Anti-IL-8 antibodies, multispecific antibodies against PD-L1 and IL-8 and uses

Abstract

The invention relates to an anti-IL-8 antibody, a PD-L1 and IL-8 targeting multispecific antibody and application of the anti-IL-8 antibody and the multispecific antibody in anti-tumor treatment. Specifically, an anti-PD-L1 antibody and an anti-IL-8 antibody are connected to form a multispecific antibody, and the antibody can target PD-L1 molecules on the surface of a tumor cell and IL-8 in a tumor tissue at the same time. Experimental results show that the multispecific antibody can effectively inhibit the growth of tumor cells and enhance the infiltration of immune cells in tumor tissues, so that the killing effect on the tumor cells is promoted.

Description

Technical Field

[0001] This invention belongs to the biomedical field of tumor treatment, and specifically relates to the structure and use of isolated anti-IL-8 antibodies or their antigen-binding fragments, and multispecific antibody molecules (e.g., bispecific antibodies) against PD-L1 and IL-8. Background Technology

[0002] Programmed death receptor ligand 1 (PD-L1) is an important immune checkpoint molecule highly expressed on the surface of various tumor cells. PD-L1 binds to the T cell surface receptor PD-1, transmitting negative regulatory signals that inhibit T cell activation, proliferation, and effector function, allowing tumor cells to evade the immune system. The PD-L1 and PD-1 pathways play a crucial role in tumor immune escape and have become important targets for tumor immunotherapy. Blocking the PD-1 / PD-L1 pathway can relieve the immunosuppression of T cells by tumor cells, restore and enhance the body's anti-tumor immune response, thereby controlling and eliminating tumors.

[0003] Interleukin-8 (IL-8) is a pro-inflammatory chemokine highly expressed in various solid tumor tissues, such as colorectal cancer, breast cancer, and lung cancer. IL-8, by binding to its receptors CXCR1 and CXCR2, recruits neutrophils and bone marrow-derived suppressor cells to infiltrate the tumor microenvironment. These cells secrete various immunosuppressive factors, such as IL-10 and TGF-β, forming an immunosuppressive microenvironment that inhibits the killing function of T cells and NK cells, promoting tumor immune escape. Furthermore, IL-8 can directly promote the proliferation, invasion, and metastasis of tumor cells. Therefore, IL-8 and its receptor signaling pathway play a crucial role in tumorigenesis, development, and immune escape, making it a potential drug target for cancer therapy.

[0004] Monoclonal antibody drugs targeting PD-L1, such as atezolizumab and durvalumab, specifically block the PD-1 / PD-L1 pathway, relieving tumor cell immunosuppression of T cells and restoring the anti-tumor immune response of T cells. They have achieved good efficacy in the treatment of various solid tumors, significantly prolonging overall survival. However, current PD-1 / PD-L1 antibody drugs still face some limitations, such as primary or secondary resistance in some patients, suggesting that blocking only a single immunosuppressive pathway may have limited effectiveness. Therefore, exploring combination therapy strategies targeting multiple immune regulatory pathways holds promise for further improving the efficacy of tumor immunotherapy.

[0005] Bispecific antibodies integrate two antigen-binding sites with different specificities into a single molecule, enabling them to simultaneously recognize two different antigenic epitopes and exert a synergistic effect. However, they face challenges such as high development difficulty and the large molecular weight of the antibody making it difficult to reach the target site.

[0006] Nanobodies are antibody fragments containing only a single heavy chain variable region (VHH domain) and smaller molecular weight, possessing advantages such as high stability, strong tissue penetration, and low immunogenicity. Combining bispecific antibodies with nanobody technology holds promise for obtaining novel antibody molecules that combine the advantages of both bispecificity and nanobodies, showing broad application prospects in the treatment of diseases such as cancer.

[0007] Although both the PD-L1 and IL-8 pathways play crucial roles in tumor immune escape, there are currently no reports of bispecific antibodies simultaneously targeting both molecules. Developing bispecific antibodies targeting PD-L1 and IL-8 holds promise for enhancing tumor immunogenicity and synergistically activating the body's anti-tumor immune response by blocking multiple immunosuppressive pathways. Compared to using PD-L1 or IL-8 antibodies alone, these bispecific antibodies may exhibit stronger anti-tumor efficacy and broader indications. Furthermore, by leveraging the tumor-targeting properties of PD-L1 and adjusting the affinity of the two domains, we can significantly increase the targeting of anti-IL-8, improve utilization, and reduce safety risks. Therefore, developing PD-L1 / IL-8 bispecific antibodies provides a new approach to improving the efficacy of tumor immunotherapy and has significant scientific and practical value. Summary of the Invention

[0008] Through experimental research, the applicant has developed an anti-IL-8 antibody, a multispecific antibody (e.g., a bispecific antibody) based on the antibody that targets programmed death receptor ligand 1 (PD-L1) and interleukin 8 (IL-8), a nucleotide sequence encoding the antibody, and the use of the antibody in the treatment or prevention of diseases such as cancer associated with PD-L1 and IL-8 activity.

[0009] In one aspect, this application provides an isolated anti-IL-8 antibody or an antigen-binding fragment thereof, comprising: HCDR1 containing the amino acid sequence shown in either SEQ ID NO:1 or 21, HCDR2 containing the amino acid sequence shown in either SEQ ID NO:2 or 22, and HCDR3 containing the amino acid sequence shown in either SEQ ID NO:3 or 23.

[0010] In another aspect, this application provides a multispecific antibody comprising: (1) a first antigen-binding portion specifically binding to IL-8; and (2) a second antigen-binding portion specifically binding to PD-L1.

[0011] In another aspect, this application relates to a polynucleotide encoding the aforementioned anti-IL-8 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, or the first antigen-binding portion.

[0012] In another aspect, this application relates to expression vectors comprising the aforementioned polynucleotides.

[0013] In another aspect, this application relates to host cells incorporating the aforementioned polynucleotides or their expression vectors.

[0014] In another aspect, this application relates to an immunoconjugate comprising the aforementioned anti-IL-8 antibody or its antigen-binding fragment, or the aforementioned multispecific antibody.

[0015] In another aspect, this application relates to a pharmaceutical composition comprising: the above-described anti-IL-8 antibody or its antigen-binding fragment, the above-described multispecific antibody or its immunoconjugate, and optionally pharmaceutically acceptable excipients.

[0016] In another aspect, this application relates to a pharmaceutical combination product comprising the aforementioned anti-IL-8 antibody or its antigen-binding fragment and anti-PD-L1 antibody or its antigen-binding fragment.

[0017] In another aspect, this application relates to a kit comprising the aforementioned anti-IL-8 antibody or its antigen-binding fragment, the aforementioned multispecific antibody or its immunoconjugate, a pharmaceutical composition, or a pharmaceutical combination product.

[0018] In another aspect, this application relates to the use of the aforementioned multispecific antibody or its immunoconjugate, pharmaceutical composition comprising the same, pharmaceutical combination product comprising the same, or kit in the preparation of a dual inhibitor of IL-8 and PD-L1. Alternatively, this application relates to the aforementioned multispecific antibody or its immunoconjugate, pharmaceutical composition, pharmaceutical combination product, or kit as a dual inhibitor of IL-8 and PD-L1. Alternatively, this application relates to a method for inhibiting both IL-8 and PD-L1, comprising administering to a subject in need the aforementioned multispecific antibody or its immunoconjugate, pharmaceutical composition comprising the same, pharmaceutical combination product comprising the same, or kit.

[0019] In another aspect, this application relates to the use of the aforementioned multispecific antibody (such as a bispecific antibody) or its immunoconjugate, pharmaceutical compositions comprising it, pharmaceutical combination products comprising it, or kits in the preparation of medicaments for the treatment or prevention of diseases associated with both the PD-1 / PD-L1 and IL-8 / CXCR1 / 2 signaling pathways. Alternatively, this application relates to a method for the treatment or prevention of diseases associated with both the PD-1 / PD-L1 and IL-8 / CXCR1 / 2 signaling pathways, comprising administering the aforementioned multispecific antibody (such as a bispecific antibody) or its immunoconjugate, pharmaceutical compositions comprising it, pharmaceutical combination products comprising it, or kits to a subject in need. Alternatively, this application relates to the aforementioned multispecific antibody (such as a bispecific antibody) or its immunoconjugate, pharmaceutical compositions comprising it, pharmaceutical combination products comprising it, or kits for the treatment or prevention of diseases associated with both the PD-1 / PD-L1 and IL-8 / CXCR1 / 2 signaling pathways.

[0020] In some implementations, the disease associated with both the PD-1 / PD-L1 signaling pathway and the IL-8 / CXCR1 / 2 signaling pathway is cancer.

[0021] Beneficial effects

[0022] The bispecific antibodies described herein include anti-PD-L1 antibodies and anti-IL-8 antibodies with different affinities, wherein the anti-IL-8 antibody has a lower affinity than the anti-PD-L1 antibody. Through research, the inventors have discovered that the combined use of anti-PD-L1 and anti-IL-8 antibodies exhibits a significant synergistic anti-tumor effect compared to using either antibody alone. This bispecific antibody first specifically binds to and accumulates in tumor tissue via its relatively high-affinity anti-PD-L1 terminus, and then blocks the pro-inflammatory and immunosuppressive effects of local IL-8 in the tumor via its relatively low-affinity anti-IL-8 terminus. This maximizes the synergistic anti-tumor effect of dual targeting of PD-L1 and IL-8, while simultaneously reducing the safety risks associated with systemic IL-8 blockade. In addition, blocking IL-8 and its receptor signaling pathway can significantly promote T cell infiltration into tumor tissue. Combining anti-IL-8 antibody with anti-PD-L1 antibody can not only relieve the immunosuppressive effect of tumor cells on T cells by blocking the PD-1 / PD-L1 pathway, but also promote more T cells to infiltrate the tumor by blocking the IL-8 pathway. The two work synergistically to enhance the body's anti-tumor immune response, thereby improving the effect of tumor immunotherapy. Attached Figure Description

[0023] Figure 1 The structures of the four PD-L1 / IL-8 bispecific antibodies designed in Example 3 are shown.

[0024] Figure 2The results of expression level and purity screening of the four bispecific antibodies designed in Example 3 are shown, as well as the preferred structure of the bispecific antibody targeting PD-L1 and IL-8 determined by screening.

[0025] Figure 3 The results of SDS-PAGE analysis of the purified and unreduced bispecific antibodies targeting PD-L1 and IL-8 in Example 3 are shown.

[0026] Figure 4 The SEC-HPLC analysis results of the purified bispecific antibody targeting PD-L1 and IL-8 selected in Example 3 are shown.

[0027] Figure 5 The blocking effect of the bispecific antibody targeting PD-L1 and IL-8 on in vitro IL-8 / CXCR1 signaling, as determined in Example 5, is shown.

[0028] Figure 6 The blocking effect of the bispecific antibody targeting PD-L1 and IL-8 on in vitro IL-8 / CXCR2 signaling, as determined in Example 5, is shown.

[0029] Figure 7 The blocking effect of the bispecific antibody targeting PD-L1 and IL-8 on in vitro PD-1 / PD-L1 signaling, as determined in Example 5, is shown.

[0030] Figure 8 The in vivo tumor-suppressing effect of the bispecific antibody targeting PD-L1 and IL-8, as determined in Example 6, in a mouse tumor-bearing model is shown, and the results are compared with the corresponding monoclonal antibodies and the combination therapy of two monoclonal antibodies. Detailed Implementation

[0031] Next, the technical solution of this application will be described in more detail through exemplary embodiments, but the protection scope of this application is not limited thereto.

[0032] Anti-IL-8 antibody or its antigen-binding fragment

[0033] This application provides an isolated anti-IL-8 antibody or its antigen-binding fragment, which can specifically bind to IL-8. The anti-IL-8 antibody can be a monoclonal antibody, polyclonal antibody, monospecific antibody, or multispecific antibody (such as a bispecific antibody), as long as the antibody can specifically recognize and bind to IL-8. The anti-IL-8 antibody or its antigen-binding fragment provided in this application, by specifically binding to IL-8, can block the signal transduction of IL-8 with its receptors CXCR1 and CXCR2, thereby potentially playing a beneficial role in the treatment of IL-8-related diseases.

[0034] In one aspect, this application provides an isolated anti-IL-8 antibody or an antigen-binding fragment thereof, comprising: HCDR1 containing the amino acid sequence shown in either SEQ ID NO:1 or 21, HCDR2 containing the amino acid sequence shown in either SEQ ID NO:2 or 22, and HCDR3 containing the amino acid sequence shown in either SEQ ID NO:3 or 23.

[0035] In some preferred embodiments, the isolated anti-IL-8 antibody or its antigen-binding fragment described in this application comprises:

[0036] (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or

[0037] (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:23.

[0038] In some preferred embodiments, the isolated anti-IL-8 antibody or its antigen-binding fragment described in this application comprises a VHH (Variable Domain of Heavy Chain of Heavy Chain Antibody). Preferably, the VHH comprises an amino acid sequence shown in any one of SEQ ID NO:4, 20, 24 and 25, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0039] In some specific embodiments, amino acids that differ from the amino acid sequences shown in any of SEQ ID NO:4, 20, 24 and 25 are predominantly (or all) present in the FR region (backbone region).

[0040] In some embodiments, the isolated anti-IL-8 antibody described in this application is selected from anti-IL-8 nanobodies or anti-IL-8 heavy chain antibodies. In some embodiments, the anti-IL-8 nanobodies have the following composition: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some embodiments, the anti-IL-8 antibody may be a camel-derived antibody or a humanized antibody. In some embodiments, the anti-IL-8 antibody or its antigen-binding fragment contains a camel-derived FR region or a heavy chain FR region derived from human immunoglobulins.

[0041] In some embodiments, the isolated anti-IL-8 antibody or its antigen-binding fragment described in this application further includes an Fc region, a heavy chain constant region, a CH2 region, or a CH3 region. In some embodiments, the anti-IL-8 heavy chain antibody comprises a VHH, a CH2 region, and / or a CH3 region; preferably, the anti-IL-8 heavy chain antibody comprises a VHH and an Fc region.

[0042] In a further preferred embodiment, the heavy chain constant region and the Fc region are selected from the constant region and Fc region of IgG1, IgG2, IgG3, and IgG4 (especially human IgG1, IgG2, IgG3, and IgG4), or variants thereof. Exemplary variants include IgG1, IgG2, or IgG4 heavy chain constant region variants with site-specific modifications and amino acid substitutions, such as the AAA mutation, DLE mutation (Shields et al., 2002; Lazar et al., 2006), YTE mutation, and LS mutation (Ghetie et al., 1997; Zalevsky et al., 2010) known in the art.

[0043] In a preferred embodiment, the Fc region is the Fc region of human IgG1 or IgG4. In some embodiments, the Fc region comprises an amino acid sequence shown in SEQ ID NO:18 or 19, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0044] In some embodiments, the isolated anti-IL-8 antibody or its antigen-binding fragment described in this application exhibits the following biological activities:

[0045] (1) The isolated anti-IL-8 antibody or its antigen-binding fragment binds to the K+ of IL-8. D Value < 5 × 10 -9 M, for example, <3×10 -9 M;

[0046] (2) The isolated anti-IL-8 antibody or its antigen-binding fragment inhibits the IL-8 / CXCR1 / 2 (IL-8 / CXCR1 or IL-8 / CXCR2) signaling pathway with an IC50 value of less than 100 nM.

[0047] Anti-PD-L1 antibody or its antigen-binding fragment

[0048] The isolated anti-PD-L1 antibody or its antigen-binding fragment described in this application can block the PD-1 / PD-L1 pathway, relieve the immunosuppression of T cells by tumor cells, restore and enhance the body's anti-tumor immune response, thereby controlling and eliminating tumors. The isolated anti-PD-L1 antibody described in this application can be a murine antibody, chimeric antibody, humanized antibody, or fully human antibody; it can be a monoclonal antibody, polyclonal antibody, monospecific antibody, or multispecific antibody, etc.

[0049] In this application, the anti-PD-L1 antibody may be any anti-PD-L1 antibody or its antigen-binding fragment (such as Fab fragment, Fab' fragment, F(ab')2 fragment, Fd fragment, Fv fragment, dAb fragment, isolated CDR region, isolated heavy chain variable region and light chain variable region, or nanobody) known in the art, such as, but not limited to, atezolizumab, durvalumab, avelumab, sugemalimab, envafolimab, adebrelimab, etc.

[0050] In some embodiments, this application provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, HCDR3 containing the amino acid sequence shown in SEQ ID NO:7, LCDR1 containing the amino acid sequence shown in SEQ ID NO:8, LCDR2 containing the amino acid sequence shown in SEQ ID NO:9, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:10.

[0051] In some embodiments, this application provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising: a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:11, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0052] In some specific embodiments, amino acids that differ from the amino acid sequence shown in SEQ ID NO:11 or 12 by at least 80% sequence identity are mainly (or all) present in the FR region (backbone region).

[0053] In some preferred embodiments, the anti-PD-L1 antibody or its antigen-binding fragment described in this application further includes a heavy chain constant region and a light chain constant region.

[0054] In a further preferred embodiment, the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4, or variants thereof, and the light chain constant region is selected from the constant regions of human κ and λ chains, or variants thereof. Exemplary variants include IgG1, IgG2, or IgG4 heavy chain constant region variants with site-specific modifications and amino acid substitutions, such as the AAA mutation, DLE mutation (Shields et al., 2002; Lazar et al., 2006), YTE mutation, and LS mutation (Ghetie et al., 1997; Zalevsky et al., 2010) known in the art.

[0055] In some embodiments, the heavy chain constant region (CH) is an IgG1 or IgG4 heavy chain constant region, preferably a human IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0056] In some embodiments, the light chain constant region is the human κ chain constant region. In some embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0057] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain and a light chain, wherein the heavy chain includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:13; and the light chain includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:14. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0058] In this paper, the isolated anti-PD-L1 antibody or its antigen-binding fragment specifically binds to the PD-L1 antigen and thereby modulates PD-1 / PD-L1 signal transduction, which can be used to treat diseases related to PD-L1 signal transduction pathways or PD-L1-mediated overdose or undesirable signal transduction.

[0059] Multispecific antibodies

[0060] The multispecific antibody of this application can bind to two targets, IL-8 and PD-L1, with high affinity, and can simultaneously block the IL-8 / CXCR1 or CXCR2 signaling pathway and the PD-1 / PD-L1 signaling pathway. By blocking two different targets, the multispecific antibody of this application may have a potential synergistic effect and stronger efficacy, and can avoid the safety risks of systemic blockade caused by anti-IL-8 antibodies alone.

[0061] This application provides a multispecific antibody comprising: (1) a first antigen-binding portion that specifically binds to IL-8; and (2) a second antigen-binding portion that specifically binds to PD-L1.

[0062] In some embodiments, the multispecific antibody may further include an additional antigen-binding portion capable of specifically binding to one or more other target antigens (which may be any antigen other than IL-8 and PD-L1).

[0063] In some embodiments, the multispecific antibody may be a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0064] Bispecific antibodies can exist in forms including, but not limited to, scFv-scFv, scFv-Fab, Fab-scFv, IgG-scFv, DVD (dual variable domain)-Ig, Quadroma, knots-into-holes, common lightchain, CrossMab, CrossFab, (SEED)body, Leucine Zipper, Duobody, IgG1 / IgG2, etc. In a preferred embodiment, the bispecific antibody described herein is a dimer with a bilaterally symmetrical structure, comprising three functional domains, thereby providing a structurally stable, highly specific, and easily prepared bispecific antibody.

[0065] In some embodiments, the first antigen-binding portion may be any anti-IL-8 antibody, as long as it is an antibody or fragment capable of inhibiting or reducing the binding of IL-8 to its receptor, including anti-IL-8 antibodies known in the prior art and anti-IL-8 antibodies to be developed in the future.

[0066] In some embodiments, the first antigen-binding portion includes: HCDR1 containing the amino acid sequence shown in either SEQ ID NO:1 or 21, HCDR2 containing the amino acid sequence shown in either SEQ ID NO:2 or 22, and HCDR3 containing the amino acid sequence shown in either SEQ ID NO:3 or 23.

[0067] In some preferred embodiments, the first antigen-binding portion includes:

[0068] (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or

[0069] (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:23.

[0070] In some preferred embodiments, the first antigen-binding portion comprises a VHH, which includes an amino acid sequence shown in any of SEQ ID NO:4, 20, 24, and 25, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the VHH.

[0071] In some specific embodiments, amino acids that are different from those shown in any of the amino acid sequences represented by SEQ ID NO:4, 20, 24 and 25 are mainly (or all) present in the FR region (backbone region).

[0072] In some embodiments, the first antigen-binding portion may include a camel-derived anti-IL-8 antibody or a humanized anti-IL-8 antibody. In some embodiments, the first antigen-binding portion includes a camel-derived FR region or a heavy chain FR region derived from human immunoglobulins.

[0073] In some embodiments, the first antigen-binding portion is an anti-IL-8 nanobody containing the VHH.

[0074] In some embodiments, the first antigen-binding portion is an anti-IL-8 heavy chain antibody containing the VHH. In some preferred embodiments, the anti-IL-8 heavy chain antibody contains the VHH, the CH2 region, and / or the CH3 region; preferably, the anti-IL-8 heavy chain antibody contains the VHH and the Fc region. In a further preferred embodiment, the Fc region is selected from the Fc regions of IgG1, IgG2, IgG3, and IgG4 (especially human IgG1, IgG2, IgG3, and IgG4), or variants thereof. In a preferred embodiment, the Fc region is the Fc region of human IgG1 or IgG4.

[0075] In some embodiments, the second antigen-binding portion can be any anti-PD-L1 antibody, as long as it is an antibody or fragment capable of inhibiting or reducing the binding of PD-L1 to PD-1, including known anti-PD-L1 antibodies in the prior art (e.g., atezolizumab, durvalumab, avelumab, sugemalimab, envorimab, adebelimab) and anti-PD-L1 antibodies to be developed in the future.

[0076] In some embodiments, the second antigen-binding portion includes: HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, HCDR3 containing the amino acid sequence shown in SEQ ID NO:7, LCDR1 containing the amino acid sequence shown in SEQ ID NO:8, LCDR2 containing the amino acid sequence shown in SEQ ID NO:9, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:10.

[0077] In some embodiments, the second antigen-binding portion includes: a VH having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 11, and a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12. In some specific embodiments, amino acids differing from those shown in SEQ ID NO: 11 or 12, with at least 80% sequence identity, are predominantly (or all) present in the FR region (backbone region).

[0078] In some embodiments, the second antigen-binding portion further includes an immunoglobulin Fc domain linked to the C-terminus of the VH. In some embodiments, the immunoglobulin Fc domain is the Fc region of human IgG, such as the human IgG1 Fc region. In some embodiments, the first antigen-binding portion further includes an immunoglobulin CH1 domain linked to the C-terminus of the VH. Further, the CH1 domain is linked to the N-terminus of the Fc domain.

[0079] In a further preferred embodiment, the second antigen-binding portion further comprises a heavy chain constant region linked to the C-terminus of the VH, the heavy chain constant region being selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4, or variants thereof. Exemplary variants include IgG1, IgG2, or IgG4 heavy chain constant region variants with site-specific modifications and amino acid substitutions, such as the AAA mutation, DLE mutation (Shields et al., 2002; Lazar et al., 2006), YTE mutation, and LS mutation (Ghetie et al., 1997; Zalevsky et al., 2010) known in the art. In some embodiments, the second antigen-binding portion further comprises a heavy chain constant region from human IgG1 linked to the C-terminus of the VH (containing the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with SEQ ID NO:15).

[0080] In some embodiments, the second antigen-binding portion further includes a human κ-chain or λ-chain constant region attached to the C-terminus of the VL, preferably a human κ-chain constant region (containing the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it).

[0081] In some preferred embodiments, the second antigen-binding portion comprises an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:13; and the light chain comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:14.

[0082] In some embodiments, the second antigen-binding portion may be in the form of a Fab molecule, (Fab')2 molecule, VH-Fc fusion peptide, scFv-Fc fusion peptide, or IgG protein.

[0083] In some embodiments, the second antigen-binding portion may be a fusion protein of the aforementioned anti-PD-L1 antibody or its antigen-binding fragment (e.g., CDR, scFv, Fab, or (Fab')2) and an immunoglobulin Fc domain (preferably, the immunoglobulin Fc domain is linked to the C-terminus of the antigen-binding fragment). In some preferred embodiments, the second antigen-binding portion is linked to the first antigen-binding portion via the included immunoglobulin Fc domain, for example, covalently, preferably via a linker. Preferably, the linker is a flexible linker peptide with a length of 5-50, preferably 5-30 (e.g., 5, 10, 15, 20, 25, 30) amino acids (e.g., glycine, serine). In some specific examples, the linker may be (G5). p Or (G4S) p , where p is an integer from 1 to 6, preferably p is 1, 2, 3 or 4, for example G4S, (G4S)2, (G4S)3, (G4S)4, G5, (G5)2, (G5)3.

[0084] In some embodiments, the second antigen-binding portion is atezolizumab.

[0085] In some embodiments, the molecular structure of the multispecific antibody described in this application is a bispecific antibody binding human IL-8 and human PD-L1. In some preferred embodiments, the bispecific antibody described in this application exists in a "2+2" structural form. In some preferred embodiments, in the bispecific antibody, the CDR of the anti-human PD-L1 antibody is located in the Fab segment (bivalent), and the CDR of the anti-human IL-8 antibody is linked to the C-terminus of the Fc of the anti-human PD-L1 antibody in the form of a bivalent nanobody.

[0086] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the heavy chain variable region, CH1 domain, and Fc domain of the aforementioned anti-PD-L1 antibody, and the anti-IL-8 nanobody; the second polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the light chain variable region and CL domain of the aforementioned anti-PD-L1 antibody. In some specific examples, the second polypeptide chain comprises VL-CL.

[0087] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain of the aforementioned anti-PD-L1 antibody and the anti-IL-8 nanobody; and the second polypeptide chain comprises the light chain of the aforementioned anti-PD-L1 antibody.

[0088] In some embodiments, the bispecific antibody further includes a linker for connecting the Fc domain and the anti-IL-8 nanobody. In some specific examples, the first polypeptide chain, from N-terminus to C-terminus, sequentially includes the heavy chain variable region of the anti-PD-L1 antibody, the CH1 domain and the Fc domain, the linker, and the anti-IL-8 nanobody (i.e., VH-CH1-Fc-linker-VHH). In some specific examples, the first polypeptide chain, from N-terminus to C-terminus, sequentially includes the heavy chain of the anti-PD-L1 antibody, the linker, and the anti-IL-8 nanobody; the second polypeptide chain includes the light chain of the anti-PD-L1 antibody. In some specific examples, the linker is connected to the N-terminus of the anti-IL-8 nanobody.

[0089] In some embodiments, the heavy chain variable region of the anti-PD-L1 antibody includes: HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:7.

[0090] In some preferred embodiments, the heavy chain variable region of the anti-PD-L1 antibody includes a VH region having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:11. In some specific embodiments, the amino acids that differ from the amino acid sequence shown in SEQ ID NO:11, having at least 80% identity, are predominantly (or all) present in the FR region (backbone region).

[0091] In some preferred embodiments, the heavy chain variable region of the anti-PD-L1 antibody includes a VH having the amino acid sequence shown in SEQ ID NO: 11.

[0092] In some embodiments, the CH1 domain and Fc domain are derived from the heavy chain constant region of human IgG, preferably human IgG1 (including the amino acid sequence shown in SEQ ID NO:15).

[0093] In some embodiments, the linker is a flexible linker peptide with a length of 5-50, preferably 5-30 (e.g., 5, 10, 15, 20, 25, or 30) amino acids (e.g., glycine or serine). In some specific examples, the linker may be (G5). p Or (G4S) p , where p is an integer from 1 to 6, preferably p is 1, 2, 3 or 4, for example the connector can be G4S, (G4S)2, (G4S)3, (G4S)4, G5, (G5)2, (G5)3.

[0094] In some embodiments, the anti-IL-8 nanobody comprises: HCDR1 containing the amino acid sequence shown in either SEQ ID NO:1 or 21, HCDR2 containing the amino acid sequence shown in either SEQ ID NO:2 or 22, and HCDR3 containing the amino acid sequence shown in either SEQ ID NO:3 or 23.

[0095] In some preferred embodiments, the anti-IL-8 nanobody comprises:

[0096] (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or

[0097] (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:23.

[0098] In some embodiments, the anti-IL-8 nanobody (also known as a "single-domain antibody" or "VHH antibody") comprises an amino acid sequence shown in any of SEQ ID NO:4, 20, 24 and 25 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0099] In some specific embodiments, amino acids that are different from those shown in any of the amino acid sequences represented by SEQ ID NO:4, 20, 24 and 25 are mainly (or all) present in the FR region (backbone region).

[0100] In some specific embodiments, the heavy chain of the anti-PD-L1 antibody includes the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0101] In some embodiments, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0102] In some embodiments, the light chain variable region of the anti-PD-L1 antibody includes: LCDR1 containing the amino acid sequence shown in SEQ ID NO:8, LCDR2 containing the amino acid sequence shown in SEQ ID NO:9, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:10.

[0103] In some preferred embodiments, the light chain variable region of the anti-PD-L1 antibody includes a VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:12. In some specific embodiments, the amino acids that differ from the amino acid sequence shown in SEQ ID NO:12, having at least 80% identity, are predominantly (or all) present in the FR region (backbone region).

[0104] In some preferred embodiments, the CL domain originates from the constant regions of the human κ and λ chains, for example, from the constant region of the human κ chain. Preferably, the CL domain comprises the amino acid sequence shown in SEQ ID NO:16.

[0105] In some embodiments, the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with it.

[0106] In some embodiments, the bispecific antibody comprises a first polypeptide chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:17, and a second polypeptide chain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO:14.

[0107] In some embodiments, the bispecific antibody comprises a first polypeptide chain shown in SEQ ID NO:17 and a second polypeptide chain shown in SEQ ID NO:14.

[0108] In a preferred embodiment, the bispecific antibody has a higher affinity for the second antigen-binding portion of PD-L1 than for the first antigen-binding portion of IL-8, ensuring that the bispecific antibody is enriched in tumor tissue and achieves a good targeting effect.

[0109] The bispecific antibody described in this application can effectively block the interaction between PD-L1 and its receptor PD-1, and between IL-8 and its receptors CXCR1 or CXCR2, at both the protein and cellular levels. In a mouse model of subcutaneous transplantation of human breast cancer MDA-MB-231 cells, treatment with the bispecific antibody described in this application can significantly inhibit tumor growth.

[0110] Polynucleotides, vectors and host cells

[0111] This application relates to a polynucleotide encoding the aforementioned anti-IL-8 antibody or its antigen-binding fragment, the aforementioned multispecific antibody (such as a bispecific antibody) or its first antigen-binding portion, an expression vector containing the aforementioned polynucleotide, and a host cell integrated with the aforementioned polynucleotide or its expression vector.

[0112] In some embodiments, the expression vector may be any expression vector capable of expressing the antibody or its antigen-binding moiety described herein, including but not limited to naked plasmids, phage particles, yeast plasmids, adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), retroviruses (such as lentiviruses), poxviruses, papillomaviruses, papillomaviruses (such as SV40), rod-shaped viruses, or baculoviruses. For ease of production and purification, the expression vector may also contain secretory signal peptides, expression tags, etc.

[0113] It is known to those skilled in the art that, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the antibody or fragment thereof of this application can be synthesized using methods known in the art (e.g., de novo solid-phase DNA synthesis, PCR amplification). Given that specific amino acid sequences have been described in this application, those skilled in the art can easily modify one or more codons of the respective coding sequences of the antibodies or their antigen-binding fragments, or bispecific antibodies of this application without altering the amino acid sequences, to prepare many different nucleic acids.

[0114] Those skilled in the art can use conventional methods known in the art to construct nucleic acids encoding the antibodies or their antigen-binding fragments, or bispecific antibodies of this application, into suitable vectors for introduction into host cells for expression of the target protein. Vector components may include, but are not limited to, signal sequences, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. In the vector, the nucleic acid encoding the target protein is operatively linked to the promoter.

[0115] In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or any other cells suitable for preparing antigen-binding constructs. In some examples, the mammalian cell is, for example, Chinese hamster (CHO) ovarian cells, CHO-S cells, 293 cells, or monkey kidney cells.

[0116] The above-described expression vector can be introduced into suitable host cells using any conventional means known in the art, such as protoplast fusion, calcium phosphate precipitation, electroporation, viral transfection, gene gun, liposome transfection, or other conventional techniques, but not limited thereto.

[0117] Under conditions suitable for the expression of the target protein, the host cells described above are cultured under conventional conditions, and then the antibodies described herein are recovered from the host cells or the culture medium of the host cells by conventional protein separation and purification methods (e.g., high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A column affinity chromatography), size exclusion chromatography, etc.).

[0118] Immunoconjugates

[0119] This application relates to immunoconjugates comprising the aforementioned anti-IL-8 antibody or its antigen-binding fragment, or the aforementioned multispecific antibody (such as a bispecific antibody).

[0120] In some embodiments, this application provides antibody-drug conjugates (ADCs) comprising the aforementioned anti-IL-8 antibody or its antigen-binding fragment conjugated to a cancer therapeutic agent, or the aforementioned multispecific antibody (such as a bispecific antibody). In some embodiments, the anti-IL-8 antibody or its antigen-binding fragment, or the multispecific antibody (such as a bispecific antibody), is directly conjugated to the therapeutic agent. In some embodiments, the anti-IL-8 antibody or its antigen-binding fragment, or the multispecific antibody (such as a bispecific antibody), is conjugated to the therapeutic agent via a linker. In the ADC, the linker for conjugating the antibody and the therapeutic agent can be a cleavable linker, such as a peptide linker, disulfide bond, or hydrazone linker, or a non-cleavable linker.

[0121] Pharmaceutical Composition

[0122] This application relates to a pharmaceutical composition comprising: the above-described anti-IL-8 antibody or its antigen-binding fragment, the above-described multispecific antibody (such as a bispecific antibody) or its immunoconjugate, and optionally pharmaceutically acceptable excipients.

[0123] In some embodiments, the pharmaceutical composition further comprises a cancer therapeutic agent, such as a chemotherapy or targeted therapy drug for cancer.

[0124] The excipients described herein can be any pharmaceutically acceptable excipient, such as, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc. For other pharmaceutically acceptable pharmaceutical excipients, please refer to, for example, *Handbook of Pharmaceutical Excipients* (4th Edition), by RC Luo et al., translated by Zheng Junmin, Chemical Industry Press, 2005.

[0125] In some embodiments, the pharmaceutical composition may be in the form of a sterile aqueous solution, microemulsion, liposome, or powder. In some embodiments, the pharmaceutical composition may be in the form of a unit dose to facilitate administration to the patient at the desired dosage.

[0126] The dosage range of the pharmaceutical compositions described herein can be determined by clinicians based on experience, taking into account factors such as the method of administration (including administration time, administration interval, and route of administration), the patient's age, weight, gender or pathological condition, diet, excretion rate, and sensitivity to the drug.

[0127] Drug combination products

[0128] This application provides a drug combination product comprising an anti-IL-8 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof. This drug combination product achieves enhanced efficacy by targeting both IL-8 and PD-L1.

[0129] In some embodiments, the anti-IL-8 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are as described above.

[0130] In some embodiments, the anti-IL-8 antibody or its antigen-binding fragment is selected from the anti-IL-8 antibody or its antigen-binding fragment described above, HuMax-IL8 or its antigen-binding fragment, or a multispecific antibody or immunoconjugate comprising the anti-IL-8 antibody or its antigen-binding fragment described above or HuMax-IL8. In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment is selected from the anti-PD-L1 antibody or its antigen-binding fragment described above, atezolizumab or its antigen-binding fragment, or a multispecific antibody or immunoconjugate comprising the anti-PD-L1 antibody or its antigen-binding fragment described above or atezolizumab.

[0131] In some embodiments, the drug combination product comprises the anti-IL-8 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment (e.g., the aforementioned anti-IL-8 antibody or its antigen-binding fragment and atezolizumab, HuMax-IL8 and atezolizumab) in a molar ratio of 100:1 to 1:100 (e.g., 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3, 2:1 to 1:2, 1:1). In some embodiments, the anti-IL-8 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment may be administered simultaneously, sequentially, or at certain intervals.

[0132] In some embodiments, the anti-IL-8 antibody or its antigen-binding fragment is packaged separately from or together with the anti-PD-L1 antibody or its antigen-binding fragment.

[0133] In some embodiments, the drug combination product also includes additional therapeutic agents, such as additional cancer therapeutic agents, such as chemotherapy or targeted therapy drugs for cancer.

[0134] medicine box

[0135] This application relates to a kit comprising the aforementioned anti-IL-8 antibody or its antigen-binding fragment, the aforementioned multispecific antibody (such as a bispecific antibody) or its immunoconjugate, a pharmaceutical composition, or a pharmaceutical combination product.

[0136] In some embodiments, the pillbox may further include instructions for use. In some embodiments, the pillbox may further include reagents for diagnosing the patient. In some embodiments, the pillbox may further include a device for administering medication to the patient, such as a syringe.

[0137] In some embodiments, the kit may further include pharmaceutical excipients, such as sterile water or saline, for assisting in administering the aforementioned anti-IL-8 antibody or its antigen-binding fragment, the aforementioned multispecific antibody or its immunoconjugate, the pharmaceutical composition, or the pharmaceutical combination product to the patient.

[0138] Uses and treatments

[0139] This application relates to the use of the aforementioned multispecific antibodies (such as bispecific antibodies) or their immunoconjugates, pharmaceutical compositions comprising them, pharmaceutical combination products comprising them, or kits in the preparation of dual inhibitors of IL-8 and PD-L1.

[0140] This application relates to the use of the aforementioned multispecific antibody (such as a bispecific antibody) or its immunoconjugate, pharmaceutical compositions comprising it, pharmaceutical combination products comprising it, or kits in the preparation of medicaments for the treatment or prevention of diseases associated with both the PD-1 / PD-L1 and IL-8 / CXCR1 / 2 signaling pathways. Alternatively, this application relates to a method for treating or preventing diseases associated with both the PD-1 / PD-L1 and IL-8 / CXCR1 / 2 signaling pathways, comprising administering the aforementioned multispecific antibody (such as a bispecific antibody) or its immunoconjugate, pharmaceutical compositions comprising it, pharmaceutical combination products comprising it, or kits to a subject in need. Alternatively, this application relates to the aforementioned multispecific antibody (such as a bispecific antibody) or its immunoconjugate, pharmaceutical compositions comprising it, pharmaceutical combination products comprising it, or kits for the treatment or prevention of diseases associated with both the PD-1 / PD-L1 and IL-8 / CXCR1 / 2 signaling pathways.

[0141] In some implementations, the diseases associated with both the PD-1 / PD-L1 signaling pathway and the IL-8 / CXCR1 / 2 signaling pathway are cancers, such as colorectal cancer, breast cancer, and lung cancer.

[0142] The multispecific antibodies (such as bispecific antibodies) or their immunoconjugates, pharmaceutical compositions, pharmaceutical combination products, or kits described in this application may be used to block the IL-8 / CXCR1 or CXCR2 and PD-1 / PD-L1 signaling pathways and inhibit the growth of tumors that highly express PDL1 and IL-8.

[0143] The anti-IL-8 antibody or its antigen-binding fragment described above, the multispecific antibody (such as a bispecific antibody) or its immunoconjugate described above, the pharmaceutical composition, the pharmaceutical combination product, or the kit described in this application may also be used to diagnose the presence of related antigens (IL-8 and / or PD-L1) in a sample.

[0144] The anti-IL-8 antibody or its antigen-binding fragment described above, the multispecific antibody (such as a bispecific antibody) or its immunoconjugate described above, the pharmaceutical composition, or the pharmaceutical combination product can be prepared into any dosage form known in the art, such as injection, suspension, solution, powder, emulsion, spray, tablet, pill, capsule, granule, ointment, suppository, gel, etc.

[0145] The anti-IL-8 antibody or its antigen-binding fragment described above, the multispecific antibody (such as a bispecific antibody) or its immunoconjugate described above, the pharmaceutical composition, or the pharmaceutical combination product described in this application are suitable for intravenous, intramuscular, intra-articular, intra-articular, subcapsular, subarachnoid, intraorbital, intracardiac, subcutaneous, parenteral, intraperitoneal, intraspinal, intranasal, or epidermal administration (e.g., by injection or infusion). The anti-IL-8 antibody or its antigen-binding fragment described above, the multispecific antibody (such as a bispecific antibody) or its immunoconjugate described above, the pharmaceutical composition, or the pharmaceutical combination product described in this application can be formulated into sterile aqueous solutions, microemulsions, liposomes, or powders.

[0146] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0147] definition

[0148] The term "antibody" in this document refers to a protein or polypeptide that can specifically recognize and bind to an antigen, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, single-domain antibodies, full-length antibodies, and antibody fragments that exhibit the desired biological activity, and can be classified into five isotypes based on heavy chain class: IgG, IgM, IgD, IgA, and IgE.

[0149] When referring to antibodies, the term "isolated" in this article means that the antibody is essentially free of other cellular components that bind to it in the natural state. For example, an isolated antibody may be an antibody that has been removed from the natural environment.

[0150] As used in this article, the term "bispecific" means that an antigen-binding construct (e.g., an antibody) contains two antigen-binding moieties (e.g., antigen-binding peptides) each with a specific binding specificity. For example, a first antigen-binding peptide and a second antigen-binding peptide may bind specifically to epitopes on a first antigen and a second antigen, respectively, or bind specifically to different epitopes of the same antigen, respectively.

[0151] The term "antigen-binding fragment" in antibody refers to a portion or segment of a full-length antibody with fewer amino acid residues than a full-length antibody, but capable of binding antigens or competing with full-length antibodies (i.e., full-length antibodies from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, single-chain Fv, diabody antibodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting full-length antibodies with papain. Furthermore, digestion of complete antibodies with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. The Fv fragment consists of VL (light chain variable region) and VH (heavy chain variable region) domains on the antibody single arm. The two domains, VL and VH, can be encoded by independent genes, but they can also be produced as a single protein chain using a recombinant approach, connecting the two domains with a synthetic linker. In this single protein chain, the VL and VH regions pair to form a single-chain Fv (scFv).

[0152] The term "scFv" in this article includes the VH and VL domains of an antibody that are present in a single polypeptide chain.

[0153] In this article, the term "heavy chain antibody" or "HcAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in 4-chain antibodies. Heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains consisting of a variable region (VHH) and other constant regions. The variable region is connected to the constant region via a hinge-like structure. Each heavy chain of camel family heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3).

[0154] The term "nanobody" in this article refers to a class of antibodies that lacks the antibody light chain and only has the variable region of the heavy chain. It is interchangeable with "single-domain antibody" and "VHH antibody".

[0155] The term “CDR” (complementarity-determining region), also known as “hypervariant region (HVR)”, as used in this article, refers to each region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. Natural antibodies typically contain three CDRs (i.e., HCDR1 to HCDR3) located in the heavy chain variable region and three CDRs (LCDR1 to LCDR3) located in the light chain variable region. Various numbering systems known in the art can be used to sequentially number the CDRs of heavy and light chains starting from the N-end. For example, Chothia is based on the three-dimensional structure of the antibody and the topology of the CDR loop; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartmentofHealth and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London) is based on antibody sequence variability; the International ImMunoGeneTics (IMGT) database (the international ImMunoGeneTics information system, http: / / imgt.cines.fr) is based on affinity propagation clustering using a large number of crystal structures; and North et al., “A New Clustering of AntibodyCDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011) is based on affinity propagation clustering using a large number of crystal structures.

[0156] Table 1. CDRs determined using different numbering systems

[0157] When referring to antibodies, the terms "variable region," "V region," or "variable domain" are used interchangeably. They refer to the structural domains of the antibody heavy or light chain that participate in the specific binding of the antibody to the antigen. They typically include an amino acid sequence arranged from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0158] The term "chimeric antibody" refers to an antibody containing sequences derived from two different antibodies (often from different species), for example, (a) altering, replacing, or exchanging a constant region or a portion thereof so that the antigen-binding site is linked to a constant region of different or altered class, effector function, and / or species origin, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that confers new properties to the chimeric antibody; or (b) altering, replacing, or exchanging a variable region or a portion thereof with a variable region of different or altered antigen specificity.

[0159] The term “humanized antibody” as used in this article refers to an antibody that retains the antigen-specific reactivity of a non-human antibody (such as an alpaca monoclonal antibody) while exhibiting low immunogenicity when administered to humans as a therapeutic agent; it typically includes CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans.

[0160] The term "epitope," also known as "antigenic determinant," as used in this article refers to an antigenic moiety that can be recognized and specifically bound by antibodies. An antigen may have multiple epitopes, which are typically composed of surface groups of a molecule, such as amino acids or sugar side chains.

[0161] The terms “flexible linker peptide”, “linker”, or “connector” used in this article refer to a short amino acid sequence composed of amino acids used for linking peptide segments, and may include glycine (G), alanine (A), threonine (T) residues, etc.

[0162] The term “operably linked” in this article refers to the linking of amino acid sequences, peptides, or proteins with different functional properties, such as linking a VH domain to a VL domain, or linking scFv to Fc, or linking scFv to a Fab or IgG domain, as described herein.

[0163] As used herein, the terms “binding” or “specific binding” mean that the binding is selective to the target antigen and can be distinguished from unwanted or nonspecific interactions. For example, an antibody that specifically binds to a target antigen means that the antibody has higher affinity, stronger binding activity, easier binding, and / or longer binding duration when binding to the target antigen compared to binding to other non-target molecules.

[0164] "Affinity" or "binding affinity" is an intrinsic binding capacity used to reflect the interaction between members of a binding pair. For example, the affinity of molecule X for its partner Y can usually be expressed by the equilibrium dissociation constant (K0). D The equilibrium dissociation constant is represented by the dissociation rate constant (K). dis or K off) and association rate constant (K a or K on The ratio of affinity to kinetic binding affinity. Affinity can be measured using common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay.

[0165] The "percentage of amino acid sequence identity" refers to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference sequence, relative to the total number of amino acid residues in the candidate sequence, after aligning the candidate sequence with a reference sequence and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. The percentage of amino acid sequence identity can be determined by aligning two or more sequences using tools known in the art, such as BLASTp, ClustalW2 (see Higgins DG et al., Methods Enzymol 1996, 266:383-402; Larkin MA et al., Bioinformatics 2007, 23:2947-2948), ALIGN, or Megalign (DNASTAR) software.

[0166] For polypeptide sequences, "conservative modification" includes the substitution, deletion, or addition of amino acids in the polypeptide sequence with other amino acids of the same class (e.g., amino acids with similar chemical properties or functions), without substantially altering the desired functional activity of the polypeptide sequence. For example, conserved substitution often results in a replacement of a certain amino acid with a chemically similar amino acid. Lists of conserved substitutions of functionally similar amino acids are well known in the art. The following are eight groups of amino acids containing mutually conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0167] The terms “subject,” “patient,” and “individual” are used interchangeably herein and include mammals or non-mammalian vertebrates (such as chickens, emus, and fish), including but not limited to domesticated animals (e.g., cattle, sheep, cats, dogs, pigs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats, guinea pigs, and hamsters), preferably humans.

[0168] The term "treating" (or "treatment") as used herein refers to reducing or alleviating a disease or symptom, slowing the onset or progression of a disease or symptom, reducing the risk of developing a disease or symptom, delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, achieving complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above. Desired therapeutic effects as described herein include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0169] As used herein, the term “optional” indicates whether the object it modifies is present or not; for example, “a pharmaceutical composition contains optional pharmaceutically acceptable excipients” means that the pharmaceutical composition may or may not contain pharmaceutically acceptable excipients.

[0170] The term "therapeutic effective amount" or "effective dose" refers to a dose or concentration that, at the required dose and for the required duration, is effective in preventing or improving symptoms associated with a disease or condition and / or reducing the severity of the disease or condition. The therapeutically effective amount of the formulations, antibodies, or antigen-binding fragments thereof, bispecific antibodies, or compositions of this application can vary depending on various factors such as disease state, individual age, sex, and weight, and the ability of the antibody or antigen-binding portion to elicit the desired response in the individual. A therapeutically effective amount can also be considered as the therapeutically beneficial effect of the formulation, antibody, or antigen-binding fragment thereof, bispecific antibody, or composition significantly outweighing any toxic or harmful effects it causes. The term "effective amount" refers to the amount of active ingredient or agent sufficient to provide a clinical benefit to a subject (including, but not limited to, improvement, relief, or reduction of a disease, condition, or related symptoms, delaying or stopping disease progression).

[0171] The terms “pharmaceutical acceptable” or “medicinally acceptable” in this article mean that the carrier, solvent, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.

[0172] In this paper, the terms "X" and "Xaa" are equivalent and refer to unspecified amino acids. Their scope is defined in the relevant descriptions. To distinguish multiple "X"s within the same amino acid sequence, each consecutive "X" is numbered (i.e., written as X...). n And define the scope they cover.

[0173] Unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (contain, contain, containing, include, include, including) used herein are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed.

[0174] Unless otherwise stated, all figures used herein to represent amounts of components, measurements, or reaction conditions should be understood to be modified in all cases by the term “about,” which means within an acceptable range of error for the corresponding value. When used with percentages, the term “about” may mean, for example, ±1%, preferably ±0.5%, more preferably ±0.1%.

[0175] Unless the context clearly indicates otherwise, singular terms in this document encompass the plural referents, and vice versa. Similarly, unless the context clearly indicates otherwise, the word "or" in this document is intended to include "and".

[0176] For purposes of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0177] Exemplary embodiments of the present invention are shown in the following numbered paragraphs, but the scope of protection of the present invention is not limited thereto:

[0178] 1. An isolated anti-IL-8 antibody or its antigen-binding fragment, comprising:

[0179] (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or

[0180] (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:23.

[0181] 2. The anti-IL-8 antibody or its antigen-binding fragment as described in paragraph 1, wherein the anti-IL-8 antibody or its antigen-binding fragment comprises an amino acid sequence shown in any one of SEQ ID NO:4, 20, 24 and 25 or an amino acid sequence having at least 80% sequence identity with it.

[0182] 3. The anti-IL-8 antibody or its antigen-binding fragment as described in paragraph 2, wherein all amino acids differing in the amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence shown in any of SEQ ID NO:4, 20, 24 and 25 are present in the FR region.

[0183] 4. The anti-IL-8 antibody or its antigen-binding fragment as described in any one of paragraphs 1-3, wherein the anti-IL-8 antibody is an anti-IL-8 nanobody or an anti-IL-8 heavy chain antibody.

[0184] 5. The anti-IL-8 antibody or its antigen-binding fragment as described in any one of paragraphs 1-4, wherein the anti-IL-8 antibody is a camel-derived antibody or a humanized antibody.

[0185] 6. The anti-IL-8 antibody or its antigen-binding fragment as described in paragraph 4, wherein the anti-IL-8 heavy chain antibody further comprises an Fc region comprising an amino acid sequence shown in SEQ ID NO: 18 or 19 or an amino acid sequence having at least 80% sequence identity with it.

[0186] 7. A multispecific antibody comprising: (1) a first antigen-binding moiety specifically binding to IL-8; and (2) a second antigen-binding moiety specifically binding to PD-L1.

[0187] 8. The multispecific antibody as described in paragraph 7, wherein the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0188] 9. The multispecific antibody as described in paragraph 7 or 8, wherein the first antigen-binding portion comprises:

[0189] (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or

[0190] (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:23.

[0191] 10. The multispecific antibody as described in any one of paragraphs 7-9, wherein the first antigen-binding portion comprises an amino acid sequence shown in any one of SEQ ID NO:4, 20, 24 and 25 or an amino acid sequence having at least 80% sequence identity with it.

[0192] 11. The multispecific antibody as described in paragraph 10, wherein all amino acids differing in the amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence shown in any of SEQ ID NO:4, 20, 24 and 25 are present in the FR region.

[0193] 12. The multispecific antibody as described in any one of paragraphs 7-11, wherein the first antigen-binding portion comprises a camel-derived anti-IL-8 antibody or a humanized anti-IL-8 antibody.

[0194] 13. The multispecific antibody as described in any one of paragraphs 7-12, wherein the first antigen-binding portion is an anti-IL-8 nanobody or an anti-IL-8 heavy chain antibody.

[0195] 14. The multispecific antibody as described in paragraph 13, wherein the anti-IL-8 heavy chain antibody further comprises an Fc region comprising an amino acid sequence shown in SEQ ID NO: 18 or 19 or an amino acid sequence having at least 80% sequence identity with it.

[0196] 15. The multispecific antibody as described in any one of paragraphs 7-14, wherein the second antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, HCDR3 containing the amino acid sequence shown in SEQ ID NO:7, LCDR1 containing the amino acid sequence shown in SEQ ID NO:8, LCDR2 containing the amino acid sequence shown in SEQ ID NO:9, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:10.

[0197] 16. The multispecific antibody as described in any one of paragraphs 7-15, wherein the second antigen-binding portion comprises: VH having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:11, and VL having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0198] 17. The multispecific antibody as described in paragraph 16, wherein all amino acids that differ in the amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:11 or 12 are present in the FR region.

[0199] 18. The multispecific antibody as described in any one of paragraphs 7-17, wherein the second antigen-binding portion further comprises:

[0200] The heavy chain constant region of human IgG1 containing the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:15; and / or

[0201] Human κ chain constant region containing the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80% sequence identity with it.

[0202] 19. The multispecific antibody as described in any one of paragraphs 7-18, wherein the second antigen-binding portion comprises an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:13, and the light chain comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:14.

[0203] 20. The multispecific antibody as described in any one of paragraphs 7-19, wherein the second antigen-binding portion is connected to the first antigen-binding portion by a linker.

[0204] 21. The multispecific antibody as described in paragraph 20, wherein the linker is (G5). p Or (G4S) p , where p is an integer from 1 to 6.

[0205] 22. The multispecific antibody as described in any one of paragraphs 7-21, wherein the second antigen-binding portion is atezolizumab.

[0206] 23. The multispecific antibody as described in paragraph 7, wherein the multispecific antibody is a bispecific antibody, and the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain:

[0207] The first polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the heavy chain variable region of the anti-PD-L1 antibody, the CH1 domain and the Fc domain, as well as the anti-IL-8 nanobody.

[0208] The heavy chain variable region of the anti-PD-L1 antibody includes: HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:7.

[0209] The anti-IL-8 nanobody comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:23;

[0210] The second polypeptide chain comprises, from the N-terminus to the C-terminus, the light chain variable region of the anti-PD-L1 antibody and the CL domain, wherein the light chain variable region of the anti-PD-L1 antibody includes: LCDR1 containing the amino acid sequence shown in SEQ ID NO:8, LCDR2 containing the amino acid sequence shown in SEQ ID NO:9, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:10.

[0211] 24. The multispecific antibody as described in paragraph 23, wherein the CH1 domain and Fc domain are derived from the heavy chain constant region of human IgG1, and the CL domain is derived from the human κ chain constant region.

[0212] 25. The multispecific antibody as described in paragraph 23 or 24, wherein the heavy chain variable region of the anti-PD-L1 antibody comprises: a VH having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:11; or

[0213] The light chain variable region of the anti-PD-L1 antibody includes a VL that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0214] 26. The multispecific antibody as described in paragraph 25, wherein all amino acids that differ in the amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence shown in SEQ ID NO:11 or 12 are present in the FR region.

[0215] 27. The multispecific antibody as described in any one of paragraphs 23-26, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain of the anti-PD-L1 antibody and the anti-IL-8 nanobody, and the second polypeptide chain comprises the light chain of the anti-PD-L1 antibody;

[0216] The heavy chain of the anti-PD-L1 antibody includes the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence having at least 80% sequence identity with it.

[0217] The anti-IL-8 nanobody comprises an amino acid sequence shown in any one of SEQ ID NO:4, 20, 24, and 25, or an amino acid sequence having at least 80% sequence identity with it.

[0218] The light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 80% sequence identity with it.

[0219] 28. The multispecific antibody as described in any one of paragraphs 23-27, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain of the anti-PD-L1 antibody, a linker, and the anti-IL-8 nanobody, and the second polypeptide chain comprises the light chain of the anti-PD-L1 antibody, wherein the linker is (G5). p Or (G4S) p , where p is an integer from 1 to 6.

[0220] 29. The multispecific antibody as described in any one of paragraphs 23-28, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence having at least 80% sequence identity with it, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 80% sequence identity with it.

[0221] 30. A polynucleotide encoding an anti-IL-8 antibody or an antigen-binding fragment thereof, as described in any one of paragraphs 1-6, or a multispecific antibody or a first antigen-binding portion thereof, as described in any one of paragraphs 7-29.

[0222] 31. An expression vector comprising the polynucleotides described in paragraph 30.

[0223] 32. A host cell integrated with the polynucleotide described in paragraph 30 or the expression vector described in paragraph 31.

[0224] 33. An immunoconjugate comprising any one of paragraphs 1-6 of an anti-IL-8 antibody or an antigen-binding fragment thereof, or any one of paragraphs 7-29 of a multispecific antibody.

[0225] 34. A pharmaceutical composition comprising: an anti-IL-8 antibody or an antigen-binding fragment thereof as described in any one of paragraphs 1-6, a multispecific antibody as described in any one of paragraphs 7-29, or an immunoconjugate as described in paragraph 33, and optionally pharmaceutically acceptable excipients.

[0226] 35. A kit comprising any one of paragraphs 1-6 of an anti-IL-8 antibody or an antigen-binding fragment thereof, any one of paragraphs 7-29 of a multispecific antibody, any one of paragraphs 33 of an immunoconjugate, or any one of paragraphs 34 of a pharmaceutical composition.

[0227] 36. Use of any of the multispecific antibodies described in paragraphs 7-29, the immunoconjugates described in paragraph 33, the pharmaceutical compositions described in paragraph 34, or the kits described in paragraph 35 in the preparation of dual inhibitors of IL-8 and PD-L1.

[0228] 37. Use of any of the multispecific antibodies described in paragraphs 7-29, the immunoconjugates described in paragraph 33, the pharmaceutical compositions described in paragraph 34, or the kits described in paragraph 35 in the preparation of medicaments for the treatment or prevention of diseases associated with both the PD-1 / PD-L1 signaling pathway and the IL-8 / CXCR1 / 2 signaling pathway.

[0229] Example

[0230] The present invention will now be described in further detail through embodiments, but the present invention is not limited to these embodiments.

[0231] Example 1: Combination therapy of anti-PD-L1 antibody and anti-IL-8 antibody to alleviate anti-PD-L1 antibody resistance

[0232] In a C-NKG mouse model of human breast cancer MDA-MB-231 cells reconstituted by hPBMCs, we evaluated the antitumor effect of the combination of anti-PD-L1 antibody (atezolizumab) and anti-IL-8 antibody (HuMax-IL8, BMS-986253).

[0233] We first identified the tumor cell line MDA-MB-231, which highly expresses PD-L1 and IL-8, through screening.

[0234] We cultured MDA-MB-231 cells in an in vitro monolayer using L-15 medium supplemented with 15% fetal bovine serum and 1% antibacterial / antifungal agent (purchased from GIBCO, catalog number: 15240-112), at 37°C in a 0% CO2 incubator. When the cell saturation reached 80%-90% and the desired number was achieved, we collected the MDA-MB-231 cells and resuspended them in serum-free L-15 medium to a concentration of 1×10⁶ cells / mL. 8 The concentration of cells / mL was increased, and then an equal volume of matrix gel (purchased from BD, catalog number: 356234) was added and mixed well to obtain an MDA-MB-231 cell suspension. Each mouse was injected subcutaneously with 0.2 mL of the MDA-MB-231 cell suspension above the right scapula.

[0235] Three days before inoculating tumor cells, hPBMCs were inoculated: frozen hPBMCs were thawed, and the collected hPBMCs were filtered through a 70 μm filter membrane and the concentration was adjusted to 2.5 × 10⁻⁶ using serum-free RPMI-1640 medium. 7 / mL, to obtain hPBMCs cell suspension, and each mouse was inoculated with 0.2mL of the hPBMCs cell suspension via tail vein.

[0236] Tumors were measured using vernier calipers when the average tumor volume in mice reached ~100 mm. 3 Mice were randomly assigned to groups based on tumor volume. The model group served as a negative control, and mice in this group were administered a blank solvent (1×PBS solution (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, pH 7.4), purchased from Sigma-Aldrich, product number: P3813). The anti-PD-L1 antibody-only group served as a positive control. The grouping and administration regimens are shown in Table 2.

[0237] Table 2. Animal Experiment Grouping and Dosing Regimens

[0238]

[0239] Tumor volume measurement: Measured twice weekly using vernier calipers. The tumor volume is calculated using the formula V = 0.5 × L × W × W, where L and W represent the major and minor diameters of the tumor, respectively.

[0240] The antitumor efficacy of the test substance was evaluated by tumor growth inhibition rate (TGI) (%) or relative tumor proliferation rate (T / C) (%). The formula for calculating TGI (%) is: TGI (%) = [1 - (mean tumor volume at the end of treatment group administration - mean tumor volume at the start of treatment group administration) / (mean tumor volume at the end of model group administration - mean tumor volume at the start of control group administration)] × 100%.

[0241] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100% (T) RTV : Mean tumor volume in the treatment group; C RTV (mean tumor volume of the negative control group), T RTV With C RTV Take data from the same day.

[0242] The inhibitory effect of the test substance on tumor growth in the MDA-MB-231 xenograft immune reconstitution model was calculated based on the tumor volume measurement on day 24 after administration, and the results are shown in Table 3.

[0243] Table 3. Tumor growth inhibition status

[0244]

[0245] The experimental results showed that the combination of anti-PD-L1 antibody and anti-IL-8 antibody had a significantly better tumor-suppressive effect (TGI = 51.28%) than the single administration of PD-L1 antibody (TGI = 39.13%), indicating that the combination of anti-PD-L1 antibody and anti-IL-8 antibody has an enhanced therapeutic effect compared with anti-PDL1 antibody alone. Therefore, it can be expected that the two can show a synergistic effect.

[0246] Example 2: Screening of anti-IL-8 nanobodies

[0247] The IL-8 protein required for constructing the IL-8 nanobody library was purchased from Acro. Alpacas were immunized with the IL-8 protein every two weeks, and venous blood was collected every week to measure the titer. When the immunization titer reached the library construction standard, 10 mL of venous blood was collected for library construction.

[0248] Yeast display library construction: Total RNA was extracted from lymphocytes and tissues using an RNA extraction kit provided by QIAGEN. The extracted RNA was reverse transcribed into cDNA using the SuperScript III FIRST STRANDSUPERMIX kit according to the manufacturer's instructions. Nested PCR was used to amplify the nucleic acid fragment encoding the variable region of the heavy chain antibody. The target single-domain antibody nucleic acid fragment was recovered and cloned into the yeast display vector pYD1 using restriction endonucleases BamHI and NotI (purchased from NEB). The product was then electroporated into EBY100 electroporated competent yeast cells to construct an immune single-domain antibody yeast display library, which was then validated. The library size was calculated to be 1.54 × 10⁻⁶ cells after serial dilution plating. 9To determine the insertion rate of the library, 200 clones were randomly selected for sequencing. 198 clones showed correct foreign fragment insertion, resulting in an accuracy rate of 99%. Analysis and alignment of the DNA and amino acid sequences of the sequenced clones confirmed that all sequences were completely different and belonged to the expected Camelidae VHH sequence, exhibiting 100% diversity.

[0249] The IL-8 nanobody library was transformed into yeast cells and induced to grow in SGCAA liquid medium for 16 h. Then, yeast negative panning was performed, using magnetic separation to remove non-specifically bound yeast cells by incubation with washed magnetic beads. Next, biotin-labeled IL-8 protein (purchased from Acro, catalog number: IL8-H82H3) was incubated with magnetic beads to capture yeast cells that specifically bound IL-8 protein. The specifically bound yeast cells were washed and collected, and then amplified in SDCAA medium.

[0250] Yeast culture filtered with magnetic beads was induced to express anti-IL-8 antibody in SGCAA medium for 16 h. The cells were collected and washed with PBS. The induced yeast cells were incubated with IL-8 protein for 1 h to allow the anti-IL-8 antibody to bind to the IL-8 protein. Unbound cells were washed away. PE-SA was added to bind to the IL-8 protein for 1 h. After washing, IL-8-positive bacteria were detected and sorted by flow cytometry. IL-8-positive single colonies were picked, cultured in SDCAA medium, and then induced to express anti-IL-8 antibody in SGCAA medium for 16 h. The cells were collected, incubated with IL-8 antigen for 1 h, and unbound cells were washed away. PE-SA was added and incubated for 1 h. Finally, after washing, flow cytometry was used to identify high-affinity IL-8 positive strains.

[0251] After screening, we obtained IL-8 nanobodies with good affinity and in vitro blocking activity. These nanobodies were then humanized to obtain humanized nanobodies, which were then linked to an IgG4 Fc fragment (SEQ ID NO:19) to construct an expression vector. Eukaryotic transient transfection was performed to express the recombinant nanobodies (also known as "heavy chain antibodies"). The amino acid sequence of the humanized nanobodies is as follows:

[0252]

[0253]

[0254] After obtaining the recombinant nanobodies described above, we conducted in vitro functional experiments for further screening. The experimental steps are as follows:

[0255] The recombinant anti-IL-8 nanobody was serially diluted, starting from the highest concentration of 200 μg / mL, and then serially diluted 3-fold (110 μL of the test sample stock solution was added to 220 μL of DMEM + 1% FBS, and then serially diluted), for a total of 9 dilutions. 100 μL of each diluted antibody was added to a 96-well plate, with two replicates for each dilution (wells near the edge were left undiluted). Then, 50 μL of 1.6 μg / mL IL-8 protein (final concentration 0.4 μg / mL) was added to each well. After mixing, the plates were incubated at 37°C for 1 hour.

[0256] Cell count, aspirate 4 × 10⁻⁶ cells. 6 One set of 293F-Tango-CXCR2-Luc cells in logarithmic growth phase was transferred to a 15 mL centrifuge tube, mixed well, and centrifuged at 500 g for 5 min. After centrifugation, the cells were resuspended in 10 mL of IL-8-free DMEM + 1% FBS medium, mixed thoroughly, and 50 μL of the cell suspension was added to a 96-well plate. Simultaneously, 50 μL of cell suspension was added to the blank wells of the 96-well plate, followed by 150 μL of DMEM + 1% FBS medium, as a control group.

[0257] The co-cultured 96-well plates were transferred to a cell culture incubator and cultured at 37°C and 5% CO2 for 18 hours. After 18 hours of co-culture, the 96-well plates were removed, and 100 μL of Bright-Glo™ assay reagent (including the blank background wells of the control group) was added. Air bubbles were avoided during sample addition. The plates were kept in the dark and incubated at room temperature for 5 minutes. The Luciferase fluorescence values ​​were read using a Tecan M1000 multi-mode microplate reader. The maximum inhibition rate and IC50 were calculated based on the fluorescence values. 50 (μg / mL), the results are shown in Table 4.

[0258] Through in vitro activity screening, the affinity blocking activity of the recombinant nanobody corresponding to the 9-G5-HM2 nanobody was slightly weaker than that of the positive BMS-98 6253. Therefore, the 9-G5-HM2 nanobody was selected as the candidate nanobody to facilitate the formation of a target action mechanism with the PD-L1 antibody atezolizumab, i.e., the affinity of the PD-L1 binding end (1.33E-11M) is greater than that of the IL-8 antibody end (2.132E-9M).

[0259] Table 4. Nanobody Screening

[0260]

[0261] Example 3: Design and expression of anti-PD-L1 and IL-8 bispecific antibodies

[0262] Based on previous experimental results, we will combine anti-IL-8 nanobody sequences with anti-PD-L1 antibodies (selected for lower affinity than anti-PD-L1 antibodies) to design bispecific antibodies. To avoid interference between structural domains, the structures of the four bispecific antibodies we designed are as follows: Figure 1 As shown, eukaryotic transient transduction expression was performed.

[0263] First, the DNA sequences of four target bispecific antibodies were designed, optimized, and synthesized. The amino acid sequence of the anti-IL-8 nanobody portion is shown in SEQ ID NO:4, and the heavy and light chain amino acid sequences of the anti-PD-L1 portion are shown in SEQ ID NO:13 and 14, respectively. The anti-IL-8 nanobody portion is linked to the variable or constant region of the anti-PD-L1 portion via a (G4S)3 linker. Subsequently, these four complete DNA sequences were subcloned into the pcDNA3.4 vector, successfully constructing four recombinant plasmids: pcDNA3.4-DS1, pcDNA3.4-DS2, pcDNA3.4-DS3, and pcDNA3.4-DS4. These recombinant plasmids were prepared for transfection of HD CHO-S cells. Next, the cells were cultured in HD CHO-S cell culture medium (purchased from Pronosei) and maintained at 37°C in Corning Erlenmeyer flasks with 8% CO2 using a VWR Scientific orbital shaker. One day before transfection, cells were seeded into Corning Erlenmeyer flasks at an appropriate density. On the day of transfection, four recombinant plasmids and the transfection reagent Lipofectamine 3000 (purchased from Thermo) were mixed according to the instructions and then added to the cell culture flasks to be transfected. The recombinant plasmid encoding the target antibody was transiently co-transfected into suspended HD CHO S cell cultures. On day 10, the cell culture medium was centrifuged and filtered, and the cell culture supernatant was collected for purification. The filtered cell culture supernatant was loaded into MabSelect SuRe... TM On LX, after washing with phosphate-buffered saline (PBS; 10 mM phosphate, 140 mM NaCl, pH 7.4) and eluting with citrate at pH 3.0, the eluates were combined and the final formulation buffer (PBS) was replaced using molecular sieve chromatography (Sephadex G100). The purified protein was analyzed by SDS-PAGE and HPLC to determine its molecular weight and purity. Protein concentration was determined using the A280 method.

[0264] We determined the optimal structure of the bispecific antibody through expression level and purity screening, such as... Figure 2As shown in the figure. Subsequently, we further analyzed the bispecific antibody against this structure using reducing and non-reducing SDS-PAGE and SEC-HPLC methods, with results as shown in the figure. Figure 3 and Figure 4 As shown.

[0265] Example 4: Detection of the affinity of bispecific antibodies for PD-L1 and IL-8

[0266] To further evaluate the druggability of the bispecific antibody obtained in Example 3, we used a biacore T200 to detect the affinity of the bispecific antibody for the two target antigens. We used a Preotein A chip to capture the candidate bispecific antibody at a working concentration, and then used different concentrations of PD-L1 and IL-8 antigens to detect the affinity of the candidate bispecific antibody for the two targets.

[0267] Affinity data, after double subtraction (i.e., subtracting the control channel signal from the experimental channel signal in each cycle, and then subtracting the blank signal from the sample signal), were fitted using Biacore T200 Evaluation software, version 3.1, with a "1:1 binding" model. The equilibrium dissociation constant KD = kd / ka. In this experiment, the antigen binding response was controlled within 30-100 RU. After data fitting, the parameters satisfied: √(Chi^2) / Rmax < 0.1, U-value < 15, Tc range: 10. 5 -10 20 Table 5. Affinity of the bispecific antibody in Example 3 to PD-L1 and IL-8

[0268]

[0269] It can be seen that the affinity of the bispecific antibody obtained in Example 3 for the target antigen at both ends meets the design requirements.

[0270] Example 5: In vitro blocking activity of bispecific antibodies against PD-L1 and IL-8

[0271] To further evaluate the druggability of the bispecific antibody obtained in Example 3, we used cAMP assays (IL-8 to CXCR1 binding block assay) and luciferase reporter gene assays (IL-8 to CXCR2 binding block assay and PD-L1 to PD-1 binding block assay) to evaluate the in vitro activity of the bispecific antibody.

[0272] The following target cells were cultured first: CHO-K1 / Gα15 / CXCR1, 293T Tango Cxcr Luc, GS-C3 / PD-L1, and GS-J2B / PD-1.

[0273] CXCR1 and IL-8 binding blockade assay - cAMP method experimental procedure:

[0274] 1. Collect target cells CHO-K1 / Gα15 / CXCR1 by centrifugation and resuspend them in complete culture medium (90% F-12 + 10% FBS).

[0275] 2. Prepare working solutions of the test sample (at twice the concentrations used in the detection) and working solutions of the reference (Humax-IL-8 and human IgG1) using the complete culture medium.

[0276] 3. Adjust the target cell density with the complete culture medium and transfer the target cell suspension (3000 cells / well, 5 μL / well) to a 384-well detection plate.

[0277] 4. Transfer the test sample working solution, reference working solution, or complete culture medium to the corresponding well of the 384-well test plate at a rate of 5 μL / well, and incubate the test plate at room temperature for 30 minutes.

[0278] 5. According to the instructions of the HTRF cAMP detection kit (purchased from Unimicron, catalog number: 62AM9PEB), prepare the detection working solution using Lysis / detection buffer. Add 10 μL of detection working solution to each well and incubate at room temperature in the dark for 1 hour.

[0279] 6. Use an ELISA reader to read the HTRF signal value.

[0280] 7. Data Analysis

[0281] Raw experimental data were exported via the PHERAstar FSX system and analyzed using Microsoft Office Excel 2021 and GraphPad Prism 6 software. The ratio of acceptor to donor emission signals for each well was calculated using the following formula:

[0282] Ratio = Signal 665nm / Signal 620nm ×10 4 ;

[0283] To plot the sample concentration, the relative value of IC50 can be obtained using the following four-parameter equation:

[0284] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)×HillSlope)).

[0285] Where X = Log sample concentration, Y = HTRF ratio.

[0286] The results are as follows Figure 5 As shown.

[0287] CXCR2 and IL-8 binding blocking assay - reporter gene assay steps:

[0288] 1. Centrifuge to collect target cells 293T Tango Cxcr Luc and resuspend in cell culture medium (99% DMEM + 1% FBS).

[0289] 2. Prepare the working solution of the test sample (at a concentration twice that used in the detection) and the working solution of the reference (Humax-IL-8 and human IgG1) (at a concentration twice that used in the detection) using the cell culture medium, and add 100 μL of each solution to a 96-well detection plate.

[0290] 3. Prepare IL-8 protein working solution (4×) using the cell culture medium, and add 50 μL to each well of the 96-well detection plate.

[0291] 4. After mixing thoroughly, place the 96-well plate in an incubator (37℃, 5% CO2) and incubate for 1 hour.

[0292] 5. Adjust the target cell density with the cell culture medium, transfer the target cell suspension (20,000 cells / well, 50 μL / well) to the 96-well detection plate, and continue to incubate the 96-well detection plate in an incubator (37°C, 5% CO2) for 18 hours.

[0293] 6. After incubation, remove the 96-well detection plate, transfer Bright-Lite™ luciferase detection working solution (50 μL / well) to the corresponding wells of the detection plate, and incubate at room temperature for 5-10 minutes.

[0294] 7. Use an enzyme-linked immunosorbent assay (ELISA) reader to read the chemiluminescence value.

[0295] 8. Data Analysis

[0296] Raw experimental data were exported via the PHERAstar FSX system and analyzed using Microsoft Office Excel 2021 and GraphPad Prism 6 software. The relative values ​​of EC50 were obtained using the following four-parameter equation:

[0297] Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)*HillSlope)).

[0298] Where X = Log sample concentration, and Y = fluorescence signal value.

[0299] The results are as follows Figure 6 As shown.

[0300] PD-L1 / PD-1 binding blockade assay - reporter gene assay steps:

[0301] 1. Collect target cells (GS-C3 / PD-L1) by centrifugation and resuspend them in complete culture medium (90% F-12 + 10% FBS).

[0302] 2. Adjust the target cell density and seed 20 μL / well (5000 cells / well) into the experimental plate and transfer the experimental plate to a carbon dioxide incubator (37℃, 5% CO2) for 16-20 hours.

[0303] 3. Prepare reference (tecentriq Biosimilar) and human IgG1 working solutions (at a concentration twice that used in the test) and test sample working solutions (at a concentration twice that used in the test) using cell culture medium (RPMI 1640 + 10% FBS).

[0304] 4. Collect effector cells (GS-J2B / PD-1) by centrifugation, resuspend the cells in the cell culture medium and adjust the effector cell density.

[0305] 5. Remove the experimental plate containing the target cells from the CO2 incubator and remove the culture medium.

[0306] 6. Transfer the reference working solution or test working solution (20 μL / well) and the effector cell suspension (15,000 cells / well, 20 μL / well) sequentially into the experimental plate.

[0307] 7. Transfer the experimental plate to a carbon dioxide incubator (37℃, 5% CO2) and incubate for 6 hours.

[0308] 8. Remove the incubated experimental plate from the CO2 incubator, transfer the Bio-Lite™ luciferase assay working solution into the corresponding wells of the experimental plate, and incubate at room temperature for 5-10 minutes.

[0309] 9. Use an enzyme-linked immunosorbent assay (ELISA) reader to read the chemiluminescence value.

[0310] 10. Data Analysis

[0311] Raw experimental data were exported via the PHERAstar FSX system and analyzed using Microsoft Office Excel 2021 and GraphPad Prism 6 software. The relative values ​​of EC50 were obtained using the following four-parameter equation:

[0312] Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)*HillSlope)).

[0313] Where X = Log sample concentration, and Y = fluorescence signal value.

[0314] The results are as follows Figure 7 As shown.

[0315] As can be seen from the above experiments, the bispecific antibody of this application can effectively block the binding of IL-8 to its receptors CXCR1 and CXCR2, and can also effectively block the binding of PD-L1 to PD-1.

[0316] Example 6: In vivo efficacy study of bispecific antibodies

[0317] To verify the in vivo efficacy of the bispecific antibody obtained in Example 3, we used the same experimental methods as in Example 1 to evaluate its in vivo efficacy. The groups are shown in Table 6, where, as controls, the anti-PD-L1 antibody was atezolizumab (Tecentriq Biosimilar), and the anti-IL-8 antibody was HuMax-IL8.

[0318] Table 6. Mouse grouping and administration details in in vivo efficacy studies

[0319]

[0320] Tumor volume measurement in tumor-bearing mice: Measurements were taken twice weekly using vernier calipers. The tumor volume was calculated using the formula V = 0.5 × L × W × W, where L and W represent the major and minor axes of the tumor, respectively.

[0321] The results are as follows Figure 8 As shown, the bispecific antibody demonstrated superior in vivo therapeutic efficacy compared to single-drug anti-PD-L1 and anti-IL-8 antibodies, and also superior to the combined use of anti-PD-L1 and anti-IL-8 antibodies. Compared to the solvent group, the bispecific antibody treatment group of this invention exhibited a significant difference in in vivo tumor suppression.

Claims

1. An isolated anti-IL-8 antibody or its antigen-binding fragment, comprising: (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

23.

2. The anti-IL-8 antibody or its antigen-binding fragment as described in claim 1, wherein, The anti-IL-8 antibody or its antigen-binding fragment comprises an amino acid sequence shown in any one of SEQ ID NO:4, 20, 24 and 25 or an amino acid sequence having at least 80% sequence identity with it; Preferably, all amino acids that differ from the amino acid sequences shown in any of SEQ ID NO:4, 20, 24 and 25, having at least 80% sequence identity, are present in the FR region; Preferably, the anti-IL-8 antibody is an anti-IL-8 nanobody or an anti-IL-8 heavy chain antibody; Preferably, the anti-IL-8 antibody is a camel-derived antibody or a humanized antibody; Preferably, the anti-IL-8 heavy chain antibody further comprises an Fc region, the Fc region comprising an amino acid sequence shown in SEQ ID NO:18 or 19 or an amino acid sequence having at least 80% sequence identity with it.

3. A multispecific antibody, comprising: (1) It specifically binds to the first antigen-binding portion of IL-8; And (2) specifically binds to the second antigen-binding portion of PD-L1; Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

4. The multispecific antibody as described in claim 3, wherein, The first antigen-binding portion includes: (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

23. Preferably, the first antigen-binding portion comprises an amino acid sequence shown in any one of SEQ ID NO:4, 20, 24 and 25 or an amino acid sequence having at least 80% sequence identity with it; Preferably, all amino acids that differ from the amino acid sequences shown in any of SEQ ID NO:4, 20, 24 and 25, having at least 80% sequence identity, are present in the FR region; Preferably, the first antigen-binding portion comprises a camel-derived anti-IL-8 antibody or a humanized anti-IL-8 antibody; Preferably, the first antigen-binding portion is an anti-IL-8 nanobody or an anti-IL-8 heavy chain antibody; Preferably, the anti-IL-8 heavy chain antibody further comprises an Fc region, the Fc region comprising an amino acid sequence shown in SEQ ID NO:18 or 19 or an amino acid sequence having at least 80% sequence identity with it.

5. The multispecific antibody as described in claim 3 or 4, wherein, The second antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, HCDR3 containing the amino acid sequence shown in SEQ ID NO:7, LCDR1 containing the amino acid sequence shown in SEQ ID NO:8, LCDR2 containing the amino acid sequence shown in SEQ ID NO:9, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

10. Preferably, the second antigen-binding portion comprises: VH having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:11, and VL having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:12; Preferably, all amino acids that differ from the amino acid sequence shown in SEQ ID NO:11 or 12, having at least 80% sequence identity, are present in the FR region; Preferably, the second antigen-binding portion further includes: The heavy chain constant region of human IgG1 containing the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO:15; and / or Human κ chain constant region containing the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80% sequence identity with it; Preferably, the second antigen-binding portion comprises an anti-PD-L1 antibody, the anti-PD-L1 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:13, and the light chain comprises a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

14. Preferably, the second antigen-binding portion is connected to the first antigen-binding portion via a linker; Preferably, the connector is (G5). p Or (G4S) p , where p is an integer from 1 to 6; Preferably, the second antigen-binding portion is atezolizumab.

6. The multispecific antibody as described in claim 3, wherein, The multispecific antibody is a bispecific antibody, and the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain: The first polypeptide chain, from the N-terminus to the C-terminus, sequentially comprises the heavy chain variable region of the anti-PD-L1 antibody, the CH1 domain and the Fc domain, as well as the anti-IL-8 nanobody. The heavy chain variable region of the anti-PD-L1 antibody includes: HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

7. The anti-IL-8 nanobody comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO:1, HCDR2 containing the amino acid sequence shown in SEQ ID NO:2, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:3; or HCDR1 containing the amino acid sequence shown in SEQ ID NO:21, HCDR2 containing the amino acid sequence shown in SEQ ID NO:22, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:23; The second polypeptide chain comprises, from the N-terminus to the C-terminus, the light chain variable region of the anti-PD-L1 antibody and the CL domain, wherein the light chain variable region of the anti-PD-L1 antibody includes: LCDR1 containing the amino acid sequence shown in SEQ ID NO:8, LCDR2 containing the amino acid sequence shown in SEQ ID NO:9, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

10. Preferably, the CH1 domain and Fc domain are derived from the heavy chain constant region of human IgG1, and the CL domain is derived from the human κ chain constant region; Preferably, the heavy chain variable region of the anti-PD-L1 antibody includes: a VH having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:11; or The light chain variable region of the anti-PD-L1 antibody includes a VL having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:12; Preferably, all amino acids that differ from the amino acid sequence shown in SEQ ID NO:11 or 12, having at least 80% sequence identity, are present in the FR region; Preferably, the first polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain of the anti-PD-L1 antibody and the anti-IL-8 nanobody, and the second polypeptide chain comprises the light chain of the anti-PD-L1 antibody; The heavy chain of the anti-PD-L1 antibody includes the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence having at least 80% sequence identity with it. The anti-IL-8 nanobody comprises an amino acid sequence shown in any one of SEQ ID NO:4, 20, 24, and 25, or an amino acid sequence having at least 80% sequence identity with it. The light chain of the anti-PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 80% sequence identity with it; Preferably, the first polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain of the anti-PD-L1 antibody, a linker, and the anti-IL-8 nanobody, and the second polypeptide chain comprises the light chain of the anti-PD-L1 antibody, wherein the linker is (G5). p Or (G4S) p , where p is an integer from 1 to 6; Preferably, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence having at least 80% sequence identity with it, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 80% sequence identity with it.

7. An immunoconjugate comprising the anti-IL-8 antibody of claim 1 or 2 or its antigen-binding fragment, or the multispecific antibody of any one of claims 3-6.

8. A pharmaceutical composition comprising: The anti-IL-8 antibody of claim 1 or 2 or its antigen-binding fragment, the multispecific antibody of any one of claims 3-6 or the immunoconjugate of claim 7, and optionally pharmaceutically acceptable excipients.

9. A kit comprising the anti-IL-8 antibody of claim 1 or 2 or its antigen-binding fragment, the multispecific antibody of any one of claims 3-6, the immunoconjugate of claim 7, or the pharmaceutical composition of claim 8.

10. Use of the multispecific antibody of any one of claims 3-6, the immunoconjugate of claim 7, the pharmaceutical composition of claim 8, or the kit of claim 9 in the preparation of a dual inhibitor of IL-8 and PD-L1, or in the preparation of a medicament for the treatment or prevention of diseases associated with both the PD-1 / PD-L1 signaling pathway and the IL-8 / CXCR1 / 2 signaling pathway.

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