ light chain.

[0131] Within the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0132] The term "variable region" or "variable domain" in this disclosure refers to a domain of the heavy or light chain of an antibody involved in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0133] The term "variable" in this disclosure refers to the fact that certain segments of the variable domain are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the variable domains of the light and heavy chains. The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are held together tightly by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD (1991)). Constant domains do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0134] The term "hypervariant region" or "HVR" in this disclosure refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the "complementarity-determining region (CDR)," the amino acid residues from the CDR having the highest sequence variability and / or being involved in antigen recognition.

[0135] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system, or combinations thereof. For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. The correspondence between different numbering systems is well known to those skilled in the art, and some commonly used software can be used to define HCDR1-3 and LCDR1-3. For example, the Kabat antibody numbering system, an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1991). The Chothia antibody numbering system, an immunoglobulin numbering system proposed by Chothia et al., is a classic rule for identifying CDR region boundaries based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883; Al-Lazikani et al., 1997, JMB 273:927-948). The IMGT antibody numbering system is based on the international ImMunoGeneTics information (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. The Martin numbering system is a new numbering scheme recommended by Martin et al., focusing on the structural alignment of different frame regions of unconventional lengths; it can be completed using the Chothia numbering system corrected by ABnum software. The Contact numbering scheme defines the CDR region based on existing antibody complex crystal structure data. The Honegger (also known as the AHo numbering scheme) is based on structural alignment of 3D structures covering observed length variations of immunoglobulin variable regions, allowing the definition of structurally conserved Cα positions, thus deriving appropriate FR regions and CDR lengths.

[0136] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0137] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0138] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0139] "Humanized antibodies" have an amino acid sequence that corresponds to that of antibodies produced by humans or human cells, or derived from non-human antibodies using sequences encoded by human antibody libraries or other human antibodies. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0140] As used herein, the term "substitution" or "replacement" of amino acids can refer to the substitution of a conserved amino acid residue, wherein the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another respect, a string of amino acids can be conservatively replaced by a structurally similar string that differs in the order and / or composition of its side chain family members.

[0141] The terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” in this disclosure refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0142] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0143] After obtaining the DNA fragments encoding the VH and / or VL of the antibody, these DNA fragments can be further manipulated using recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding the VH and / or VL is operatively linked to another DNA fragment encoding a different protein, such as the antibody constant region or a flexible linker. As used herein, the term "operatively linked" means that two DNA fragments are joined together such that the amino acid sequences encoded by both fragments remain within the reading frame.

[0144] By operatively linking DNA encoding the VH region to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are well known in the art (see, for example, Kabat, BA et al. (1991), Sequences of Proteins of Immunologicl Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments including these regions can be obtained by PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but IgG1 or IgG2a constant regions are most preferred. For Fab fragment heavy chain genes, the DNA encoding the VH region can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0145] By operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are well known in the art (see, for example, Kabat, BA et al. (1991), Sequences of Proteins of Immunologic Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments including these regions can be obtained by PCR amplification. The light chain constant region can be either a κ or λ constant region, but a κ constant region is preferred.

[0146] To generate the scFv gene, the DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as continuous single-stranded proteins with their VH and VL regions linked by the flexible linker.

[0147] The terms "antigen-binding domain" or "antigen-binding site" used in this disclosure refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, referred to as an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0148] The term "antigenic determinant" in this disclosure is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this invention can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0149] "Specific binding" refers to the selective binding to an antigen, distinguishable from unwanted or nonspecific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to unrelated proteins is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. In some embodiments, the dissociation constant (Kd) of the antigen-binding molecule is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10). -7 M or lower, such as 10 -7 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0150] "Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Binding affinity is usually expressed by the dissociation constant (Kd), which is the sum of the dissociation rate constant and the association rate constant (K0, K ... off and K on The ratio of the rate constants is used. Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

[0151] The term "isolated" nucleic acid molecule or polynucleotide in this disclosure refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding a polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this invention further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0152] The terms "vector" or "expression vector" and "expression construct" used herein are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector comprises a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. The term "expression cassette" in this disclosure refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in the target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter.

[0153] The term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or its antibody- or antigen-binding fragment) chemically linked to one or more chemical drugs. In a preferred embodiment, an ADC comprises a binding protein, a drug, and a connector linking the binding protein to the drug. These conjugates are also referred to as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are called "immunotoxins." Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., killing). Examples include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucoside, epipodophyllotoxin thiophene glycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, scintillans, actinomycin D, L-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), causative agents (e.g., nitrogen mustard, thioepa, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozomycin, mitomycin C, and cis-dichlorodiamineplatin(II)(D)). DP (cisplatin), amiodarone derivatives (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., actinomycin D, bleomycin, scintillan, and atrazodine (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies or antigen-binding fragments thereof disclosed herein include buprofen, scutellarin, metansin, auristatin, and their derivatives.

[0154] Cytotoxins can be conjugated to antibodies or antigen-binding fragments thereof disclosed herein using adapter techniques available in the art. Examples of adapter types already used for conjugating cytotoxins to antibodies or antigen-binding fragments thereof include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing adapters. Alternatively, adapters may be selected that are readily cleaved at low pH or readily cleaved by proteases, such as proteases preferentially expressed in tumor tissues, like cathepsins (e.g., cathepsins B, C, D).

[0155] As used herein, the term "radioisotope" refers to an antibody or its antigen-binding fragment that can be conjugated for diagnostic or therapeutic use, including but not limited to iodine. 131 ,indium 111 ,yttrium 90 Helu 177 Methods for preparing radioimmunoconjugates are known in the art.

[0156] The term "chimeric antigen receptor" or "CAR" refers to a receptor that possesses desired antigen specificity and signal transduction domains to propagate intracellular signals upon antigen binding. For example, T lymphocytes recognize specific antigens via the interaction of T cell receptors (TCRs) with short peptides presented by class I or II major histocompatibility complex (MHC) molecules. For initial activation and clonal expansion, naïve T cells depend on antigen-presenting cells (APCs) that provide additional co-stimulatory signals. In some embodiments, monocytes and macrophages can be engineered to express, for example, chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment, where they act as potent immune effectors by infiltrating the tumor and killing target cancer cells. CARs may include antigen-binding domains, transmembrane domains, and intracellular domains. The antigen-binding domain binds to the antigen on the target cell. Examples of cell surface markers that can be used as antigens binding to the antigen-binding domain of a CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0157] The term "modified immune cell" refers to an immune cell that has been genetically modified to express a CAR. In some embodiments, the immune cell is a T cell or a cell derived therefrom. In some embodiments, the immune cell is a natural killer (NK) cell or a cell derived therefrom. In some embodiments, the immune cell is a B cell or a cell derived therefrom. In some embodiments, the immune cell is a monocyte or macrophage, or a cell derived therefrom.

[0158] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes the amount sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0159] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug eliminates, mitigates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0160] The terms "individual" or "subject" refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human.

[0161] The term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antibodies or antigen-binding fragments of the present disclosure, along with other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0162] The term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0163] Examples of suitable aqueous or non-aqueous carriers that can be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable flowability can be maintained, for example, by applying a coating material such as lecithin, in the case of a dispersion, by maintaining the desired particle size, and by applying a surfactant.

[0164] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of microorganisms can be prevented by the sterilization procedures described above or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, and phenolic sorbic acid. Isotonic agents, such as sugars and sodium chloride, may also be required in the composition. Additionally, prolonged absorption of injectable drugs can be achieved by including delayed absorption agents, such as aluminum monostearate and gelatin.

[0165] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the provisional preparation of sterile injections or dispersions. The use of these media and reagents for the application of pharmaceutically active substances is well known in the art. Additional active compounds may also be incorporated into the composition.

[0166] Therapeutic compositions must generally be sterile and stable under the conditions of preparation and storage. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, polyethylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, by using a coating, such as lecithin, appropriate flowability can be maintained by maintaining the desired particle size in the case of a dispersant, and by using a surfactant. In many cases, the composition preferably contains an isotonic agent, such as sugars, polyols such as mannitol, sorbitol, or sodium oxide. Prolonged absorption of injectable drugs can be achieved by adding delayed absorption agents, such as monostearate and gelatin, to the composition.

[0167] Sterile injection solutions can be prepared by mixing the active compound in a suitable solvent in the required amount, and adding one or a combination of the components listed above as needed, followed by aseptic microfiltration. Dispersants are typically prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other desired components listed above. For sterile powders used to prepare sterile injection solutions, preferred preparation methods include vacuum drying and freeze-drying (lyophilization), yielding a powder containing the active ingredient plus any additional desired components from the aforementioned aseptically filtered solution.

[0168] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose form varies depending on the subject being treated and the specific route of administration. The amount of active ingredient that can be combined with a carrier material to prepare a single-dose form is generally the amount of the composition that produces the therapeutic effect. Typically, this amount, in 100% terms, ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0169] The term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antibody thereof or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody thereof or an antibody thereof or an antigen-binding fragment thereof, to a patient having one or more diseases or symptoms, and the therapeutic agent having a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0170] The term "parenteral" administration, as used in this article, refers to a mode of administration other than enteral and local administration, typically via injection, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions described herein, can be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasally, via airway instillation, or via intrapleural instillation. The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions described herein, can also be administered in aerosol or spray form, such as nasally; or intrathecally, intramedullaryly, or intraventricularly; or transdermally, percutaneously, locally, enterically, intravaginally, sublingually, or rectally. The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions described herein, can be formulated into various dosage forms as needed, and the physician can determine the beneficial dosage for the patient based on factors such as patient type, age, weight, general disease condition, and route of administration.

[0171] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0172] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for the alignment of protein and nucleotide sequences. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the BLAST website of the Center for Biotechnology Information (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0173] Those skilled in the art will understand that the reference herein to having “at least 85% sequence identity” compared to a sequence is intended to include all sequences with more than 85% sequence identity, such as including at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity.

[0174] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0175] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0176] Example

[0177] Example 1: Production of mouse anti-SLC15A4 antibody

[0178] 1. Preparation of immunogen and tag protein

[0179] The immunogen and antigen-specific B cell-enriched tag proteins used in this disclosure were constructed.

[0180] (1) Primers were designed based on Genbank accession number XM_011537895.2 (SLC15A4 mRNA), and the primer sequences are shown in Table 1:

[0181] Table 1

[0182]

[0183] (2) Construction of expression vectors for immunogen and antigen-specific B cell-enriched tag proteins

[0184] First, let's take pFastBac TM Using Dual (Invitrogen) as a template, reverse PCR was performed according to the PrimeSTAR HSDNA Polymerase (TaKaRa) instructions, using primers HF1 and HR1 respectively, to obtain the PCR product pFastBac. TM Dual.TEV.his full-length clip.

[0185] The PCR conditions were as follows: pre-denaturation, 98℃, 2 minutes; denaturation, 98℃, 30 seconds; annealing, 58℃, 30 seconds; extension, 72℃, 2 minutes 30 seconds; a total of 20 cycles were performed, with an additional 10 minutes of denaturation time at the end.

[0186] The PCR products were digested with DpnI enzyme (Thermo Fisher Scientific) for 1–4 hours, and the digested products were transformed into E. coli TOP10 strains to construct the vector pFastBac. TM Dual.TEV.his.

[0187] Then, using synthesized and codon-optimized human SLC15A4 DNA as a template, the human SLC15A4 (32-558) fragment was amplified with primers 15F2 and 15R, and the human SLC15A4 (32-558) fragment with an N-terminal flag tagged was amplified with primers 15F3 and 15R. The PCR products of the amplified human SLC15A4 (32-558) fragment and the SLC15A4 (32-558) fragment with an N-terminal flag tagged were recovered. The PCR products and pFastBac enzymes digested with the same enzymes were digested with BamHI-HF and HindIII-HF. TMThe Dual.TEV.his plasmid was recovered and ligated to obtain expression vectors for human SLC15A4(32-558) and human SLC15A4(32-558) with a flag tag attached to the N-terminus. The recombinant plasmids were transformed into DH10-Bac competent cells and plated onto plates containing kanamycin, tetracycline, gentamicin, x-gal, and IPTG. After static incubation at 37°C for 3 days, white single clones were picked and transferred to liquid culture medium. After expansion culture, baculovirus (Bacmid) was extracted the following day and named Bacmid-SLC15A4(32-558) and Bacmid-flag-SLC15A4(32-558), respectively.

[0188] (3) Immunogen and tag protein expression

[0189] The Sf9 cell concentration was diluted to 0.5 × 10⁻⁶. 6 After spreading the sample at a density of 1 / mL, evenly distribute 2 mL per well in a 6-well plate and incubate at 27°C for 30 minutes to ensure complete adhesion. Take a sterile EP tube and dilute 10 μL of Bacmid-SLC15A4(32-558) and Bacmid-flag-SLC15A4(32-558) with 90 μL of Grace medium (Thermo Fisher Scientific). Take another sterile EP tube and dilute 6 μL of transfection reagent cellfectin (Thermo Fisher Scientific) with 100 μL of Grace medium. Mix the two solutions thoroughly and incubate at room temperature for 30 minutes. Add 800 μL of Grace medium to the above mixture; aspirate the medium from the 6-well plate and add 2 mL of Grace medium to wash the cells; add the transfection solution dropwise into the cells and incubate at 27°C for 5 hours; aspirate the transfection solution and add 2 mL of ESF921 insect cell culture medium (Expression Systems) containing 10% fetal bovine serum; after incubation at 27°C for 72 hours, collect the supernatant, which is the P1 virus. Since the virulence of the P1 virus is relatively weak, further amplification of the viral particles is needed for subsequent large-scale expression. Add 10 mL of 2×10⁻⁶ g / mL of the transfection solution to the cells. 6 Add 5% P1 virus to Sf9 insect cells per mL, incubate at 27°C with shaking for 72 hours, and collect the supernatant to obtain P2 virus. Continue this process until P4 virus is finally used as the expression virus.

[0190] (4) Protein expression and purification

[0191] Following the instructions for the Bac-to-Bac baculovirus expression system (Invitrogen), human SLC15A4 (32-558) or human SLC15A4 (32-558) with an N-terminal flag tag was expressed alone in Sf9 insect cells. Sf9 insect cells were grown at a density of 2–2.5 × 10⁻⁶ cells / year. 6 Cells were infected with 2% (v / v) P4 baculovirus at a concentration of cells / mL and cultured with shaking at 27°C for 72 hours before centrifugation to collect the cells. The cells were then resuspended in lysis buffer supplemented with 1 mM PMSF (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM imidazole, 10% glycerol) and homogenized by sonication on ice.

[0192] The protein was incubated at 4°C with gentle agitation for 2 hours using a final concentration of 1% (w / v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) and 0.1% (w / v) Cholesteryl Hemisuccinate Tris Salt (CHS, Anatrace). After centrifugation at 18,000 rpm for 30 minutes at 4°C to remove insoluble fractions, the supernatant was incubated with equilibrated nickel column stock and gently stirred for 1 hour. The resin was then washed with lysis buffer supplemented with 0.06% glyco-diosgenin (GDN, Anatrace). The target protein was then eluted with elution buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 mM imidazole, 10% glycerol, 0.12% GDN) and concentrated to 750 μL using a 50 kDa stop-volume concentrate. The protein was then further purified by gel filtration chromatography (SEC) on a Superdex 200, and the peak of the target protein was collected. After SDS-PAGE verification, the target proteins were pooled and concentrated to 5-10 mg / ml and stored at -80°C for later use.

[0193] 2. Immunization of Balb / C mice

[0194] The SLC15A4(32-558) protein obtained in step 1 was used as an immunogen. 20 μg of SLC15A4(32-558) protein was mixed with 200 μg of MnJ(β) adjuvant (Qimeng Biotechnology) to form a 0.2 mL suspension. Six-week-old Balb / c mice were immunized via subcutaneous injection at multiple sites. Immunization was repeated monthly for a total of three times. A final booster immunization was performed using 20 μg of unadjuvanted SLC15A4 protein, and single-cell sequencing analysis of the B-cell receptor (BCR) was performed 7 days later.

[0195] according to Figure 1An exemplary procedure is shown for immunizing mice, screening antibodies, and expressing and purifying them.

[0196] Figure 1 B shows an SDS-PAGE gel image of an SLC15A4 (32-558) protein sample after Ni-NTA resin affinity chromatography and size exclusion chromatography. The apparent molecular weight is approximately 60 kDa, and the sample exhibits good homogeneity and high purity.

[0197] 3. Single-cell BCR sequencing analysis

[0198] The immunized mice were sacrificed, and their spleens and draining lymph node cells were removed and sealed. The specific steps were as follows:

[0199] The isolated mouse cells were then mixed with blocking solution (PBS + 2mM EDTA + 10% mouse serum + TruStain FcX). TM The cells were co-incubated with anti-mouse CD16 / 32 antibody (1:100, Biolegend) for 20 minutes, then the blocked cells were co-incubated with human SLC15A4 (32-558) protein carrying the flag tag for 30 minutes. The cells were washed once with FACS buffer (PBS + 2mM EDTA + 2% BSA), centrifuged, and stained with secondary antibody, specifically CD3e (APC-eFluor). TM 780, Invitrogen), B220 (PerCP-Cy5.5, BioLegend), CD19 (PE-Cy7, BioLegend), GL7 (Alexa Fluor TM 488, Invitrogen), CD38 (BV421, BioLegend), DYKDDDDY Tag (PE, BioLegend) and LIVE / DEAD TM Stain with Fixable Violet DeadCell Stain Kit (Yellow, Invitrogen) for 30 minutes, wash once with FACS buffer, centrifuge, resuspend, and sort CD3+ using BD ArialIII. - B220 + CD19 + CD38 - GL7 + Flag + B cells were analyzed using single-cell BCR sequencing (10xgenomic). Sequencing data were analyzed using Cell Ranger Loupe VDJ Browser 4.0.0 software to select target sequences.

[0200] 4. Target antibody expression

[0201] The light and heavy chains of the target antibody selected in the previous step were subjected to codon optimization analysis, and the optimized sequences of the variable regions of the light and heavy chains were ligated into the pTT3 (Addgene) vector containing the constant region of the mouse light chain κ chain and the constant region of the heavy chain IgG2a, respectively. The mouse antibody constant region sequence was derived from the in vitro hybridoma cells JL2 (Xuyuan Zhang et al. The binding of a monoclonal antibody to the apical region of SCARB2 blocks EV71 infection. PROTEIN & CELL, 2017.8, 8(8):590~600.). The above antibody expression vectors were synthesized at Sangon Biotech and BGI Genomics.

[0202] The synthesized antibody expression vector was extracted using an endotoxin-free plasmid mini-extraction kit (purchased from Tiangen Biotech). The antibody light and heavy chains were then mixed in a 1:1 ratio with PEI transfection reagent and allowed to stand. The mixture was then added to 293F cells (purchased from ATCC) and cultured at 37°C for 5 days in a 5% CO2 shaker. The supernatant was then collected by centrifugation.

[0203] Protein A packing material (Captiva™ PriMAB, cat: CA-PRI-1000) was packed into the column, and the antibody was adsorbed through the affinity chromatography column. The bound antibody was eluted from the affinity chromatography column with 0.1M glycine (pH 2.7) elution buffer. The antibody in the collection tube was used to determine the protein concentration. The liquids in the tubes containing protein were combined, the antibody was diluted with PBS, added to a 30KD ultrafiltration centrifuge tube, and concentrated by centrifugation at 3000 rpm and 4°C. The solution in which the antibody was dissolved was replaced with PBS.

[0204] Example 2: Identification of functional antibodies against SLC15A4 in mice

[0205] 1. Construction of the detection vector

[0206] An antibody detection protein was constructed.

[0207] Primers were designed based on GenBank accession number XM_011537895.2 (SLC15A4 mRNA), and the primer sequences are shown in Table 2:

[0208] Table 2

[0209]

[0210] Using a synthetic human SLC15A4 fragment as a template, the full-length human SLC15A4 fragment (amino acid sequence shown in SEQ ID NO:1) was amplified using 15-F and 15-R. The IRES-GFP fragment was then amplified using the pIRES2-EGFP-puro vector (Addgene) as a template via IGF and IGR. Fusion PCR was then performed using 15-F and IGR, and the fusion PCR product was ligated to the pTT3 vector (Addgene) to obtain the plasmid pTT3-hSLC15A4-IRES-GFP. In this plasmid, the EGFP gene is located downstream of the SLC15A4 sequence; an EGFP signal can be observed when SLC15A4 is expressed. Subsequent SLC15A4 mutant expression vectors were constructed using pTT3-hSLC15A4-IRES-GFP as a template.

[0211] 2. Screening using SLC15A4 antibodies bound to the cell membrane surface

[0212] Using Lipofectamine transfection reagent (Thermo Fisher), pTT3-hSLC15A4-IRES-GFP, pTT3-hSLC15A4(32-558)-IRES-GFP, and pTT3-hSLC15A4(L14A / L15A)-IRES-GFP plasmids were transfected into 293T cells (purchased from ATCC). Twenty-four hours after transfection, cells expressing human SLC15A4, GFP-FL (SLC15A4-WT, primarily lysosomal localization), and its membrane-localized mutant cells GFP-LL / AA (SLC15A4-L14A / L15A, membrane localization) and GFP-32-558 (SLC15A4(32-558), membrane localization) were obtained. These cells were treated with PBS containing 2 mM EDTA. The cell suspension was then added to 96-well U-shaped culture plates and incubated for 10 hours. 5 Cells / 100 μl / well. Centrifuge at 2200 rpm for 3 minutes, discard the supernatant, add 1-2 μg / mL of the antibody obtained in Example 1, incubate at 4°C for 30 minutes, wash twice with FACS buffer (PBS + 2 mM EDTA + 2% BSA), centrifuge, stain with PE goat anti-mouse IgG antibody (Biolegend), wash once with FACS buffer. After centrifugation and resuspending, analyze the GFP and PE signals of the cells using flow cytometry (BD LSR Fortessa). GFP and PE double-positive wells are antibody-positive wells that bind to the hSLC15A4 membrane surface.

[0213] Figure 2A shows that the XY_107 antibody (Ab107) can effectively bind to SLC15A4 expressed on the cell membrane surface. Notably, wild-type SLC15A4, primarily located on the endolysosomal membrane, can also be detected by the XY_107 antibody as being expressed on the cell membrane, indicating that this molecule may be cyclically expressed between the endolysosomal membrane and the cell membrane.

[0214] 3. XY_107 antibody-mediated SLC15A4 endocytosis

[0215] To verify whether the XY_107 antibody can bind to SLC15A4 on the cell membrane surface and mediate endocytosis to exert an inhibitory function, the human monocyte cell line THP1 expressing SLC15A4 was first co-incubated with Fc receptor binding inhibitor antibody (Invitrogen) for 20 minutes, and then co-incubated with 1 μg / mL AF647-labeled XY_107 antibody for different times. The results were analyzed by flow cytometry and confocal microscopy.

[0216] Figure 2 B-2D shows that at 37°C, the ability of AF647-labeled XY_107 antibody to bind to THP1 cells is significantly enhanced. Confocal microscopy images show that there is also AF647 signal inside the cells, indicating that AF647-labeled XY_107 antibody can bind to a small amount of SLC15A4 expressed on the surface of THP1 cells and mediate endocytosis.

[0217] The amino acid sequences of the heavy chain variable region, light chain variable region, and heavy chain CDR1-3 and light chain CDR1-3 of antibody XY_107 are shown in Table 3 below. The nucleic acid sequences of the heavy chain variable region and light chain variable region of antibody XY_107 are shown in SEQ ID NO:19 and 20, respectively.

[0218] Table 3

[0219]

[0220] Example 3: Mouse anti-SLC15A4 antibody XY_107 inhibits the activation of the TLR7 / 8 signaling pathway in THP1

[0221] 1. Preparation of XY_107 antibody Fab fragment

[0222] To eliminate the influence of the antibody Fc fragment, a Pierce Fab kit (Thermo Scientific) was used. TM The XY_107 antibody Fab fragment was prepared and concentrated to ~10 mg / mL, aliquoted, and cryopreserved. The XY_107 Fab antibody used for functional experiments was processed using Toxin Eraser. TMEndotoxin Removal Kit (GenScript) removes endotoxins.

[0223] 2. The XY_107 antibody Fab fragment inhibits the activation of TLR7 / 8 signaling in THP1, leading to cytokine production.

[0224] THP1 was used at a rate of 1-2 × 10 5 80 μL of SLC15A4 antibody XY_107Fab fragment was seeded into 96-well plates, followed by incubation overnight with 80 μL of different dilutions. The next day, 40 μL of R848 (InvivoGen) was added to the cell culture wells to a final concentration of 5 μg / ml, and the cells were cultured for another 24 hours. The cell supernatant was then collected by centrifugation, and the expression of TNFα and IL6 was detected using a Human TNF ELISA kit (no. 3512-1H-20, MABTECH) and a Human IL-6 ELISA kit (no. 3460-1H-20, MABTECH) according to the kit instructions. The results are shown below. Figure 3 A and Figure 3 B in.

[0225] like Figure 3 As shown in Figures A to 3B, the SLC15A4 monoclonal antibody XY_107 effectively inhibited the expression of IL6 and TNFα induced by R848-induced TLR7 / 8 signal activation in THP1. Furthermore, the degree of inhibition was positively correlated with the concentration of the XY_107 antibody Fab fragment.

[0226] In addition, THP1 was 1-2×10 5 80 μL of the antibody was seeded into 96-well plates, followed by 80 μL of a 40 μg / ml diluted SLC15A4 antibody XY_107Fab fragment, and incubated overnight. The next day, 40 μL of different concentrations of R848 (InvivoGen) were added to the cell culture wells, and the cells were cultured for another 24 hours. The cell supernatant was then collected by centrifugation, and the expression of TNFα and IL6 was detected using the Human TNF ELISA Kits for Human (no. 3512-1H-20, MABTECH) and the Human IL-6 ELISA Kit (no. 3460-1H-20, MABTECH) according to the kit instructions. The results are shown below. Figure 3 C and Figure 3 D.

[0227] like Figure 3 As shown in C to 3D, the SLC15A4 monoclonal antibody XY_107 at different concentrations stimulated by R848 can effectively inhibit the expression of IL6 and TNFα induced by TLR7 / 8 signal activation in THP1.

[0228] In addition, THP1 was used at 5×105 200 μL of SLC15A4 antibody XY_107Fab fragment was seeded into 24-well plates, followed by incubation overnight with 200 μL of different dilutions of the antibody. The next day, 100 μL of R848 (InvivoGen) was added to the cell culture wells to bring the final concentration to 5 μg / ml, and the cells were cultured for another 1.5 hours. Cells were then collected by centrifugation for IFNB1 expression detection.

[0229] Specifically, total RNA was extracted from collected cells using TRIzol (Invitrogen), and the RNA was treated with RQ1 RNase-FreeDNase (Promega, M6101) to eliminate the influence of genomic DNA. Subsequently, cDNA was synthesized using Oligo(dT) and MMLV reverse transcriptase (Promega, M1705) according to the manufacturer's instructions. Using the cDNA as a template, real-time PCR quantification was performed on a QuantStudio Q7 (AppliedBiosystems) instrument using a PowerUp SYBRGreen Master Mix (Applied Biosystems, A25742). The results are shown below. Figure 3 In E, the primer sequences involved are shown in Table 4 below (using Actinβ as an internal reference).

[0230] Table 4

[0231]

[0232] like Figure 3 As shown in Figure E, the SLC15A4 monoclonal antibody XY_107 can effectively inhibit the expression of IFNB1 induced by R848-induced TLR7 / 8 signal activation in THP1. Furthermore, the degree of inhibition is positively correlated with the concentration of the XY_107 antibody Fab fragment.

[0233] In summary, as Figure 3 As shown in A-3E, the mouse anti-SLC15A4 antibody XY_107 inhibits the activation of the TLR7 / 8 signaling pathway in THP1, resulting in the production of cytokines.

[0234] Example 4: Mouse anti-SLC15A4 antibody XY_107 inhibits the activation of TLR7 / 8 / 9 signaling pathway in human PBMCs

[0235] This embodiment tested the inhibitory effect of SLC15A4 antibody XY_107 on the TLR7 / 8 / 9 signaling pathway in human PBMCs.

[0236] Human PBMCs (Peripheral blood mononuclear cells) were directly isolated and purified using density gradient centrifugation (Ficoll, Haoyang Biotechnology Co., Ltd.). Specifically, concentrated leukocytes were transferred to 50mL centrifuge tubes, each containing no more than 17.5mL, and then an equal volume of PBS-EDTA (PBS + 2mM EDTA) was added and mixed well. Using a 15mL pipette, 14mL of lymphocyte separation medium (Haoyang Biotechnology Co., Ltd.) was taken and inserted along the tube wall to the bottom. The lymphocyte separation medium was slowly added, rising slowly as it was added, until 11mL was added. Centrifuged at room temperature, 2500 rpm for 30 minutes, with the centrifuge set to slow acceleration and descent speed set to 1 for slow deceleration. After centrifugation, a narrow band of white membrane layer, mainly composed of mononuclear cells, was observed at the interface between the two liquid layers. The upper layer of liquid was aspirated, and the white membrane layer was removed using a pipette. Transfer the cells to a new 50 mL centrifuge tube, add PBS-EDTA to a final volume of 50 mL, centrifuge at 2000 rpm for 10 minutes, discard the supernatant, resuspend the pellet in PBS-2 (PBS + 2 mM EDTA + 2% FBS) to a final volume of 50 mL, centrifuge at 1000 rpm for 10 minutes, and repeat once. Discard the supernatant, resuspend the cells in PBS-2, transfer to a 15 mL centrifuge tube, and take a small number of cells for counting.

[0237] PBMC at 1×10 6 200 μL of SLC15A4 antibody XY_107Fab fragment was seeded into each well of a 24-well plate, followed by incubation overnight with 200 μL of different dilutions of the antibody. The next day, 100 μL of R848 (InvivoGen) or CpGA (InvivoGen) was added to each well to a final concentration of 5 μg / mL, and the cells were cultured for another 24 hours. The culture supernatant was then collected by centrifugation for cytokine detection.

[0238] The collected cell supernatant was used to detect IFN-α expression using a human IFN-α ELISA kit (no. 3425-1H-20, MABTECH). The ELISA experiment was performed according to the kit's instructions. The results are shown in... Figure 3 F and Figure 3 G in.

[0239] like Figure 3 As shown in F and 3G, the SLC15A4 monoclonal antibody XY_107Fab fragment can effectively inhibit the activation of IFN-α signaling in human PBMCs induced by the TLR7 / 8 agonist R848 or the TLR9 agonist CpGA.

[0240] Example 5: Mouse anti-SLC15A4 antibody XY_107 inhibits R848-induced THP1 activation and adhesion.

[0241] 1. Slc15A4 - / - Construction of THP1 cells

[0242] The SLC15A4 gRNA sequence (forward(F): caccgCTAACGACAGGATCGCTCCC (SEQ ID NO:#) and reverse(R): aaacGGGAGCGATCCTGTCGTTAGc (SEQ ID NO:#)) was annealed and then ligated into the lentiCRISPR v2 vector (Addgene) purified by BsmBI-V2 (NEB) via homologous recombination. The vector was then packaged into a lentivirus for infection into THP1 cells using standard lentiviral packaging methods. After selection with puromycin, the infected THP1 cells were cultured as single clones. Sequencing confirmed the frameshift mutation in the single clones as Slc15A4. - / - THP1 cells.

[0243] 2. XY_107 antibody Fab fragment inhibits R848-induced THP1 activation and adhesion.

[0244] Human monocyte cell line THP1 cells undergo morphological changes upon activation, transforming from round monocytes to an adherent state or further differentiating into macrophages. This process is accompanied by increased expression levels of CD14 and HLA-DR. To further demonstrate the inhibitory effect of XY_107 on the TLR7 / 8 pathway, THP1 cells or Slc15A4 cells were... - / - THP1 cells were used at a rate of 4 × 10 5 200 μL of the antibody XY_107Fab fragment diluted 40 μg / ml was seeded into 24-well plates, followed by 200 μL of the same antibody overnight. The next day, 100 μL of R848 (InvivoGen) was added to the cell culture wells to bring the final concentration to 5 μg / ml, or 100 μL of culture medium was added as a control. The cells were cultured for another 24 hours. The culture plates were then photographed under white light using a Nikon Eclipse Ti2 microscope, or the cells were cultured for another 4 days to detect the expression levels of HLA-DR and CD14 on the cell membrane surface.

[0245] like Figure 4 As shown in Figure A, the SLC15A4 monoclonal antibody XY_107Fab fragment can effectively inhibit R848-induced THP1 cell adhesion. Figure 4As shown in B and 4C, the SLC15A4 monoclonal antibody XY_107Fab fragment can effectively inhibit the expression levels of HLA-DR and CD14 induced by R848.

[0246] Example 6: Detection of the binding domain of SLC15A4 monoclonal antibody XY_107 to human SLC15A4

[0247] This embodiment tested the binding of the SLC15A4 monoclonal antibody XY_107 to the domain of human SLC15A4. Variants were constructed using n×GS to replace different regions of human SLC15A4: extracellular loop regions △LL1-2(63-72) (SEQ ID NO:25), △LL3-4(125-154) (SEQ ID NO:26), △LL5-6(216-222) (SEQ ID NO:27), △LL7-8(348-366) (SEQ ID NO:28), △LL9-10(429-453) (SEQ ID NO:29), and △LL11-12(511-532) (SEQ ID NO:30). These variants were expressed in HEK293T cells according to the method in Example 2.

[0248] HEK293T cells expressing the extracellular loop regions ΔLL1-2 (63-72), ΔLL3-4 (125-154), ΔLL5-6 (216-222), ΔLL7-8 (348-366), ΔLL9-10 (429-453), and ΔLL11-12 (511-532) were fixed with 2% paraformaldehyde. The fixed cells were then permeabilized with 1x Intracellular Fixation & Permeabilization Buffer (eBioscience) according to the manufacturer's instructions. After permeabilization, the cells were added to 96-well U-shaped culture plates and cultured for 10 days. 5Cells / 100 μl / well. Centrifuge at 2200 rpm for 3 minutes, discard the supernatant, add 1 μg / ml XY_107 antibody, resuspend, and incubate at 4°C for 30 minutes. Wash cells twice with 1x Intracellular Fixation & Permeabilization Buffer. Then incubate with 100 μL / well of PE goat anti mouse IgG antibody (BioLegend) at 4°C in the dark for 30 minutes. Wash cells once with 1x Intracellular Fixation & Permeabilization Buffer, centrifuge and resuspend, and analyze GFP and PE signals using an Attune NxT Flow cytometer (Thermo Fisher Scientific). Analyze the binding of the SLC15A4 monoclonal antibody XY_107 to HEK293T cells (FL) expressing human SLC15A4 and HEK293T cells expressing the above SLC15A4 mutant.

[0249] The results are as follows Figure 5 A and Figure 5 As shown in B: The SLC15A4 monoclonal antibody XY_107 does not bind to HEK293T cells expressing human SLC15A4TM7-TM8 replaced with GS (△LL7-8) and HEK293T cells expressing human SLC15A4TM9-TM10 replaced with GS (△LL9-10). The binding level of HEK293T cells expressing human SLC15A4TM11-TM12 replaced with GS (△LL11-12) is reduced by about half. The binding level of HEK293T cells expressing human SLC15A4TM1-TM2 replaced with GS (△LL1-2), HEK293T cells expressing human SLC15A4TM3-TM4 replaced with GS (△LL3-4), and HEK293T cells expressing human SLC15A4TM5-TM6 replaced with GS (△LL5-6) is basically unaffected. It is speculated that the XY_107 antibody binds to the C-terminal domain (CTD) of SLC15A4.

[0250] Example 7: SLC15A4 monoclonal antibody XY_107 binds to SLC15A4 monomer but not to SLC15A4-TASL complex

[0251] 1. Constructing TASL expression vectors

[0252] A chimeric TASL protein coding sequence optimized for insect cell codons was synthesized (amino acid sequence shown in SEQ ID NO: 31), in which amino acids 1-18 at the N-terminus were replaced with the human TASL sequence, while the mouse TASL protein with the random sequence of amino acids 35-203 was deleted. This sequence was digested with BamHI-HF and HindIII-HF enzymes and then compared with pFastBac, which carries a C-terminal 2xStrep tag, using the same enzyme digestion. TM Dual plasmids were recovered and ligated to obtain expression vectors for the chimeric TASL.

[0253] 2. Expression of SLC15A4-TASL complex protein

[0254] Following the method in Example 1, Sf9 insect cells were prepared at a density of 2–2.5 × 10⁻⁶. 6 Cells were inoculated at a concentration of 2% (v / v) SLC15A4 and TASL P4 baculovirus, and cultured with shaking at 27°C for 72 hours before centrifugation to collect the cells. After two-step affinity chromatography (Ni-NTA resin and Strep-Tactin resin), the protein was further purified using Superdex 200. After SDS-PAGE validation, peak proteins were combined and concentrated to 5–10 mg / ml, then frozen at -80°C for later use.

[0255] 3. Detect the binding ability of SLC15A4 monoclonal antibody XY_107 to SLC15A4 protein and its complex.

[0256] To determine the binding affinity of the SLC15A4 monoclonal antibody XY_107 to the SLC15A4 protein, purified SLC15A4 (~6 mg / mL) was incubated with 1.2-fold excess of the XY_107 Fab fragment (Fab107) on ice for 30 min in SEC buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, and 0.01% GDN). The sample was then loaded onto a Superdex G200 Increase 10 / 300GL column equilibrated with SEC buffer, and peak tips were collected for SDS-PAGE validation. The results are shown in Figures 1-2. Figure 6 A and Figure 6 C.

[0257] like Figure 6 A and Figure 6 As shown in C, the SLC15A4 monoclonal antibody XY_107Fab fragment (Fab107) can effectively bind to the SLC15A4 protein expressed alone.

[0258] To determine the binding affinity between the SLC15A4 monoclonal antibody XY_107 and the SLC15A4-TASL complex, purified SLC15A4-TASL complex (~6 mg / mL) was incubated with a 1.2-fold excess of the Fab fragment (Fab107) of the monoclonal antibody XY_107 on ice for 30 min in SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, and 0.01% GDN). The sample was then loaded onto a Superdex G200 Increase 10 / 300GL column equilibrated with SEC buffer, and peak tips were collected for SDS-PAGE validation. The results are shown in Figures 1-2. Figure 6 B to Figure 6 C.

[0259] like Figure 6 B and Figure 6 As shown in C, the SLC15A4 monoclonal antibody XY_107Fab fragment (Fab107) cannot effectively bind to the SLC15A4-TASL complex protein.

[0260] Example 8: Binding assay of SLC15A4 homeoline antibody

[0261] 1. Antibody sequence information

[0262] Ten antibodies (108-117) from the same germline as XY_107 were synthesized using the method described in Example 1. The amino acid sequences of the variable region of the heavy chain of the SLC15A4 monoclonal antibody XY_107 and its germline-derived antibodies were analyzed by single-cell BCR sequencing using the method described in Example 2. Sequence alignment analysis was performed using BioEdit version 7.2.5. The results are as follows. Figure 7 and Figure 8 As shown.

[0263] from Figure 7 It can be seen that the amino acid sequences of the heavy chain of antibodies XY_108 to XY_117 (SEQ ID NO:32 to 41) have different degrees of amino acid mutations compared to the heavy chain amino acids of antibody XY_107.

[0264] like Figure 8 As shown, the light chain amino acid sequences (SEQ ID NO: 42-47) of antibodies XY_109, XY_110, XY_112, XY_113, XY_114, and XY_116 all exhibit varying degrees of amino acid mutations compared to the light chain amino acid sequence of antibody XY_107. However, the light chain amino acid sequences of antibodies XY_108, XY_111, XY_115, and XY_117 are identical to those of antibody XY_107.

[0265] The heavy chain HCDR1-3 and light chain LCDR1-3 of antibodies XY_108-XY_117 are shown in Table 5 below:

[0266] Table 5: Sequences of the heavy chain HCDR1-3 and light chain LCDR1-3 of antibodies XY_108-XY_117.

[0267]

[0268]

[0269] 2. Antibody function testing

[0270] The antibody was expressed using the method described in Example 1, and its ability to bind to SLC15A4 and inhibit TLR7 / 8 / 9 signal activation was tested. As shown in Table 6 below, antibodies XY_108 to XY_117 were able to bind to SLC15A4 and inhibit TLR7 / 8 / 9 signal activation. The results indicate that changes in some amino acids did not affect antibody binding and function.

[0271] Table 6

[0272] Heavy chain Light chain hSLC15A4 combined TLR7 / 8 / 9 inhibition 107H 107L +++ +++ 108H 108L +++ +++ 109H 109L +++ +++ 110H 110L +++ +++ 111H 111L +++ +++ 112H 112L +++ +++ 113H 113L +++ +++ 114H 114L +++ +++ 115H 115L +++ +++ 116H 116L ++ ++ 117H 117L ++ ++

[0273] Example 9: The SLC15A4 monoclonal antibody XY_107 binds to the C-terminal domain (CTD) of human SLC15A4, stably forming an outward (intralysosomal) opening conformation.

[0274] 1. Assembly and purification of Nanodisc (nanophospholipid disks)

[0275] The purified SLC15A4 protein, the Fab fragment of the SLC15A4 antibody XY_107, membrane scaffold protein 1D1 (MSP1D1, amino acid sequence as shown in SEQ ID NO:81), and phospholipid POPG (Avanti Polar Lipids, 268550-95-4) were mixed at a molar ratio of 1:1.1:2.2:120 and then gently stirred and incubated on ice for 1 hour. Bio-beads were added to a final concentration of 100 mg / mL and the mixture was continuously rotated overnight at 4°C. The next day, the Bio-beads were removed, and the protein was purified using a Superdex 200 buffer pre-equilibrated with Nanodiscbuffer. The peak protein was collected, and after SDS-PAGE analysis, it was concentrated to 15 mg / mL using a 10 kDa cutoff concentration tube for cryo-electron microscopy sample preparation.

[0276] 2. Cryo-electron microscopy sample preparation and data collection

[0277] Turn on the Vitrobot Mark IV (Thermo Fisher Scientific) power supply, replace the filter paper, and set the temperature to 8°C and the humidity to 100%. Place the copper cup, cold bridge, sample box, and other tools into the sample preparation foam box, and pre-cool with liquid nitrogen for 20 minutes. Slowly introduce ethane gas into the copper cup. Due to the low temperature, the ethane will slowly liquefy. When the liquefied ethane almost fills the copper cup, quickly close the ethane valve. Using tweezers, pick up the grid and place it face up in the Salarus glow discharge instrument. After evacuating the vacuum, perform glow discharge on the grid using oxygen and argon. After 30 seconds, stop the glow discharge, release the vacuum, and remove the grid. When the liquid ethane in the copper cup becomes a solid-liquid mixture due to the low temperature, use tweezers to pick up the grid and fix it onto the Vitrobot Mark IV. Pipette 3 μL of protein sample from the Vitrobot onto the grid and prepare the sample under the conditions of wait time 5 s, blot force -6 to 2, and blot time 3 to 5 s. Quickly transfer the grid from the ethane to a cryostat pre-cooled in liquid nitrogen, and then store the cryostat in a liquid nitrogen tank.

[0278] Samples were initially screened using Talos 120 and Talos 200 electron microscopes. Extensive data collection was then performed on samples that performed well in the initial screening using a Talos Arctica 200kV FEG (Thermo Fisher Scientific). Data collection conditions were: voltage 200kV; pixel size... Electron Dosage The underfocus range is 0.8-1.2μm.

[0279] 3. Cryo-electron microscopy sample preparation

[0280] Motion correction for SLC15A4-Fab107 electron microscope images was performed using the MotionCor2 program. Micrographs unsuitable for further data processing were manually deleted, and the remaining images were then processed using cryoSPARC v3, with CTF estimation performed using CTFFIND or GCTF scripts. Thousands of particles were selected for 2D classification using a blobpicker, generating 2D averages for further template selection. A total of 1,202,084 particles were selected using the template picker. After two rounds of 2D classification, 407,127 particles with clear details were obtained. Next, 50,000 particles from the 2D classification were reconstructed in 3D, yielding a resolution of [resolution missing]. The structure. Subsequent CTF refinement and Bayesian polishing will increase the resolution to [missing information]. After processing the other two batches of data in a similar manner, 173,320 and 146,232 particles were obtained respectively. The REFINE3D dataset, after merging all particles, yielded a resolution of [resolution missing]. The structure was evaluated using ResMap and Golden Standard Fourier Shell Correlation, and all parameters were found to be normal.

[0281] 4. The SLC15A4 monoclonal antibody XY_107Fab fragment (Fab107) binds to the SLC15A4 CTD.

[0282] like Figure 9 A to Figure 9 As shown in E, in the hSLC15A4-Fab107 composite structure, Fab107 binds to the intralysosomal surface of the C-terminal domain of hSLC15A4, forming a relatively extensive interaction interface (~1000). The structure shows that the interaction region between Fab107 and hSLC15A4 mainly consists of two interaction surfaces, one of which is... Figure 9 The light chain of Fab107 shown in C interacts with the main side chains of loop(LL)7-8 and loop(LL)9-10 on the lysosomal side of SLC15A4 via hydrogen bonds. Specifically, this is achieved at Q27 of the light chain CDR1. L N28 L and N32 L The side chains (light and heavy chains are indicated by superscript, L represents the light chain and H represents the heavy chain) form hydrogen bonds with T360 and N441 of SLC15A4, respectively, and N92 of the light chain CDR3. L The main chain forms hydrogen bonds with T438 of SLC15A4. Secondly... Figure 9 The heavy chain of Fab107, as shown in D and 9E, forms hydrogen bonds and hydrophobic interactions with loops (LL)7-8, (LL)9-10, and (LL)11-12 on the lysosomal side of SLC15A4. That is... Figure 9 The side chain H52 of the Fab107 heavy chain shown in D H and W33 H Hydrogen bonds are formed between K431, E432, and T434 on loop (LL)9-10 on the lysosomal side of SLC15A4; E57 is a side chain of the Fab107 heavy chain. H D106 H and D108 H It forms hydrogen bond interactions with the side chains R350, N526, N436, and H445 of SLC15A4; and Figure 9 E shows the W107 of the Fab107 heavy chain CDR3. HY102 H Y103 H and Y105 H It forms a hydrophobic interaction with F524 of SLC15A4.

[0283] 5. The SLC15A4 point mutation further confirms the existence of the aforementioned key binding sites.

[0284] This embodiment is to further confirm... Figure 9 A to Figure 9 Based on the key binding site of XY_107 to hSLC15A4 shown in the structure obtained from D, point mutation expression vectors for SLC15A4 were designed according to the conventional mutant construction protocol, including R350A / P361A, N436A / T438A / N441A / H445A, F524S, and N526A. The primer sequences used for construction are shown in Table 7.

[0285] Table 7

[0286]

[0287] Following the method in Example 6, the binding level of the SLC15A4 monoclonal antibody XY_107 to the HEK293T cells expressing the hSLC15A4 point mutation was detected. Figure 9 As shown in F, the F524S mutation has little effect on the binding level of hSLC15A4 to XY_107 antibody, while the N436A / T438A / N441A / H445A, R350A / P361A and N526A mutants all affect the binding of hSLC15A4 to XY_107 to varying degrees.

[0288] 6. The SLC15A4 monoclonal antibody XY_107 stabilizes SLC15A4 in an outward (inner lysosomal cavity) opening state.

[0289] like Figure 9 As shown in G, the structures are stacked around the CTD, showing that the CTD of hSLC15A4 with an inward open (cytoplasmic) conformation has some conflicts with the XY_107Fab fragment (Fab107). The main conflicts are between LL7-8, LL9-10 and TM9 of hSLC15A4 and the H and L chains of Fab107.

[0290] The CTD of the inward-open conformation of SLC15A4 conflicts with the binding of Fab107, providing a structural explanation for the reduced binding between Fab107 and the SLC15A4-TASL complex. Biochemical and structural results indicate that the tight interaction between Fab107 and hSLC15A4 enables Fab107 to stabilize SLC15A4 in an outward-open state, inhibiting SLC15A4 recruitment of TASL and blocking the SLC15A4-TASL-mediated TLR7 / 8 / 9 pathway.

[0291] Example 10: Changes in key amino acids of SLC15A4 monoclonal antibody significantly affect antibody binding and function.

[0292] 1. Antibody sequence information

[0293] Based on the complex structure information of SLC15A4 protein and XY_107 monoclonal antibody Fab fragment in Example 8, mutants of the heavy chain variable region and light chain variable region of the antibody were constructed, respectively. The names of the mutants and their mutation sites are shown in Table 8 below.

[0294] Table 8

[0295]

[0296] 2. Antibody function testing

[0297] The antibody was expressed using the method described in Example 1, and its ability to bind to SLC15A4 and inhibit TLR7 / 9 signaling activation was detected. As shown in Table 9, XY_107 monoclonal antibodies with mutations at key sites in either the heavy or light chain significantly affected the antibody's ability to bind to SLC15A4 and inhibit TLR7 / 8 / 9 signaling activation.

[0298] Table 9

[0299] Heavy chain Light chain hSLC15A4 combined TLR7 / 8 / 9 inhibition 107H 107L +++ +++ Mut1-H 107L + / - - mut2-H 107L + / - - Mut3-H 107L + / - - Mut4-H 107L + / - - 107H mut1-L + / - - 107H mut2-L + / - - 107H mut3-L + / - -

[0300] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that binds to SLC15A4, characterized in that, The antibody or its antigen-binding fragment inhibits the binding of SLC15A4 to TASL. Preferably, the antibody or its antigen-binding fragment binds to all or part of the CTD domain of human SLC15A4, stabilizing SLC15A4 in an outwardly opening conformation. Preferably, the antibody or its antigen-binding fragment binds to the region corresponding to amino acids 348-532 of human SLC15A4. Preferably, the antibody or its antigen-binding fragment binds to the region corresponding to amino acids 348–366, 429–453, and / or 511–532 of human SLC15A4.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes: (1) Heavy chain variable region complementarity-determining region (HCDR): HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in any one of SEQ ID NO:11 and 32-41, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region. HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any one of SEQ ID NO:11 and 32-41, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region; and HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any one of SEQ ID NO:11 and 32-41, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or (2) Complementarity-determining region (LCDR) of light chain variable region: LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in any one of SEQ ID NO:12 and 42-47, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region. LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in any one of SEQ ID NO:12 and 42-47, or having an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region; and LCDR3, having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in any one of SEQ ID NO:12 and 42-47, or having an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region. The three HCDRs contained in the heavy chain variable region and / or the three LCDRs contained in the light chain variable region are defined by rules of Kabat, Chothia, IMGT, Martin, Contact, Honegger, Gelfand or combinations thereof.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment includes: Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:11, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:32, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:33, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:42, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:34, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:43, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:35, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:36, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:44, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:37, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:45, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:38, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:46, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:39, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or Having the amino acid sequence of HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:40, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence of LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:47, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region; and / or The amino acid sequence having HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:41, or having HCDR1, HCDR2, and HCDR3 with one or more amino acid substitutions, deletions, or additions compared to the heavy chain variable region; and / or having the amino acid sequence LCDR1, LCDR2, and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:12, or having LCDR1, LCDR2, and LCDR3 with one or more amino acid substitutions, deletions, or additions compared to the light chain variable region. The three HCDRs contained in the heavy chain variable region and / or the three LCDRs contained in the light chain variable region are defined by rules of Kabat, Chothia, IMGT, Martin, Contact, Honegger, Gelfand or combinations thereof.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes: HCDR1 has an amino acid sequence as shown in GX1SFTX2Y; HCDR2 has an amino acid sequence as shown in X3HPX4DSE; HCDR3, which has features such as CAX5WGX6X7X8X9X 10 DX 11 DX 12 FDX 13 The amino acid sequence shown in W; LCDR1, which has features such as KASQNVGX 14 NVX 15 The amino acid sequence shown; LCDR2, having the amino acid sequence shown in SASYRYS (SEQ ID NO:17); and, LCDR3, which has features such as CQQYNX 16 X 17 The amino acid sequence shown in PYTF, where X1 to X 17 It does not exist and / or is selected from any natural amino acid residues. Preferably, X1 is selected from Y, F, W, or H, more preferably from Y or F; and / or Preferably, X2 is selected from R, N, K, H, G, Q, S, T, Y, or C, more preferably from R or N; and / or Preferably, X3 is absent or selected from A, V, L, I, F, P, M, or W, preferably I; and / or Preferably, X4 is selected from G, N, Q, S, T, Y, or C, more preferably from S or T; and / or Preferably, X5 is selected from K, R, H, I, A, V, L, I, F, P, M, or W, more preferably from R or I; and / or Preferably, X6 is selected from G, N, Q, S, T, Y, C, A, V, L, I, F, P, M, or W, more preferably from A or S; and / or Preferably, X7 is selected from Y, F, W or H, more preferably from Y or F; and / or Preferably, X8 is selected from G, N, Q, S, T, Y, C, Y, F, W or H, more preferably from Y, F or N; and / or Preferably, X9 is selected from K, R, H, A, V, L, I, F, P, M, or W, more preferably from K, R, or I; and / or Preferably, X 10 Selected from Y, F, W, H, G, N, Q, S, T, Y or C, more preferably selected from Y or N; and / or preferably, X 11 Selected from Y, F, W, H, G, N, Q, S, T, Y or C, more preferably selected from W or R; and / or Preferably, X 12 Selected from Y, F, W, H, G, N, Q, S, T, Y, C, D, or E, more preferably selected from Y, F, S, or D; and / or Preferably, X 13 Selected from G, N, Q, S, T, Y, or C, more preferably selected from S or Y; and / or Preferably, X 14 Selected from G, N, Q, S, T, Y, C, R, K, or H, more preferably selected from T, R, or K; and / or Preferably, X 15 Selected from A, V, L, I, F, P, M, W, D, or E, more preferably selected from V or D; and / or Preferably, X 16 Selected from G, N, Q, S, T, Y, or C, more preferably selected from N or S; and / or Preferably, X 17 Selected from A, V, L, I, F, P, M, W, Y, F or H, more preferably selected from F or Y.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes: HCDR1, having an amino acid sequence as shown in SEQ ID NO:13, 48 or 49, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to it; HCDR2, having an amino acid sequence as shown in SEQ ID NO:14, 50 or 51, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to it; HCDR3, having an amino acid sequence as shown in any one of SEQ ID NO:15, 52-61, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions. LCDR1, having an amino acid sequence as shown in SEQ ID NO:16, 62 or 63, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to it; LCDR2, having the amino acid sequence shown in SEQ ID NO:17, or having an amino acid sequence with one or more amino acid substitutions, deletions, or additions; and LCDR3 having an amino acid sequence as shown in SEQ ID NO:18, 64 or 65, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence as shown in any one of SEQ ID NO: 11 and 32-41, or an amino acid sequence having at least 85% sequence identity with such sequence; and / or, the light chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence as shown in any one of SEQ ID NO: 12 and 42-47, or an amino acid sequence having at least 85% sequence identity with such sequence. Preferably, the antibody or its antigen-binding fragment comprises any one or more of the following: The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:11, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:32, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:33, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:42, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:34, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:43, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:35, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:36, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:44, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:37, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:45, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:38, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:46, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:39, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:40, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:47, or an amino acid sequence having at least 85% sequence identity with it. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:41, or an amino acid sequence having at least 85% sequence identity with it; and / or, the light chain variable region has an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity with it.

7. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is of type IgA, IgD, IgE, IgG, or IgM. Preferably, the antibody is of type IgG, more preferably of type IgG1, IgG2, IgG3, or IgG4. Preferably, the antigen-binding fragment includes scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, and dsFv. Preferably, the antibody is a bispecific antibody, a bispecific antibody, or a multispecific antibody. Preferably, the antibody or antigen-binding fragment includes murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

8. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. The expression vector according to claim 9, characterized in that, The expression vectors include prokaryotic expression vectors and eukaryotic expression vectors. Preferably, the eukaryotic expression vector includes a yeast expression vector, a mammalian expression vector, or an insect expression vector.

11. A host cell comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, the nucleic acid molecule as described in claim 7, or the expression vector as described in claim 9 or 10.

12. A chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7.

13. A modified immune cell comprising the chimeric antigen receptor of claim 12.

14. An antibody-drug conjugate comprising: The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 7; and A drug covalently linked to the antibody or its antigen-binding fragment.

15. A detection kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7.

16. A pharmaceutical composition comprising: an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7; and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition may also include other small molecule drugs, antibodies, or peptides. Preferably, the route of administration of the pharmaceutical composition is parenteral, injection, oral, or topical. Preferably, the pharmaceutical composition is in the form of an aqueous solution, suspension, powder, tablet, capsule, granule, injection, or infusion.

18. A method for diagnosing, treating, and / or preventing SLC15A4-related disease, the method comprising administering to a subject in need an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, a nucleic acid molecule as described in claim 8, an expression vector as described in claim 9 or 10, a host cell as described in claim 11, a chimeric antigen receptor as described in claim 12, an immune cell as described in claim 13, and / or an antibody-drug conjugate as described in claim 14.

19. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the expression vector of claim 9 or 10, the host cell of claim 11, the chimeric antigen receptor of claim 12, the immune cell of claim 13, and / or the antibody-drug conjugate of claim 14 in the diagnosis, treatment and / or prevention of SLC15A4-related diseases.

20. The method of claim 18 or the use of claim 19, wherein the SLC15A4-related diseases include inflammatory diseases. Preferably, the inflammatory disease is caused by elevated expression of type I interferon or inflammatory cytokines. Preferably, the SLC15A4-related diseases include tumors, systemic lupus erythematosus, colitis, inflammatory bowel disease, type II diabetes, multiple sclerosis, psoriasis, autoimmune thyroid disease, rheumatoid arthritis, lupus, and glomerulonephritis.

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