Bispecific recombinant protein targeting CCR8 and CTLA-4 and application thereof

Abstract

The invention discloses a bispecific recombinant protein targeting CCR8 and CTLA-4 and an application of the bispecific recombinant protein. The bispecific recombinant protein comprises one or more first binding domains which bind to CCR8 and one or more second binding domains which bind to CTLA-4; the first binding domain includes a heavy chain variable region and a light chain variable region. The bispecific recombinant protein retains the targeted antigen binding capacity of a parent monoclonal antibody, has good ADCC activity and the capacity of effectively activating T cells, has a better tumor inhibition effect compared with the parent monoclonal antibody, and has a clinical application prospect.

Description

[0001] This application claims priority to Chinese patent application 2024110853413, filed on August 8, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to a bispecific recombinant protein targeting CCR8 and CTLA-4 and its applications. Background Technology

[0003] Regulatory T cells (Tregs) are an immunosuppressive subset of CD4+ T cells, playing a crucial role in maintaining self-tolerance and immune homeostasis. However, in tumor immunology, Tregs can suppress the body's anti-tumor immune response through various mechanisms. Increased Treg cell numbers and decreased Teff / Treg ratios in the tumor microenvironment (TME) are associated with poor prognosis in various tumors. Therefore, suppressing tumor-infiltrating Tregs is considered an important direction for tumor immunotherapy.

[0004] Antitumor therapies targeting Tregs aim to restore the body's antitumor immune response while maintaining peripheral tolerance. There are seven main strategies: (1) clearing tumor-infiltrating Tregs, (2) inhibiting tumor infiltration of Tregs, (3) increasing the sensitivity of tumor-infiltrating Tregs to immune checkpoint inhibitors, (4) targeting the co-stimulatory receptors of tumor-infiltrating Tregs, (5) targeting inhibitory cytokines secreted by Tregs, (6) altering Treg fragility, and (7) targeting Treg metabolism. Among these, targets related to clearing tumor-infiltrating Tregs include CCR8 and CTLA-4.

[0005] Chemokine receptor 8 (CCR8), a member of the β-chemokine receptor family, is a seven-transmembrane protein similar to G protein-coupled receptors. It is primarily expressed on tumor-infiltrating Tregs and plays a crucial role in Treg-mediated immunosuppression. Clinical studies have shown that CCR8 expression is increased on intratumoral Tregs in various tumors, including clear cell renal cell carcinoma, breast cancer, and bladder cancer, while expression is minimal on peripheral blood Tregs. Research has also indicated that high levels of CCR8+ Tregs are associated with immune tolerance and poor prognosis in patients with breast and bladder cancer. Therefore, CCR8 is considered a promising target for cancer therapy, and the development of anti-CCR8 antibodies could selectively clear Tregs from the tumor microenvironment (TME) and enhance anti-tumor immune responses.

[0006] Although no antibody drugs targeting CCR8 have been approved for marketing yet, many pharmaceutical companies at home and abroad have already made plans for this target, and the earliest has entered Phase II clinical trials. Therefore, differentiated and innovative development targeting CCR8 is imperative.

[0007] Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a member of the immunoglobulin superfamily expressed on the surface of activated T cells, responsible for transmitting inhibitory signals to T cells. CTLA-4 inhibits T cell proliferation and activation by competitively binding to B7 molecules on the surface of antigen-presenting cells with the T cell coactivator CD28. Clinical trial results have fully demonstrated the efficacy of CTLA-4-targeting antibodies. The marketed ipilimumab is approved as a monotherapy for melanoma and as a combination therapy for renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, esophageal cancer, and malignant pleural mesothelioma. Another marketed antibody, tremelimumab, is also approved for the treatment of hepatocellular carcinoma and non-small cell lung cancer. Initially, the mechanism of action of CTLA-4 antibodies was generally believed to be "immune checkpoint blockade," that is, blocking the interaction between CTLA-4 and B7 to release the immune brake, thereby enhancing immunity and killing tumors. However, studies have shown that CTLA-4 antibodies can also effectively eliminate tumor-infiltrating Treg cells that highly express CTLA-4 through the Fc receptor-mediated ADCC effect, thereby relieving the immunosuppression of Tregs and achieving an anti-tumor effect.

[0008] As a widely studied immune checkpoint, only two monoclonal antibodies have been approved for marketing, mainly due to limited single-drug response rates and the potential for causing immune-related side effects. Therefore, research on CTLA-4-targeted antibody drugs focuses primarily on improving therapeutic efficacy in tumor immunotherapy and reducing related immune side effects.

[0009] Although existing technologies have disclosed several bispecific recombinant protein drugs targeting CTLA-4, no bispecific recombinant protein targeting both CCR8 and CTLA-4 has yet entered clinical trials. There is still an urgent need to develop high-quality anti-CCR8×CTLA-4 bispecific recombinant proteins to enrich the methods and approaches of tumor immunotherapy. Summary of the Invention

[0010] The technical problem to be solved by the present invention is the lack of high-quality anti-CCR8×CTLA-4 bispecific recombinant protein for tumor treatment in the prior art. The present invention provides a bispecific recombinant protein targeting CCR8 and CTLA-4 and its application.

[0011] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0012] A first aspect of the present invention provides a bispecific recombinant protein comprising one or more first binding domains for binding CCR8 and one or more second binding domains for binding CTLA-4; the first binding domains comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to Kabat.

[0013] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13 or having 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%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:13.

[0014] In some embodiments of the present invention, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14 or having 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%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:14.

[0015] In some embodiments of the present invention, the first binding domain is an antigen-binding fragment that binds to CCR8; the second binding domain is an antigen-binding fragment that binds to CTLA-4.

[0016] In some embodiments of the present invention, the number of the first binding domains in the bispecific recombinant protein is 1, 2, or 4; and the number of the second binding domains is 1, 2, or 4.

[0017] In some specific embodiments of the present invention, the number of the first binding structural domains is 2, and the number of the second binding structural domains is 1 or 2.

[0018] In some embodiments of the present invention, one or more of the first binding domains are connected to each other, and the first binding domains are connected to the second binding domains by peptide linkers having amino acid sequences as shown in any of SEQ ID NO:20-25.

[0019] In some specific embodiments of the present invention, the first binding domain and the second binding domain are connected by a peptide linker having an amino acid sequence as shown in any of SEQ ID NO:20-25.

[0020] In some specific embodiments of the present invention, one or more of the first binding domains are connected by a peptide linker having an amino acid sequence as shown in SEQ ID NO:21.

[0021] In some embodiments of the present invention, the antigen-binding fragment is selected from Fab, Fab'-SH, scFv, Fv or (Fab')2.

[0022] In some embodiments of the present invention, in the first binding structural domain, the heavy chain variable region and the light chain variable region are directly bound to CCR8.

[0023] In some embodiments of the present invention, in the second binding structural domain, the heavy chain variable region and the light chain variable region are directly bound to CTLA-4 or connected through a connector before being bound to CTLA-4.

[0024] In some embodiments of the present invention, in the first binding structural domain, the heavy chain variable region and the light chain variable region are directly bound to CCR8; in the second binding structural domain, the heavy chain variable region and the light chain variable region are connected by connectors and then bound to CTLA-4.

[0025] In this invention, the linker can be conventional in the art, such as the GS linker. In some instances, the linker has an amino acid sequence as shown in SEQ ID NO:20.

[0026] In some embodiments of the present invention, the first binding domain is Fab and the second binding domain is scFv.

[0027] In other embodiments of the present invention, the first binding domain is Fv and the second binding domain is Fab.

[0028] In other embodiments of the present invention, the first binding domain and the second binding domain are Fab.

[0029] In some embodiments of the present invention, when the second binding structural domain is scFv, the C end of the heavy chain variable region is connected to the N end of the light chain variable region through a connector.

[0030] In other embodiments of the present invention, when the second binding structural domain is scFv, the C end of the light chain variable region is connected to the N end of the heavy chain variable region via a connector.

[0031] In some embodiments of the present invention, the bispecific recombinant protein further includes Fc.

[0032] In some embodiments of the present invention, the Fc is the Fc of human immunoglobulin, preferably the Fc of human IgG, and more preferably the Fc of human IgG1.

[0033] In some specific embodiments of the present invention, the Fc has an amino acid sequence as shown in SEQ ID NO:19.

[0034] In some embodiments of the present invention, the first binding structural domain is located at the N end of the Fc, and the second binding structural domain is connected to the N end or the C end of the Fc.

[0035] In some embodiments of the present invention, the second binding domain is connected to the C-terminus of the Fc via a peptide linker, the peptide linker having, for example, the amino acid sequence shown in SEQ ID NO:20 or 25.

[0036] In some embodiments of the present invention, the number of the first binding structural domains is 2, the number of the second binding structural domains is 1, the C-end of one of the first binding structural domains and the second binding structural domain are respectively connected to the N-end of the Fc, and the C-end of the other first binding structural domain is connected to the N-end of the first binding structural domain or the N-end of the second binding structural domain connected to the N-end of the Fc.

[0037] In some embodiments of the present invention, the first binding structural domain is connected to the N end of the Fc, and the second binding structural domain is connected to the C end of the Fc.

[0038] In other embodiments of the present invention, the C-terminus of the second bonding domain is connected to the N-terminus of the Fc, and the C-terminus of the first bonding domain is connected to the N-terminus of the second bonding domain.

[0039] In some specific embodiments of the present invention, the first binding structural domain is Fab, the second binding structural domain is scFv, the C-terminus of the first binding structural domain is connected to the N-terminus of the Fc, and the N-terminus of the second binding structural domain is connected to the C-terminus of the Fc.

[0040] In some other specific embodiments of the present invention, the first binding domain is Fab, the second binding domain is scFv, and the first binding domain, the second binding domain, and the Fc are sequentially connected from the N end to the C end.

[0041] In some other specific embodiments of the present invention, the first binding structural domain is Fv, the second binding structural domain is Fab, and the first binding structural domain, the second binding structural domain, and Fc are sequentially connected from the N end to the C end.

[0042] In some other specific embodiments of the present invention, the first bonding domain and the second bonding domain are Fab, and the first bonding domain, the second bonding domain and the Fc are sequentially connected from the N end to the C end.

[0043] In some other specific embodiments of the present invention, the first bonding structural domain and the second bonding structural domain are Fab, the C-terminus of the first bonding structural domain is connected to the N-terminus of the Fc, and the C-terminus of the Fc is connected to the N-terminus of the second bonding structural domain.

[0044] In some other specific embodiments of the present invention, the first bonding structural domain and the second bonding structural domain are Fab, the number of the first bonding structural domain is 2, the number of the second bonding structural domain is 1, the C-end of one of the first bonding structural domains and the second bonding structural domain are respectively connected to the N-end of the Fc, and the C-end of the other first bonding structural domain is connected to the N-end of the first bonding structural domain or the N-end of the second bonding structural domain connected to the N-end of the Fc.

[0045] In some embodiments of the present invention, the second binding domain includes a heavy chain variable region (VH') and a light chain variable region (VL'), wherein the heavy chain variable region comprises amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and the light chain variable region comprises amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to Kabat.

[0046] In some embodiments of the present invention, the heavy chain variable region of the second binding domain comprises an amino acid sequence as shown in SEQ ID NO:15 or having 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%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:15.

[0047] In some embodiments of the present invention, the light chain variable region of the second binding domain comprises an amino acid sequence as shown in SEQ ID NO:16 or having 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%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:16.

[0048] In some embodiments of the present invention, the bispecific recombinant protein further includes a heavy chain constant region 1 (CH1) and a light chain constant region (CL).

[0049] In some embodiments of the present invention, the heavy chain constant region 1 and the light chain constant region are connected to the heavy chain variable region and the light chain variable region in a CrossMab configuration.

[0050] In some embodiments of the present invention, the heavy chain constant region 1 and the light chain constant region are the heavy chain constant region and the light chain constant region of a person.

[0051] In some embodiments of the present invention, the heavy chain constant region 1 is selected from CH1 of IgG1, IgG2, IgG3 and IgG4.

[0052] In some embodiments of the present invention, the light chain constant region is a kappa chain or a lambda chain.

[0053] In some specific embodiments of the present invention, the heavy chain constant region 1 has an amino acid sequence as shown in SEQ ID NO:17.

[0054] In some specific embodiments of the present invention, the light chain constant region is a kappa chain having an amino acid sequence as shown in SEQ ID NO:18.

[0055] In some specific embodiments of the present invention, the bispecific recombinant protein comprises polypeptide chain 1 and polypeptide chain 2. The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1 - heavy chain variable region of the second binding domain - linker - light chain variable region of the second binding domain; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; wherein, Fc1 is paired to form Fc.

[0056] In the above embodiments, the peptide linker used has an amino acid sequence as shown in SEQ ID NO:25.

[0057] In some other specific embodiments of the present invention, the bispecific recombinant protein comprises polypeptide chain 1 and polypeptide chain 2. The structure of polypeptide chain 1 from N-terminus to C-terminus is: heavy chain variable region (VH) of the first binding domain - heavy chain constant region 1 - Fc1 - light chain variable region (VL') of the second binding domain - linker - heavy chain variable region (VH') of the second binding domain; the structure of polypeptide chain 2 from N-terminus to C-terminus is: light chain variable region (VL) of the first binding domain - light chain constant region; wherein, Fc1 is paired to form Fc.

[0058] In the above embodiments, the peptide linker used has an amino acid sequence as shown in SEQ ID NO:20.

[0059] In some embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-CH1-Fc1-VH'-linker-VL';

[0060] The structure of the polypeptide chain 2 from the N-terminus to the C-terminus is: VL-CL.

[0061] In other embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-CH1-Fc1-VL'-linker-VH';

[0062] The structure of the polypeptide chain 2 from the N-terminus to the C-terminus is: VL-CL.

[0063] In some other specific embodiments of the present invention, the bispecific recombinant protein comprises polypeptide chain 1 and polypeptide chain 2, wherein the structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - light chain variable region of the second binding domain - linker - heavy chain variable region of the second binding domain - Fc1; and the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region.

[0064] In the above embodiments, the peptide linker used has an amino acid sequence as shown in SEQ ID NO:20.

[0065] In some embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-CH1-VL'-linker-VH'-Fc1;

[0066] The structure of the polypeptide chain 2 from the N-terminus to the C-terminus is: VL-CL.

[0067] In some other specific embodiments of the present invention, the bispecific recombinant protein comprises polypeptide chain 1 and polypeptide chain 2. The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain variable region of the second binding domain - heavy chain constant region 1 - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain variable region of the second binding domain - light chain constant region; wherein, Fc1 is paired to form Fc.

[0068] In the above embodiments, the peptide linker used in the heavy chain has an amino acid sequence as shown in SEQ ID NO:22, and the peptide linker used in the light chain has an amino acid sequence as shown in SEQ ID NO:23.

[0069] In some embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-VH'-CH1-Fc1;

[0070] The structure of the polypeptide chain 2 from the N-terminus to the C-terminus is: VL-VL'-CL.

[0071] In some other specific embodiments of the present invention, the bispecific recombinant protein comprises polypeptide chain 1, polypeptide chain 2, and polypeptide chain 3. The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - heavy chain variable region of the second binding domain - light chain constant region - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from the N-terminus to the C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; wherein, Fc1 is paired to form Fc.

[0072] In the above embodiments, the peptide linker used has an amino acid sequence as shown in SEQ ID NO:24.

[0073] In some embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-CH1-VH'-CL-Fc1;

[0074] The structure of polypeptide chain 2 from the N-terminus to the C-terminus is: VL-CL;

[0075] The structure of the polypeptide chain 3 from the N-terminus to the C-terminus is: VL'-CH1.

[0076] In some other specific embodiments of the present invention, the bispecific recombinant protein comprises polypeptide chain 1, polypeptide chain 2, and polypeptide chain 3. The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1 - heavy chain variable region of the second binding domain - light chain constant region; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from the N-terminus to the C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; wherein, Fc1 is paired to form Fc.

[0077] In the above embodiments, the peptide linker used has an amino acid sequence as shown in SEQ ID NO:20.

[0078] In some embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-CH1-Fc1-VH'-CL;

[0079] The structure of polypeptide chain 2 from the N-terminus to the C-terminus is: VL-CL;

[0080] The structure of the polypeptide chain 3 from the N-terminus to the C-terminus is: VL'-CH1.

[0081] In other specific embodiments of the present invention, the bispecific recombinant protein comprises polypeptide chain 1, polypeptide chain 2, polypeptide chain 3, and polypeptide chain 4. The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - heavy chain variable region of the second binding domain - light chain constant region - Fc2; the structure of polypeptide chain 4 from the N-terminus to the C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; wherein, Fc1 and Fc2 together form Fc.

[0082] In the above embodiments, the peptide linker used has an amino acid sequence as shown in SEQ ID NO:21.

[0083] In some embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-CH1-Fc1;

[0084] The structure of polypeptide chain 2 from the N-terminus to the C-terminus is: VL-CL;

[0085] The structure of polypeptide chain 3 from the N-terminus to the C-terminus is: VH-CH1-VH'-CL-Fc2;

[0086] The structure of the polypeptide chain 4 from the N-terminus to the C-terminus is: VL'-CH1.

[0087] In other specific embodiments of the present invention, the structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from the N-terminus to the C-terminus is: heavy chain variable region of the second binding domain - light chain constant region - Fc2; the structure of polypeptide chain 4 from the N-terminus to the C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; wherein, Fc1 and Fc2 together form Fc.

[0088] In the above embodiments, the peptide linker used has an amino acid sequence as shown in SEQ ID NO:21.

[0089] In some embodiments of the present invention, the structure of the polypeptide chain 1 from the N-terminus to the C-terminus is: VH-CH1-VH-CH1-Fc1;

[0090] The structure of polypeptide chain 2 from the N-terminus to the C-terminus is: VL-CL;

[0091] The structure of polypeptide chain 3 from the N-terminus to the C-terminus is: VH'-CL-Fc2;

[0092] The structure of the polypeptide chain 4 from the N-terminus to the C-terminus is: VL'-CH1.

[0093] In this invention, Fc1 and Fc2 together form Fc, and Fc1 and Fc2 are connected, for example, via KiH.

[0094] A second aspect of the present invention provides an isolated nucleic acid molecule that encodes a bispecific recombinant protein as described in the first aspect.

[0095] A third aspect of the present invention provides a recombinant vector comprising nucleic acid molecules as described in the second aspect.

[0096] In some embodiments of the present invention, the recombinant vector is an expression vector.

[0097] A fourth aspect of the present invention provides a transformant comprising a nucleic acid molecule as described in the second aspect or a recombinant vector as described in the third aspect;

[0098] Alternatively, the transformant expresses the bispecific recombinant protein as described in the first aspect.

[0099] A fifth aspect of the present invention provides a composition comprising a bispecific recombinant protein as described in the first aspect, a nucleic acid molecule as described in the second aspect, a recombinant vector as described in the third aspect and / or a transformant as described in the fourth aspect, and a second reagent.

[0100] In some embodiments of the present invention, the composition is a pharmaceutical composition, and the second reagent is a pharmaceutically acceptable carrier and / or excipient.

[0101] A sixth aspect of the present invention provides a method for preparing a bispecific recombinant protein, the method comprising culturing a transformant as described in the fourth aspect and obtaining the bispecific recombinant protein from the culture.

[0102] The seventh aspect of the present invention provides the use of a bispecific recombinant protein as described in the first aspect, a nucleic acid molecule as described in the second aspect, a recombinant vector as described in the third aspect, a transformant as described in the fourth aspect, or a composition as described in the fifth aspect in the preparation of a medicament for the prevention and / or treatment of cancer.

[0103] In some embodiments of the present invention, the cancer is a cancer infiltrated by Treg cells.

[0104] In some embodiments of the present invention, the cancer is selected from colorectal cancer, melanoma, malignant pleural mesothelioma, renal cancer, liver cancer, esophageal cancer, non-small cell lung cancer, and glioblastoma.

[0105] An eighth aspect of the present invention provides a method for preventing and / or treating cancer, the method comprising administering to a subject in need an effective amount of a bispecific recombinant protein as described in the first aspect, a nucleic acid molecule as described in the second aspect, a recombinant vector as described in the third aspect, a transformant as described in the fourth aspect, or a composition as described in the fifth aspect.

[0106] In some embodiments of the present invention, the cancer is a cancer infiltrated by Treg cells.

[0107] In some embodiments of the present invention, the cancer is selected from colorectal cancer, melanoma, malignant pleural mesothelioma, renal cancer, liver cancer, esophageal cancer, non-small cell lung cancer, and glioblastoma.

[0108] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0109] The reagents and raw materials used in this invention are all commercially available.

[0110] The positive and progressive effects of this invention are as follows:

[0111] The bispecific recombinant protein of this invention retains the antigen-binding ability of the parental monoclonal antibody, exhibits good ADCC activity and the ability to effectively activate T cells. The bispecific recombinant protein of this invention has a better tumor-suppressing effect than the parental monoclonal antibody and shows promise for clinical application. Attached Figure Description

[0112] Figure 1 : Schematic diagram of the structure of the bispecific recombinant protein targeting CCR8 and CTLA-4. AH are schematic diagrams of the structures of the bispecific recombinant proteins BsAb Format1, 2, 3, 4, 5, 9, 16 and 28, respectively.

[0113] Figure 2 SDS-PAGE gel image of bispecific recombinant protein.

[0114] Figure 3 Flow cytometry was used to analyze the binding activity of the bispecific recombinant protein to the CHOK1-hCCR8 cell line overexpressing hCCR8.

[0115] Figure 4 Flow cytometry was used to analyze the binding activity of the bispecific recombinant protein to the CHOK1-hCTLA-4 cell line overexpressing hCTLA-4.

[0116] Figure 5 Flow cytometry was used to analyze the blocking activity of bispecific recombinant proteins. A: Blocking activity of BsAb on the binding of CHOK1-hCTLA-4 to CD80; B: Blocking activity of BsAb on the binding of CHOK1-hCTLA-4 to CD86.

[0117] Figure 6A Purity analysis of the fucose-free bispecific recombinant protein BsAb Format16_AF by HPLC-SEC at a UV wavelength of 280 nm.

[0118] Figure 6B Purity analysis of the fucose-free bispecific recombinant protein BsAb Format28_AF was performed by HPLC-SEC at a UV wavelength of 280 nm.

[0119] Figure 7 : DSF determination of Tm for defucosylated bispecific recombinant protein. A: Real-time melting curves of BsAb Format16_AF and BsAb Format28_AF as a function of temperature; B: Normalized derivatives of the antibody real-time melting curves.

[0120] Figure 8 Affinity detection of BLI for desalinated trehalose-type bispecific recombinant proteins and parental monoclonal antibodies. A: Real-time shift signal of the interference spectrum generated by the binding of parental monoclonal antibody ipilimumab to hCTLA-4-his; B: Real-time shift signal of the interference spectrum generated by the binding of BsAb Format16_AF to hCTLA-4-his; C: Real-time shift signal of the interference spectrum generated by the binding of BsAb Format28_AF to hCTLA-4-his.

[0121] Figure 9 : Flow cytometry analysis of the dual targeting of defucosylated bispecific recombinant proteins.

[0122] Figure 10 Analysis of ADCC activity of defucosylated bispecific recombinant protein in Jurkat-hFcγRIIIa / NFAT-luc cells and CHOK1-hCCR8 cells overexpressing hCCR8.

[0123] Figure 11 Analysis of ADCC activity of defucosylated bispecific recombinant protein in Jurkat-hFcγRIIIa / NFAT-luc cells and CHOK1-hCTLA-4 cells overexpressing hCTLA-4.

[0124] Figure 12 Analysis of T cell activation capacity of BsAb Format16_AF and BsAb Format28_AF using SEB stimulation assay.

[0125] Figure 13 Analysis of the inhibitory activity of the fucose-de-form bispecific recombinant protein BsAb Format16_AF on MC38 growth in hCCR8 transgenic C57BL / 6 mice. A represents tumor volume change, and B represents body weight change.

[0126] Figure 14 Analysis of the inhibitory activity of the fucose-de-form bispecific recombinant protein BsAb Format16_AF on MC38 growth in hCTLA-4 transgenic C57BL / 6 mice. A represents tumor volume change, and B represents body weight change.

[0127] Figure 15 In hCCR8 / hCTLA-4 transgenic C57BL / 6 mice, the inhibitory activity of the defucosylated bispecific recombinant protein BsAb Format16_AF and its parental antibody on MC38 growth was compared. A represents tumor volume change, and B represents body weight change.

[0128] Figure 16 Pharmacokinetic analysis of bispecific recombinant protein in cynomolgus monkeys. Detailed Implementation

[0129] Terminology Definition

[0130] As used herein, the term "recombinant protein" refers to an artificially designed / constructed protein, rather than a naturally occurring protein. The "recombinant" in "recombinant protein" of this invention does not represent its production method; it is used only to indicate that the "recombinant protein" does not exist naturally. The recombinant protein of this invention can be an expressed protein or an assembled protein.

[0131] As used herein, the terms "antibody" or "immunoglobulin" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains. The amino acid composition and sequence of the constant regions of the immunoglobulin heavy chains differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0132] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are known as the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FWR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FWR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0133] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0134] As used herein, the term "antigen-binding fragment" or "Fab" comprises a variable region (VL) of the light chain, a constant region (CL) of the light chain, a variable region (VH) of the heavy chain, and a constant region 1 (CH1) domain of the heavy chain, which can bind to an antigen. When referring to the direct connection or linkage between the variable and constant regions of the antigen-binding fragment via a linker sequence, the constant region refers to either the constant region (CL) of the light chain or the constant region 1 (CH1) of the heavy chain.

[0135] As used herein, the term "Fab'" comprises a portion of a light chain and a heavy chain containing the VH and CH1 domains, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab' segments to form the F(ab')2 molecule. "F(ab')2" comprises two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab')2 segment consists of two Fab' segments held together by disulfide bonds between the two heavy chains.

[0136] As used in this article, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0137] In this invention, the scFv (single chain antibody fragment) can be a conventional single chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids. The VL and VH domains enable them to pair as linkers to form monovalent molecules as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating G4S amino acid sequences or variants thereof. For example, linkers with the amino acid sequences (G4S)4 or (G4S)3 can be used, but variants thereof can also be used.

[0138] As used herein, the term “Fc” (fragment crystallizable) consists of the constant CH2 and CH3 domains and hinge region of an immunoglobulin such as IgG.

[0139] As used herein, the term "knobs-into-holes" or "KiH" technology refers to the use of genetic engineering techniques to introduce different mutations into the two CH3 domains of a heavy chain to induce heterodimerization of the heavy chain. A knot is formed on one heavy chain and a hole is formed on the other, and the two then preferentially bind together to form an asymmetric antibody (Ridgway JB, et al. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996, 9(7): 617-621). As those skilled in the art know, multiple knots and / or holes can be formed on one heavy chain, and correspondingly, multiple holes and / or knots can be formed on the other heavy chain.

[0140] As used herein, the terms “linker” or “peptide linker” refer to an amino acid sequence that connects different functional binding fragments (such as a first binding domain and a second binding domain, or a first binding domain or a second binding domain and an Fc), or connects different domains within the same functional binding fragment.

[0141] As used herein, the term "host cell" is used to prepare a transformant and typically includes a single cell, cell line, or cell culture that may be, or is already, a recipient of a subject plasmid or vector, containing the polynucleotides disclosed in this application, or expressing a protein heterodimer (e.g., a heterodimeric protein) of this application. The host cell may include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (morphologically or in terms of total genomic DNA complementarity). The host cell may include cells transfected in vitro with the vectors disclosed in this application. The host cell may be a bacterial cell (e.g., *E. coli*), yeast cell, or other eukaryotic cell, such as HEK293 cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, or myeloma cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a CHO cell.

[0142] As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. This term may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for the replication of DNA or RNA, and expression vectors for the transcription and / or translation of DNA or RNA. It also includes vectors that provide more than one of the aforementioned functions. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally means a suitable host cell containing an expression vector capable of producing the desired expression yield.

[0143] As used herein, the terms "treatment" and "therapeutic method" are used interchangeably. The term "treatment" includes controlling the progression of a disease, symptom, condition, and associated symptoms, preferably reducing the impact of the disease, symptom, condition, or alleviating one or more symptoms of the disease, symptom, condition. This term includes curing the disease or completely eliminating the symptoms. This term includes symptom relief. This term also includes, but is not limited to, non-curative palliative treatment. The term "treatment" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein or fusion protein of the present invention to prevent or delay, reduce or alleviate the progression of a disease, symptom, condition, or the impact of one or more symptoms of the disease, symptom, condition.

[0144] As used herein, the term "administration" refers to the delivery of a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein or fusion protein of the present invention to a subject. Administration can be systemic or local. Administration can be performed using an administration device, such as a syringe. Methods of administration include, but are not limited to, implantation, nasal inhalation, spraying, and injection. Routes of administration include inhalation, intranasal administration, oral administration, intravenous administration, subcutaneous administration, or intramuscular administration.

[0145] Molecular conformation of bispecific recombinant proteins

[0146] A schematic diagram of the molecular structure of the bispecific recombinant protein is shown below. Figure 1As shown. The antibody sequence binding to CTLA-4 can be found in the marketed ipilimumab. The bispecific recombinant protein BsAb Format1 has a bivalent CCR8 binding domain in Fab form, located at the N-terminus of the molecule; and a monovalent CTLA-4 binding domain in Fab form. The CH1 and CL positions of the Fab domain are interchanged using CrossMab technology to prevent mismatches. The CTLA-4 binding domain is tandemly linked to the C-terminus of the CCR8 binding domain via (GGGGS)2 (SEQ ID NO:21). The bispecific recombinant protein BsAb Format2 has a bivalent CCR8 binding domain in Fab form, interconnected via (GGGGS)2 (SEQ ID NO:21); and a monovalent CTLA-4 binding domain in Fab form. The CH1 and CL positions of the Fab domain are interchanged using CrossMab technology to prevent mismatches. The bispecific recombinant protein BsAb Format3 adopts a DVD-Ig conformation, with a CCR8 binding domain located at the N-terminus and a CTLA-4 binding domain tandemly connected to the C-terminus of the CCR8 binding domain. The peptide linkers used for the heavy and light chains are ASTKGP (SEQ ID NO:22) and TVAAP (SEQ ID NO:23), respectively. The bispecific recombinant protein BsAb Format4 has a bivalent CCR8 binding domain in Fab form, located at the N-terminus, and a bivalent CTLA-4 binding domain. The CH1 and CL positions of the Fab domain are interchanged using CrossMab technology to prevent mismatches. The CTLA-4 binding domain is tandemly connected to the C-terminus of the CCR8 binding domain via (GGGGS)3 (SEQ ID NO:24). The bispecific recombinant protein BsAb Format5 has a bivalent CCR8 binding domain in Fab form located at the N-terminus of the molecule, and a bivalent CTLA-4 binding domain. The CH1 and CL of the Fab are interchanged using CrossMab technology to prevent mismatch. The CTLA-4 binding domain is tandemly linked to the C-terminus of Fc via (GGGGS)4 (SEQ ID NO:20).Bispecific recombinant proteins BsAb Format9 and BsAb Format16 possess a bivalent CCR8-binding domain in Fab form, located at the N-terminus of the molecule; and a bivalent CTLA-4-binding domain in scFv form. The scFv domains of BsAb Format9 are tandemly linked in a VH-VL sequence, while those of BsAb Format16 are tandemly linked in a VL-VH sequence. The CTLA-4-binding domains of BsAb Format9 and BsAb Format16 are tandemly linked to the C-terminus of the Fc region via peptide linkers GGGGSGGGGTGGGGS (SEQ ID NO:25) and (GGGGS)4 (SEQ ID NO:20), respectively. Bispecific recombinant protein BsAb Format28 possesses a bivalent CCR8-binding domain in Fab form, located at the N-terminus of the molecule; and a bivalent CTLA-4-binding domain in scFv form, tandemly linked to the C-terminus of the CCR8-binding domain via (GGGGS)4 (SEQ ID NO:20). The antibody subtype is human IgG1.

[0147] The antibody that binds to CCR8 is antibody B9B11, which was previously applied for by the inventors. The amino acid sequence of the antibody used is shown in Table 1 below.

[0148] Table 1 Sequences of CCR8-binding and CTLA-4-binding antibodies

[0149]

[0150]

[0151] The ipilimumab was mutated to VH G44C and VL Q100C during use.

[0152] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0153] Example 1: Protein expression of bispecific recombinant protein

[0154] The heavy and light chain plasmids of the quality-controlled bispecific recombinant protein were sterilized by filtration through a 0.22 μm filter, mixed in an appropriate ratio, and co-transfected into HEK293-6E cells via PEI-mediated transfection. Cell viability and number were monitored every other day. When cell viability decreased to approximately 60%, the protein supernatant was collected by centrifugation and purified using Protein A affinity chromatography. Antibodies were eluted with glycine, neutralized, and concentrated using ultrafiltration tubes. The storage buffer was then replaced with PBS. Preliminary analysis of the purity and size of the bispecific recombinant protein was performed by reducing SDS-PAGE. Results are as follows: Figure 2 As shown, the heavy and light chain sizes of the eight bispecific recombinant proteins obtained were consistent with expectations.

[0155] Example 2: Binding activity of bispecific recombinant protein at the cellular level

[0156] The full-length human CCR8 gene (UniProt ID: P51685) and the full-length human CTLA-4 gene (UniProt ID: P16410) were inserted into vectors used to construct stable cell lines. Plasmids were transfected into CHOK1 cells using transfection reagents. Positive clones were screened using puromycin after 24 to 48 hours. After obtaining single-clone cells, the binding activity of bispecific recombinant proteins was assessed using CHOK1 cells overexpressing hCCR8 and hCTLA-4, respectively. Logarithmically growing CHOK1-hCCR8 cells and CHOK1-hCTLA-4 cells were centrifuged, the supernatant was discarded, and the cells were washed three times with FACS Buffer, resuspended, and counted at 2 × 10⁻⁶. 5 Cells were seeded per well in 96-well plates. The bispecific recombinant protein and parental control antibody were serially diluted and added to the corresponding wells, mixed, and incubated at 4°C for 1 hour. After centrifugation and discarding the supernatant, the cells were washed three times with FACS Buffer, and then a 1:1000 dilution of APC anti-human IgG Fc secondary antibody (Jackson ImmunoResearch, catalog number 109-135-098) was added. The mixture was incubated at 4°C in the dark for 0.5 hours. The cells were washed three times with FACS Buffer, resuspended in an appropriate amount of FACS Buffer, and analyzed using iQue Screener PLUS. The binding curves of the bispecific recombinant protein to CHOK1-hCCR8 and CHOK1-hCTLA-4 are shown below. Figure 3 and Figure 4 As shown, higher antibody concentrations result in stronger average fluorescence signals, indicating that binding activity is concentration-dependent. The maximum binding signal values ​​and EC50 values ​​of the bispecific recombinant protein and parental monoclonal antibody with CCR8 and CTLA-4 are also shown. 50As shown in Table 2, the results indicate that among the eight bispecific recombinant proteins, BsAb Format2 exhibited poor binding activity to CCR8, while the other recombinant proteins maintained the activity of the parental monoclonal antibody B9B11 relatively well. However, in binding to CHOK1-hCTLA-4, only BsAb Format16 and BsAb Format28 showed relatively good activity.

[0157] Table 2

[0158]

[0159] Example 3: Blocking activity of bispecific recombinant protein at the cellular level

[0160] CHOK1-hCTLA-4 cells in logarithmic growth phase were centrifuged, the supernatant was removed, and the cells were washed three times with FACS Buffer. After resuspending, the cells were counted at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in 96-well plates. The bispecific recombinant protein and parental control antibody were serially diluted and added to the corresponding wells, along with either biotin-CD80 (Sinochem, catalog number 10698-H02H) or biotin-CD86 (Sinochem, catalog number 10698-H02H). The mixture was incubated at 4°C for 1 hour. After centrifugation and discarding the supernatant, the cells were washed three times with FACS Buffer. APC Streptavidin (BioLegend, catalog number 405207) diluted 1:1000 was added, and the mixture was incubated at 4°C in the dark for 1 hour. The cells were resuspended in an appropriate amount of FACS Buffer and analyzed using iQue Screener PLUS. The blocking ability of the bispecific recombinant protein against the interaction between CHOK1-hCTLA-4 and CD80 or CD86 was as follows: Figure 5 As shown, higher antibody concentrations correlate with stronger blocking activity. Table 3 shows the maximum blocking percentages of the bispecific recombinant protein and the parental monoclonal antibody. The results indicate that the blocking activity of the bispecific recombinant protein was weaker than that of the parental antibody ipilimumab, with BsAbFormat16 and BsAbFormat28 showing better blocking activity.

[0161] Table 3

[0162]

[0163]

[0164] Example 4: HPLC-SEC analysis of the purity of bispecific recombinant protein

[0165] The heavy and light chains of BsAb Format16 and BsAb Format28 were transfected into HEK293-6E (Fut8- / -) (fucosyltransferase 8 knockout) cells at a ratio of 2:3. The supernatant was purified by Protein A affinity assay, and the purity of the bispecific recombinant protein was determined by HPLC-SEC. The results are as follows: Figure 6A and Figure 6B As shown in the chromatogram, the absorption signal of the bispecific recombinant protein at UV 280 nm was detected at approximately 11 min after injection. The purities of the defucosylated bispecific recombinant proteins BsAbFormat16_AF and BsAbFormat28_AF were approximately 95% and 99%, respectively, indicating high purity.

[0166] Example 5: Determination of Tm value of bispecific recombinant protein

[0167] The Tm value of the bispecific recombinant protein was determined using differential scanning fluorometry (DSF). 2 μM of the bispecific recombinant protein was mixed with 10× SYPRO Orange fluorescent dye and placed in a quantitative real-time PCR instrument. The reaction program was run, increasing the temperature from 25℃ to 95℃, with each cycle increasing the temperature by 0.3℃. The fluorescence signal was detected in the HEX channel. Results are as follows: Figure 7 As shown in Figure A, the fluorescence intensity of the bispecific recombinant protein changes with increasing temperature. Figure 7 B represents the normalized derivative of the real-time dissolution curve of the bispecific recombinant protein. In this detection, the bispecific recombinant protein exhibited two fluorescence absorption transition peaks, with corresponding Tm values ​​shown in Table 4. The results indicate that both bispecific recombinant proteins possess good thermal stability.

[0168] Table 4

[0169]

[0170] Example 6: Affinity determination of bispecific recombinant protein

[0171] The affinity of a bispecific recombinant protein for the target antigen hCTLA-4 was detected using biomembrane interference technology on a ForteBio Octet Red96 label-free molecular interaction analyzer. hCTLA-4-his protein (Sino Biotech, catalog number 11159-H08H) was serially diluted seven times at two-fold concentrations starting from 100 nM. The bispecific recombinant protein to be tested and its parental monoclonal antibody ipilimumab were diluted to 10 μg / ml and immobilized on a protein A probe. As the antigen in solution binds to the immobilized antibody, the thickness of the probe biomembrane changes, resulting in a detectable change in spectral wavelength. Results are as follows: Figure 8The results show that as hCTLA-4-his gradually binds to the antibody, a real-time shift signal occurs in the interference spectrum. The kinetic parameters of the binding of the parental antibody ipilimumab, the bispecific recombinant proteins BsAb Format16_AF and BsAb Format28_AF to hCTLA-4-his are shown in Table 5. The results indicate that the bispecific recombinant proteins have good binding activity to hCTLA-4-his, but their dissociation rate is faster than that of the parental antibody.

[0172] Table 5

[0173] Antibody KD(M) kon(1 / Ms) kdis(1 / s) Full R^2 Ipilimumab 1.111E-09 1.93E+05 2.14E-04 0.9958 BsAb Format16_AF 5.483E-09 1.34E+05 7.33E-04 0.9953 BsAb Format28_AF 3.241E-09 1.97E+05 6.39E-04 0.9954

[0174] Example 7: Dual targeting of bispecific recombinant proteins

[0175] CHOK1-hCCR8 cells in logarithmic growth phase were centrifuged, the supernatant was removed, and the cells were washed three times with FACS Buffer. After resuspending, the cells were counted at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in 96-well plates. Serially diluted bispecific recombinant protein and parental control antibody were added to the corresponding wells, mixed, and incubated at 4°C for 1 hour. After centrifugation and discarding the supernatant, the cells were washed three times with FACS Buffer, and then serially diluted biotin-hCTLA-4-his was added. The mixture was then incubated at 4°C in the dark for 1 hour. Cells were washed three times with FACS Buffer, and then a 1:1000 dilution of APC streptavidin (BioLegend, catalog number 405207) was added. The mixture was then incubated at 4°C in the dark for 1 hour. Cells were resuspended in an appropriate amount of FACS Buffer and analyzed by flow cytometry using iQue Screener PLUS. Results are shown below. Figure 9 As shown, the bispecific recombinant protein can bind to both target antigens hCCR8 and hCTLA-4 simultaneously.

[0176] Example 8: ADCC activity of bispecific recombinant protein

[0177] CHOK1-hCCR8 or CHOK1-hCTLA-4 cells were digested and centrifuged, and then diluted to 0.5 × 10⁻⁶ cells with Assay Buffer preheated to 37°C. 6 Jurkat-hFcγRIIIa / NFAT-luc cells were seeded at 4000 cells / well in 96-well white plates (Corning, catalog number 3917). Serially diluted bispecific recombinant protein and its parental monoclonal antibody were added, mixed, and incubated at 37°C in a 5% CO2 incubator for 1 hour. Jurkat-hFcγRIIIa / NFAT-luc cells were collected, centrifuged, and diluted to 3.75 × 10⁻⁶ cells / mL with AssayBuffer preheated to 37°C. 6Cells / mL, at 1.5 × 10⁻⁶ 5 Add cells / well to the corresponding well, mix well, and incubate at 37°C, 5% CO2 for 6 hours. Add an equal volume of Bio-Lite™ Luciferase Assay Buffer (Vazyme, catalog number DD1201-03) to each well, incubate at room temperature for 5 minutes, and then detect the luminescence value using Spectra M5e. Results are as follows. Figure 10 and Figure 11 The results showed that the defucosylated bispecific recombinant protein exhibited concentration-dependent ADCC activity. EC50 values ​​of ADCC activity of the bispecific recombinant protein and the parental monoclonal antibody were compared. 50 As shown in Table 6.

[0178] Table 6

[0179]

[0180] Example 9: T cell activation effect of bispecific recombinant protein

[0181] Human PBMCs are divided into 1×10 5 Cells were placed in 96-well plates, and serially diluted bispecific recombinant protein was added. The plates were incubated at 37°C for 30 min, followed by the addition of staphylococcal enterotoxin B (SEB) at a final concentration of 400 ng / mL. The plates were then incubated at 37°C in a 5% CO2 incubator for 96 hours. The IL-2 content in the culture supernatant was then measured. Results are as follows: Figure 12 As shown, the bispecific recombinant protein can effectively activate T cells.

[0182] Example 10: Activity of bispecific recombinant protein in hCCR8 transgenic mice

[0183] Six- to eight-week-old female CCR8 humanized mice (C57BL / 6-hCCR8, purchased from Biocytogen, catalog number 110096) were selected, and MC38 cells (5 × 10⁶ cells) were subcutaneously inoculated into the right rib area of ​​the mice. 5 / 0.1mL / animal), when the tumor volume grows to 80-100mm 3 The drug administration began at a specific time. The experiment was conducted in two groups of seven animals each, administered intraperitoneally once a week. The doses of the fucose-deformed bispecific recombinant protein BsAb Format16_AF and isotype_AF (human IgG1 isotype control antibody) were 6.66 mg / kg and 5 mg / kg, respectively. Results are as follows: Figure 13 The results showed that the bispecific recombinant protein could effectively inhibit the growth of tumor cells in mice, demonstrating a good antitumor effect.

[0184] Example 11: Activity of bispecific recombinant protein in hCTLA-4 transgenic mice

[0185] Six- to eight-week-old female CTLA-4 humanized mice (C57BL / 6-hCTLA-4, purchased from Biocytogen, catalog number 110011) were selected, and MC38 cells (5 × 10⁻⁶ cells) were subcutaneously inoculated into the right rib area of ​​the mice. 5 / 0.1mL / animal), the tumor volume grew to 80-100mm. 3 The drug administration was initiated at the designated time. The experiment was conducted in two groups of seven animals each, administered intraperitoneally once weekly. The doses of the defucosylated bispecific recombinant protein BsAb Format16_AF and isotype_AF were 1.33 mg / kg and 1 mg / kg, respectively. Results are as follows: Figure 14 The results showed that the bispecific recombinant protein could effectively inhibit the growth of tumor cells in mice, demonstrating a good antitumor effect.

[0186] Example 12: Activity of bispecific recombinant protein in hCCR8 / hCTLA-4 transgenic mice

[0187] Six- to eight-week-old female CCR8 and CTLA-4 dual-target humanized mice (C57BL / 6-hCCR8 / hCTLA-4, purchased from Biocytogen, catalog number 112251) were selected, and MC38 cells (5 × 10⁻⁶ cells) were subcutaneously inoculated into the right rib area of ​​the mice. 5 / 0.1mL / animal), the tumor volume grew to 80-100mm. 3 The drug administration began at a certain time. The experiment was conducted in 5 groups, with 7 animals in each group. The drugs were administered intraperitoneally, a total of 3 times during the treatment cycle. The doses of BsAb Format16_AF, B9B11_AF, ipilimumab, the combination group (B9B11_AF + ipilimumab), and isotype_AF were 0.4 mg / kg, 0.3 mg / kg, 0.3 mg / kg, 0.15 mg / kg + 0.15 mg / kg, and 0.4 mg / kg, respectively. The results are as follows: Figure 15 The results showed that the bispecific recombinant protein could effectively inhibit the growth of tumor cells in mice, exhibiting antitumor activity superior to that of the parental monoclonal antibody.

[0188] Example 13: Pharmacokinetic analysis of bispecific recombinant protein in cynomolgus monkeys

[0189] One female and one male cynomolgus monkey aged 3-5 years and weighing 2.5-3.5 kg were selected for a preliminary pharmacokinetic study of BsAbFormat16_AF. The dosage of BsAbFormat16_AF was 3 mg / kg, administered as a single dose. BsAbFormat16_AF was diluted to 0.6 mg / mL with sterile saline, and administered in a 5 mL / kg intravenous bolus over 0.5 h. Blood and serum samples were collected immediately after administration (±1 min), at 2 h (±5 min), 8 h (±10 min), 24 h (±30 min), 48 h (±30 min), 72 h (±30 min), 96 h (±30 min), 120 h (±30 min), 168 h (±30 min), 336 h (±60 min), and 504 h (±60 min), respectively. Serum antibody drug concentrations were determined using ELISA. A proportionally diluted serum sample was captured by coating an ELISA plate with goat anti-human Fab (Sigma, catalog number I5260), and then detected using HRP goat anti-human FC (Jackson ImmunoResearch, catalog number 109-035-098) or biotin-labeled hCTLA4-his. Results are as follows: Figure 16 The results showed that the bispecific recombinant protein exhibited good pharmacokinetic characteristics as expected.

Claims

1. A bispecific recombinant protein, characterized in that, The bispecific recombinant protein includes one or more first binding domains for binding CCR8 and one or more second binding domains for binding CTLA-4; the first binding domain includes a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprising amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to Kabat.

2. The bispecific recombinant protein as described in claim 1, characterized in that, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13 or having at least 90% sequence identity with SEQ ID NO:13; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14 or having at least 90% sequence identity with SEQ ID NO:14; And / or, the first binding domain is an antigen-binding fragment that binds to CCR8; the second binding domain is an antigen-binding fragment that binds to CTLA-4; And / or, in the bispecific recombinant protein, the number of the first binding domains is 1, 2, or 4; the number of the second binding domains is 1, 2, or 4; Preferably, the antigen-binding fragment is selected from Fab, Fab'-SH, scFv, Fv or (Fab')2; and / or, one or more of the first binding domains are linked by peptide linkers having amino acid sequences as shown in any of SEQ ID NO:20-25.

3. The bispecific recombinant protein as described in claim 1 or 2, characterized in that, In the first binding domain, the heavy chain variable region and the light chain variable region are directly bound to CCR8; and / or, in the second binding domain, the heavy chain variable region and the light chain variable region are directly bound to CTLA-4 or connected through a connector before being bound to CTLA-4. Preferably, in the second binding domain, the heavy chain variable region and the light chain variable region are connected by a linker before binding to CTLA-4; and / or, the linker has an amino acid sequence as shown in SEQ ID NO:20; More preferably, the first binding domain is Fab and the second binding domain is scFv; or, the first binding domain is Fv and the second binding domain is Fab; or, both the first and second binding domains are Fab. More preferably, when the second binding structural domain is scFv, the C end of the heavy chain variable region is connected to the N end of the light chain variable region through a connector, or the C end of the light chain variable region is connected to the N end of the heavy chain variable region through a connector.

4. The bispecific recombinant protein according to any one of claims 1-3, characterized in that, The bispecific recombinant protein also includes Fc; Preferably, the first binding domain is located at the N-terminus of the Fc, and the second binding domain is connected to the N-terminus or C-terminus of the Fc; and / or, the Fc is the Fc of human immunoglobulin, preferably the Fc of human IgG, more preferably the Fc of human IgG1; and / or, the Fc has the amino acid sequence shown in SEQ ID NO:

19. More preferably, the second binding domain is connected to the C-terminus of the Fc via a peptide linker, the peptide linker having, for example, the amino acid sequence shown in SEQ ID NO: 20 or 25; and / or, the number of the first binding domains is 2, the number of the second binding domains is 1, one C-terminus of the first binding domain and the second binding domain are respectively connected to the N-terminus of the Fc, and the other C-terminus of the first binding domain is connected to the N-terminus of either the first binding domain or the second binding domain connected to the N-terminus of the Fc; or, the first binding domain is connected to the N-terminus of the Fc, and the second binding domain is connected to the C-terminus of the Fc; or, the C-terminus of the second binding domain is connected to the N-terminus of the Fc, and the C-terminus of the first binding domain is connected to the N-terminus of the second binding domain; For example: The first binding domain is Fab, the second binding domain is scFv, the C-terminus of the first binding domain is connected to the N-terminus of the Fc, and the N-terminus of the second binding domain is connected to the C-terminus of the Fc; or, The first binding domain is Fab, the second binding domain is scFv, and the first and second binding domains are sequentially connected to the Fc from the N-end to the C-end; or... The first binding domain is Fv, the second binding domain is Fab, and the first binding domain, the second binding domain, and Fc are sequentially connected from the N-end to the C-end; or... The first and second bonding domains are Fab, and the first and second bonding domains are sequentially connected to the Fc from the N-end to the C-end; or... The first and second bonding domains are Fab, with the C-terminus of the first bonding domain connected to the N-terminus of the Fc, and the C-terminus of the Fc connected to the N-terminus of the second bonding domain; or, The first bonding structural domain and the second bonding structural domain are Fab. There are 2 first bonding structural domains and 1 second bonding structural domain. The C-end of one first bonding structural domain and the second bonding structural domain are respectively connected to the N-end of the Fc. The C-end of the other first bonding structural domain is connected to the N-end of the first bonding structural domain or the second bonding structural domain connected to the N-end of the Fc.

5. The bispecific recombinant protein according to any one of claims 1-4, characterized in that, The second binding domain includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains amino acid sequences HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively. The light chain variable region contains amino acid sequences LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to Kabat. Preferably, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:15 or having at least 90% sequence identity with SEQ ID NO:15; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:16 or having at least 90% sequence identity with SEQ ID NO:

16.

6. The bispecific recombinant protein according to any one of claims 1-5, characterized in that, The bispecific recombinant protein also includes a heavy chain constant region 1 (CH1) and a light chain constant region (CL); Preferably, the heavy chain constant region 1 and the light chain constant region are human heavy chain constant regions and light chain constant regions; and / or, the heavy chain constant region 1 and the light chain constant region are connected to the heavy chain variable region and the light chain variable region in a CrossMab form. More preferably, the heavy chain constant region 1 is selected from CH1 of IgG1, IgG2, IgG3 and IgG4; and / or, the light chain constant region is a kappa chain or a lambda chain; For example, the heavy chain constant region 1 has an amino acid sequence as shown in SEQ ID NO:17; and / or, the light chain constant region is a kappa chain with an amino acid sequence as shown in SEQ ID NO:

18.

7. The bispecific recombinant protein according to any one of claims 1-6, characterized in that, The bispecific recombinant protein comprises polypeptide chain 1 and polypeptide chain 2. The structure of polypeptide chain 1 from N-terminus to C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1 - heavy chain variable region of the second binding domain - linker - light chain variable region of the second binding domain; the structure of polypeptide chain 2 from N-terminus to C-terminus is: light chain variable region of the first binding domain - light chain constant region; or, The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1 - light chain variable region of the second binding domain - linker - heavy chain variable region of the second binding domain; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; or, The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - light chain variable region of the second binding domain - linker - heavy chain variable region of the second binding domain - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; or, The structure of polypeptide chain 1 from N-terminus to C-terminus is: heavy chain variable region of the first binding domain - heavy chain variable region of the second binding domain - heavy chain constant region 1 - Fc1; the structure of polypeptide chain 2 from N-terminus to C-terminus is: light chain variable region of the first binding domain - light chain variable region of the second binding domain - light chain constant region. or, The bispecific recombinant protein comprises polypeptide chain 1, polypeptide chain 2, and polypeptide chain 3. The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - heavy chain variable region of the second binding domain - light chain constant region - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from the N-terminus to the C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; or... The structure of polypeptide chain 1 from N-terminus to C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1 - heavy chain variable region of the second binding domain - light chain constant region; the structure of polypeptide chain 2 from N-terminus to C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from N-terminus to C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; or, The bispecific recombinant protein comprises polypeptide chain 1, polypeptide chain 2, polypeptide chain 3, and polypeptide chain 4. The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - heavy chain variable region of the second binding domain - light chain constant region - Fc2; the structure of polypeptide chain 4 from the N-terminus to the C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; or... The structure of polypeptide chain 1 from the N-terminus to the C-terminus is: heavy chain variable region of the first binding domain - heavy chain constant region 1 - heavy chain variable region of the first binding domain - heavy chain constant region 1 - Fc1; the structure of polypeptide chain 2 from the N-terminus to the C-terminus is: light chain variable region of the first binding domain - light chain constant region; the structure of polypeptide chain 3 from the N-terminus to the C-terminus is: heavy chain variable region of the second binding domain - light chain constant region - Fc2; the structure of polypeptide chain 4 from the N-terminus to the C-terminus is: light chain variable region of the second binding domain - heavy chain constant region 1; Fc1 is paired to form Fc, or Fc1 is paired with Fc2 to form Fc; preferably, Fc1 and Fc2 are connected by KiH.

8. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific recombinant protein as described in any one of claims 1-7.

9. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule as described in claim 8; Preferably, the recombinant vector is an expression vector.

10. A transformant, characterized in that, The transformant comprises the nucleic acid molecule as described in claim 8 or the recombinant vector as described in claim 9; Alternatively, the transformant expresses the bispecific recombinant protein as described in any one of claims 1-7.

11. A composition, characterized in that, The composition comprises the bispecific recombinant protein as described in any one of claims 1-7, the nucleic acid molecule as described in claim 8, the recombinant vector as described in claim 9, and / or the transformant as described in claim 10, and a second reagent; Preferably, the composition is a pharmaceutical composition, and the second reagent is a pharmaceutically acceptable carrier and / or excipient.

12. A method for preparing a bispecific recombinant protein, characterized in that, The method includes culturing the transformant as described in claim 10 to obtain the bispecific recombinant protein from the culture.

13. The use of the bispecific recombinant protein as described in any one of claims 1-7, the nucleic acid molecule as described in claim 8, the recombinant vector as described in claim 9, the transformant as described in claim 10, or the composition as described in claim 11 in the preparation of a medicament for the prevention and / or treatment of cancer; Preferably, the cancer is a cancer infiltrated by Treg cells; And / or, the cancer is selected from colorectal cancer, melanoma, malignant pleural mesothelioma, renal cell carcinoma, liver cancer, esophageal cancer, non-small cell lung cancer, and glioblastoma.

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