Trispecific single-chain antibodies and uses thereof

Abstract

The application provides a trispecific single-chain antibody and use thereof, wherein the trispecific single-chain antibody comprises a first antigen-binding portion specifically binding to CLEC9A, a second antigen-binding portion specifically binding to PDL1, and a third antigen-binding portion specifically binding to CTLA4. The trispecific single-chain antibody provided by the application bridges DC cells and T cells, can realize effective antigen presentation of the DC cells, and can activate T cells, while reducing antibody-mediated cytotoxicity, enhances specific killing of target cells presenting pHLA antigens, and has a broad application prospect in the field of cancer immunotherapy.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a trispecific single-chain antibody and its uses. Background Technology

[0002] Dendritic cells (DCs) are the only professional antigen-presenting cells (APCs) capable of activating naive T cells, playing a crucial role in the immune process. DCs activated under conditions such as phagocytosis of exogenous protein antigens can recruit other immune cells (e.g., macrophages (Mac), eosinophils, natural killer cells (NK), and T cells) and interact with them, thereby further activating T cells and promoting their differentiation into effector T cells or memory T cells, achieving the regulation or killing of target cells.

[0003] Immunotherapy methods such as immune checkpoint inhibition and CAR-T have made significant progress in cancer treatment, but only a portion of the population benefits from them. Tumor resistance to radiotherapy and chemotherapy, including small molecule drugs, and immune escape from immunotherapy by tumor cells limit the effectiveness of cancer treatment. T cells play a crucial role in suppressing and killing tumor cells, but effective activation of T cells requires three signals: signal 1, the interaction between pHLA (peptide-HLA) tumor-specific or related antigens presented by dendritic cells (DCs) and the T cell receptor (TCR); signal 2, the interaction between the co-stimulatory molecules CD80 and CD86 of DCs and CD28 on the surface of T cells; and signal 3, the further activation of DCs by cytokines secreted by activated DCs (such as IFN-I factors IFNα and IL-12) and IFNγ secreted by T cells, thereby enhancing T cell activity and promoting T cell proliferation.

[0004] The mechanisms involved in tumor immune escape include tumor cells downregulating the expression of human leukocyte antigen (HLA) or secreting immunosuppressive cytokines such as IL10 and TGF-β, which accelerates the exhaustion of immune cells, thereby prompting immune cells to overexpress proteins such as PD1, CTLA4, and TIM3; another mechanism is that tumor cells prevent dendritic cells (DCs) from recruiting immune cells or damage the function of DCs by overexpressing immune checkpoint proteins such as PDL1, thereby escaping the killing of innate and adaptive immune cells.

[0005] Therefore, promoting the effective recruitment of immune cells by dendritic cells (DCs) and activating DCs and recruited immune cells to achieve effective control of tumor cells has become a current research focus. Summary of the Invention

[0006] The purpose of this application is to provide a trispecific single-chain antibody that bridges DC cells and T cells and its uses.

[0007] Specifically, this application relates to the following:

[0008] 1. A trispecific single-chain antibody, wherein the trispecific single-chain antibody comprises a first antigen-binding portion that specifically binds to CLEC9A, a second antigen-binding portion that specifically binds to PDL1, and a third antigen-binding portion that specifically binds to CTLA4.

[0009] 2. The trispecific single-chain antibody according to claim 1, wherein the first antigen-binding moiety comprises VHH CLEC9A Preferably, the VHH CLEC9A The amino acid sequence is shown in SEQ ID NO: 2.

[0010] 3. The trispecific single-chain antibody according to claim 1 or 2, wherein the second antigen-binding moiety comprises VHH PDL1 Preferably, the VHH PDL1 The amino acid sequence is shown in SEQ ID NO: 3.

[0011] 4. The trispecific single-chain antibody according to any one of claims 1-3, wherein the third antigen-binding moiety comprises VHH CTLA4 Preferably, the VHH CTLA4 The amino acid sequence is shown in SEQ ID NO: 4.

[0012] 5. The trispecific single-chain antibody according to any one of claims 1-4, wherein the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion are linked to each other by short linker peptides;

[0013] Preferably, the amino acid sequence of the short linker peptide is (GGS). n The n is a natural number between 10 and 20;

[0014] More preferably, the amino acid sequence of the short linker peptide is (GGS). 10 .

[0015] 6. The trispecific single-chain antibody according to claim 5, wherein the first antigen-binding portion is attached to the N-terminus of the second antigen-binding portion, and the third antigen-binding portion is attached to the C-terminus of the second antigen-binding portion.

[0016] 7. The trispecific single-chain antibody according to any one of claims 1-6, wherein the trispecific single-chain antibody comprises a cytokine portion.

[0017] 8. The trispecific single-chain antibody according to claim 7, wherein the cytokine portion is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFNα, IFNβ, or mutants thereof;

[0018] Preferably, the cytokine portion is selected from IFNα or its mutants;

[0019] More preferably, the cytokine portion is an IFNα mutant, and the amino acid sequence of the IFNα mutant is shown in SEQ ID NO: 5.

[0020] 9. The trispecific single-chain antibody according to claim 8, wherein the cytokine portion and the third antigen-binding portion are linked to each other by a long linker peptide;

[0021] Preferably, the amino acid sequence of the long linker peptide is (GGS). n The n is a natural number between 10 and 20;

[0022] More preferably, the amino acid sequence of the long linker peptide is (GGS). 20 .

[0023] 10. The trispecific single-chain antibody according to claim 9, wherein the cytokine portion is linked to the C-terminus of the third antigen-binding portion.

[0024] 11. The trispecific single-chain antibody according to any one of claims 1-10, wherein the trispecific single-chain antibody comprises a signal peptide having the amino acid sequence of the signal peptide as shown in SEQ ID NO: 1;

[0025] Preferably, the signal peptide is located at the N-terminus of the trispecific single-chain antibody.

[0026] 12. The trispecific single-chain antibody according to any one of claims 1-11, wherein the trispecific single-chain antibody comprises a His-tagged peptide, the amino acid sequence of which is shown in SEQ ID NO: 8;

[0027] Preferably, the His-tagged peptide is located at the C-terminus of the trispecific single-chain antibody.

[0028] 13. The trispecific single-chain antibody according to any one of claims 1-12, wherein the amino acid sequence of the trispecific single-chain antibody is shown in SEQ ID NO: 9.

[0029] 14. A nucleic acid, wherein the nucleic acid comprises a trispecific single-chain antibody encoding any one of items 1-13.

[0030] 15. A host cell, wherein the host cell comprises the nucleic acid described in item 14.

[0031] 16. A method for producing a trispecific single-chain antibody, wherein the method comprises culturing the host cells described in item 15 to produce the trispecific single-chain antibody described in any one of items 1-13.

[0032] 17. A pharmaceutical composition comprising any one of claims 1-13 of the present invention.

[0033] 18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0034] 19. Use of the trispecific single-chain antibody according to any one of items 1-13 or the pharmaceutical composition according to item 17 or 18 in the preparation of a medicament for treating and / or preventing tumors.

[0035] 20. The use according to item 19, wherein the tumor is a tumor expressing a pHLA tumor-specific or related antigen.

[0036] Beneficial effects:

[0037] This application provides a trispecific single-chain antibody capable of bridging DC cells and T cells. The trispecific single-chain antibody targets CTLA4, PDL1, and CTLA4, and is linked to IFNα2 with reduced affinity. Q124R This effectively shortens the distance between dendritic cells (DCs) and T cells, inhibits the expression of immune checkpoints PDL1 and CTLA4, and activates the function of both DCs and T cells. The trispecific single-chain antibody of this application reduces antibody-mediated cytotoxicity to a certain extent and enhances the specific killing of target cells presenting pHLA antigen, showing broad application prospects in the field of cancer immunotherapy. Attached Figure Description

[0038] Figure 1 For scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Schematic diagram of the structure of a trispecific single-chain antibody;

[0039] Figure 2 For scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Schematic diagram of the structure of a four-specific single-chain antibody;

[0040] Figures 3A-3B show the antibody purity detection results. Figure 3A shows the scDB-VHH antibody purity test results. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R SDS-PAGE images of the trispecific single-chain antibody; Figure 3B shows scDB-VHH. CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 SDS-PAGE image of four specific single-chain antibodies.

[0041] Figure 4A-Figure 4B are scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The spatial structure diagrams of the three-specific single-chain antibodies are shown in Figure 4A, which is the 3D structure diagram of the antibody; and Figure 4B, which is the 3D structure diagram of the interaction between the antibody and the corresponding antigen protein.

[0042] Figure 5 For scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 3D structure diagram of a four-specific single-chain antibody.

[0043] Figure 6 CMV-induced CD8 + and CD4 + Image showing the detection results of T cell amplification.

[0044] Figures 7A-7C represent scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Figure 7 shows the results of the affinity assay between the three-specific single-chain antibody and mDC cells. Figure 7A shows the Kd values ​​of the antibody and CLEC9A and PDL1 on the surface of mDC cells; Figure 7B shows the Kd values ​​of the antibody and CLEC9A on the surface of mDC cells. D Value; Figure 7 C shows the K value of antibody and PDL1 on the surface of mDC cells. D value.

[0045] Figure 8A-Figure 8B are scDB-VHH CLEC9A -VHH PDL1-VHH CTLA4 -IFNα2 Q124R Figure 8 shows the results of the detection of affinity between the three-specific single-chain antibody and T cells. Figure 8A shows the Kd value of the antibody and CTLA4 on the surface of T cells; Figure 8B shows the Kd value of the antibody and CTLA4 on the surface of T cells. D value.

[0046] Figures 9A-9C represent scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Figure 9 shows the results of the detection of affinity between four specific single-chain antibodies and mDC cells. Figure 9A shows the Kd values ​​of the antibody and CLEC9A and CD40 on the surface of mDC cells; Figure 9B shows the Kd values ​​of the antibody and CLEC9A on the surface of mDC cells. D Value; Figure 9 C shows the K value of antibody and CD40 on the surface of mDC cells. D value.

[0047] Figures 10A-10C are scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Figure 10 shows the detection results of the affinity between four specific single-chain antibodies and T cells. Figure 10A shows the Kd values ​​of the antibody and CTLA4 and PD1 on the surface of T cells; Figure 10B shows the Kd values ​​of the antibody and CTLA4 on the surface of T cells. D Value; Figure 10 C shows the K value of antibody and PD1 on the surface of T cells. D value.

[0048] Figures 11A-11B show Elispot's detection of scDB-VHH. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Trispecific single-chain antibody and scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Figure 11 shows the results of DC cell activation of T cells mediated by four specific single-chain antibodies. Figure 11A shows the statistical results of Elispot's dot count; Figure 11B shows the actual dot count of Elispot.

[0049] Figure 12A-Figure 12B are scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Trispecific single-chain antibody and scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Figure 12 shows the detection results of T cell activation by four specific single-chain antibodies. Figure 12A shows the detection results of CD69 expression in T cells; Figure 12B shows the detection results of T cell immune checkpoint TIM3 expression.

[0050] Figures 13A-13F are scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The results of the stability assay for the trispecific single-chain antibody in mDC cell culture medium are shown in Figure 13. Figure 13A shows the antibody-mDC cell binding strength; Figure 13B shows the proportion of mDC cells binding the antibody; Figure 13C shows the expression of PDL1 on the surface of mDC cells; Figure 13D shows the proportion of cells expressing PDL1; Figure 13E shows the expression of CLEC9A on the surface of mDC cells; and Figure 13F shows the proportion of cells expressing CLEC9A.

[0051] Figures 14A-14C represent scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The results of detecting the non-targeted toxicity of the three-specific single-chain antibody against mDC cells are shown in Figure 14A. Figure 14B shows the mDC cell viability; Figure 14C shows the mDC cell number; and Figure 14C shows the mDC cell apoptosis rate.

[0052] Figure 15 To detect scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Trispecific single-chain antibody and scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4Figure showing the killing effect of T cells on CMV-loaded T2 cells mediated by four specific single-chain antibodies. Detailed Implementation

[0053] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0054] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0055] definition

[0056] The term "CLEC9A" as used in this paper (also known as DNGR1, UNQ9341, CD370, DNGR-1, member A of the C-type lectin domain family 9, or C-type lectin domain containing 9A) refers to a group V C-type lectin-like receptor (CLR) that functions as an activating receptor and is expressed on dendritic cells (DCs). CLEC9A can act as an endocytic receptor on a small subset of DCs specifically designed for the uptake and processing of material from dead cells. CLEC9A recognizes filamentous actin, is associated with actin-binding proteins, can be exposed upon cell membrane damage, and can mediate the cross-presentation of dead cell-associated antigens.

[0057] The term “PDL1” or “PD-L1” as used in this article refers to programmed death ligand-1, also known as CD279 (differentiation cluster 279), an important immunosuppressive molecule.

[0058] The term "CTLA4" used in this article refers to cytotoxic T-lymphocyte-associated protein 4, which inhibits the immune response by binding to its ligands CD80 (also known as B7-1) and CD86 (also known as B7-2). CTLA-4 inhibits the immune response in several ways: for example, 1) by competing with the T cell costimulatory receptor CD28 for its ligands CD80 and CD86, thereby blocking costimulation; and 2) by emitting negative signals that inhibit T cell activation. CTLA-4 inhibitors, by inhibiting the CTLA-4 molecule, enable T cells to proliferate rapidly and attack tumor cells.

[0059] The term “antibody” as used herein is used in the broadest sense and encompasses a wide variety of antibody structures, including not only complete (i.e., full-length) antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), their variants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies (scFV), VHH antibodies, multispecific antibodies (e.g., bispecific antibodies), multispecific single-chain antibodies (e.g., bispecific single-chain antibodies (scDB)), and any other modified configurations of immunoglobulin molecules containing antigen recognition sites of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0060] Typically, a full-length or complete antibody consists of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL), the light chain constant region including κ (Kappa) or λ (Lambda). Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant regions of the heavy chains. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0061] As used herein, the term “binding” or “specific binding” refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic determinant.

[0062] As used herein, the term "antigen-binding moiety" refers to an antipeptide molecule that specifically binds to an antigenic determinant. Specific antigen-binding moieties can be, for example, Fv, Fab, Fab', Fab'-SH, F(ab')2, biantibodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFv-Fc), nanobodies, domain antibodies, bivalent domain antibodies, or any other fragment or combination thereof of an antibody that binds to an antigen.

[0063] The term "nanobody" used in this article refers to a naturally occurring heavy chain antibody that lacks a light chain, found in animals such as camels. Cloning its variable region yields a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable Domain of Heavy Chain of Heavy Chain antibody), which is the smallest functional antigen-binding fragment.

[0064] In this article, the terms "VHH," "nanobody," and "single-domain antibody" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of a heavy chain antibody to construct a single-domain antibody consisting of only one heavy chain variable region. This is the smallest antigen-binding fragment with full function. Typically, a naturally occurring heavy chain antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0065] The term “trispecific” as used in this article means that an antibody can specifically bind to at least three different antigenic determinants.

[0066] The term “quadrispecific” as used in this article means that the antibody can specifically bind to at least four different antigenic determinants.

[0067] The terms “first,” “second,” and “third” used in this document are for convenience of distinction when there are more than one part of each type. Unless explicitly stated otherwise, they are not intended to assign a specific order or orientation.

[0068] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0069] As used herein, the terms “host cell,” “host cell line,” “host cell culture,” or related terms refer to cells (or populations thereof) in which foreign (exogenous or transgenic) nucleic acids have been introduced. The external nucleic acid may include an expression vector operatively linked to a transgene, and the host cell may be used to express nucleic acids and / or polypeptides encoded by the external nucleic acid (transgene). The host cell (or population thereof) may be cultured cells or may be extracted from the object. Regardless of the number of pathways, the host cell (or population thereof) includes primary object cells and their progeny. Progeny cells may or may not carry the same genetic material as the parent cells. The term “host cell” encompasses progeny cells. In one embodiment, “host cell” describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express antibodies as disclosed herein. In one embodiment, the host cell (or population thereof) may be incorporating an expression vector operatively linked to a nucleic acid encoding a desired antibody as described herein. The host cell and its population may carry an expression vector stably integrated into the host genome or may carry an extrachromosomal expression vector. In one implementation, the host cell and its population may carry an extrachromosomal vector that exists after several cell divisions, or exists temporarily and disappears after several cell divisions.

[0070] The term "K" as used in this article D "" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen, and is obtained as the dissociation rate constant (K). d ) Specific binding rate constant (K a (i.e., K) d / K a The ratio of antibody to antigen is considered. A smaller equilibrium dissociation constant indicates a tighter antibody-antigen binding and higher affinity between the antibody and antigen. The specific binding properties between the two molecules can be determined using methods known in the art.

[0071] As used herein, the term "pharmaceutical composition" refers to an article which is presented in a form that allows the biological activity of the active ingredient contained therein to exert its effect, and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation is to be administered.

[0072] As used herein, the term "pharmaceuticalally acceptable carrier" means a component of a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0073] The terms "tumor" and "cancer" as used herein are used interchangeably and generally refer to growths formed by the proliferation of local tissue cells under the influence of various tumorigenic factors. In one embodiment, the tumor is a tumor expressing the pHLA tumor-specific or related antigen.

[0074] In this article, the term "HLA" refers to human leukocyte antigen. HLA genes encode major histocompatibility complex (MHC) proteins in humans. MHC proteins are expressed on the cell surface and participate in the activation of the immune response. HLA class I genes encode MHC class I molecules, which are expressed on the cell surface as complexes with peptide fragments (antigens) of self or non-self proteins. MHC class I molecules interact with CD8... + Cytotoxic T cell interactions play an important role in disrupting organ transplant rejection or infected cells.

[0075] As used herein, the terms "HLA class I molecule" or "HLA class I molecule" refer to the protein product of a wild-type or variant HLA class I gene encoding an MHC class I molecule. Therefore, "HLA class I molecule" and "MHC class I molecule" are used interchangeably in this document.

[0076] The term "pHLA" as used in this article may refer to peptide-bound human leukocyte antigen.

[0077] Antibody

[0078] This application provides a trispecific single-chain antibody, wherein the trispecific single-chain antibody comprises a first antigen-binding portion that specifically binds to CLEC9A, a second antigen-binding portion that specifically binds to PDL1, and a third antigen-binding portion that specifically binds to CTLA4.

[0079] In one specific embodiment, the first antigen-binding portion includes VHH CLEC9A In one specific implementation, the VHH CLEC9A Having a sequence

[0080] SEQ ID NO: 2:

[0081] The amino acid sequence of QVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTWLQMNSLRPEDTAWYCKAFTRGDDYWGQGTQVTVSS.

[0082] In one specific embodiment, the second antigen-binding portion includes VHH PDL1In one specific implementation, the VHH PDL1 Having a sequence

[0083] SEQ ID NO: 3:

[0084] The amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS.

[0085] In one specific embodiment, the third antigen-binding portion includes VHH CTLA4 In one specific implementation, the VHH CTLA4 Having a sequence

[0086] SEQ ID NO: 4:

[0087] Amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAASGYIYSAYCMGWFRQAPGKGLEGVAAIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADVIPTETCLGGSWSGPFGYWGQGTLVTVSS.

[0088] In one specific embodiment, the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion are linked to each other by short linker peptides. In one specific embodiment, the amino acid sequence of the short linker peptide is (GGS). n Where n is a natural number between 10 and 20. In one specific embodiment, the amino acid sequence of the short linker peptide is (GGS). 10 .

[0089] In one specific embodiment, the first antigen-binding portion is attached to the N-terminus of the second antigen-binding portion, and the third antigen-binding portion is attached to the C-terminus of the second antigen-binding portion.

[0090] In one specific embodiment, the trispecific single-chain antibody includes a cytokine portion.

[0091] In one specific embodiment, the cytokine portion is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFNα, IFNβ, or mutants thereof. In one specific embodiment, the cytokine portion is selected from IFNα or a mutant thereof. In one specific embodiment, the cytokine portion is an IFNα mutant, and the IFNα mutant has a sequence...

[0092] SEQ ID NO: 5:

[0093] The amino acid sequence of CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE.

[0094] In one specific embodiment, the cytokine portion and the third antigen-binding portion are linked together by a long linker peptide. In one specific embodiment, the amino acid sequence of the long linker peptide is (GGS). n Where n is a natural number between 10 and 20. In one specific embodiment, the amino acid sequence of the long linker peptide is (GGS). 20 .

[0095] In one specific embodiment, the cytokine portion is attached to the C-terminus of the third antigen-binding portion.

[0096] In one specific embodiment, the trispecific single-chain antibody comprises a signal peptide having the amino acid sequence SEQ ID NO:1: MYRMQLLSCIALSLALVTNS. In one specific embodiment, the signal peptide is located at the N-terminus of the trispecific single-chain antibody.

[0097] In one specific embodiment, the trispecific single-chain antibody comprises a His-tagged peptide having the amino acid sequence SEQ ID NO:8: HHHHHHHHHH. In one specific embodiment, the His-tagged peptide is located at the C-terminus of the trispecific single-chain antibody.

[0098] In one specific embodiment, the trispecific single-chain antibody has

[0099] SEQ ID NO: 9:

[0100] The amino acid sequence.

[0101] Nucleic acids, host cells, and production methods

[0102] This application provides a nucleic acid, wherein the nucleic acid comprises encoding any of the aforementioned trispecific single-chain antibodies.

[0103] The nucleic acid can be obtained according to conventional methods in the art, such as obtaining the DNA sequence encoding a trispecific single-chain antibody through PCR amplification, DNA recombination or chemical synthesis techniques.

[0104] This application provides a host cell, wherein the host cell contains any of the aforementioned nucleic acids.

[0105] The method for introducing exogenous nucleic acids into the host cells can be based on conventional methods in the art. The method of introducing exogenous nucleic acids can vary depending on the host cell used. The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, for example, CHO or HEK293 cells. When the host cell is a higher eukaryotic cell, DNA transfection methods can be selected, such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, or liposome packaging.

[0106] This application provides a method for producing a trispecific single-chain antibody, wherein the method includes culturing any of the aforementioned host cells to produce any of the aforementioned trispecific single-chain antibodies.

[0107] Typically, host cells are cultured under conditions suitable for antibody expression, and then the antibody is purified using conventional immunoglobulin purification steps, such as protein A agarose purification resin, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0108] Pharmaceutical Composition

[0109] This application provides a pharmaceutical composition comprising any one of the aforementioned trispecific single-chain antibodies.

[0110] In one specific embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0111] Pharmaceutically acceptable carriers may include non-toxic buffers such as phosphoric acid, citric acid, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzyl chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoates). Parabens, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight peptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants such as Tween or polyethylene glycol (PEG), etc.

[0112] use

[0113] This application also provides the use of any of the foregoing trispecific single-chain antibodies or any of the foregoing pharmaceutical compositions in the preparation of drugs for the treatment and / or prevention of tumors.

[0114] In one specific implementation, the tumor is a tumor expressing the pHLA tumor-specific or related antigen.

[0115] In one specific embodiment, the tumor is a tumor that presents tumor-specific or related antigens through MHC class I molecules.

[0116] Based on the structure of a bispecific single-chain diabody (scDB), this application utilizes VHH and a T-cell activating factor to design a trispecific single-chain antibody that can bridge DC cells and T cells, demonstrating significant advantages in anti-tumor effects.

[0117] In this application, by detecting the activation of T cells by DC cells mediated by trispecific single-chain antibodies, it was found that trispecific single-chain antibodies can significantly increase the activation of T cells by DC cells, while increasing IFNγ secretion, and compared with scDB-VHH which also bridges DC cells and T cells. CLEC9A -VL CD40 -VH PD1 -VLPD1 -VH CD40 -VHH CTLA4 Quadrispecific single-chain antibodies are more effective.

[0118] In this application, by detecting the activation of T cells by trispecific single-chain antibodies, it can be seen that trispecific single-chain antibodies can directly activate T cells, and compared with tetraspecific single-chain antibodies, they significantly increase the expression of CD69 in T cells and reduce the expression of T cell immune checkpoint TIM3.

[0119] In this application, by detecting the non-targeted toxicity of the trispecific single-chain antibody to mDC cells, it was found that there were no significant changes in the viability, number, and apoptosis rate of mDC cells, indicating that the trispecific single-chain antibody has only mild toxicity to mDC cells.

[0120] In this application, by detecting the killing effect of T cells mediated by trispecific single-chain antibodies on CMV-loaded T2 cells, it can be seen that the killing effect of T cells mediated by trispecific single-chain antibodies on target cells is specific and has a better killing effect than that of tetraspecific single-chain antibodies.

[0121] Overall, the trispecific single-chain antibody provided in this application can present effective antigens to dendritic cells (DCs) and activate T cells. While reducing antibody-mediated cytotoxicity, it enhances the specific killing of target cells presenting pHLA antigens and has good safety profile, making it a promising candidate for tumor immunotherapy.

[0122] Example

[0123] The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0124] Example 1

[0125] 1.1 scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Trispecific single-chain antibody structural design

[0126] Designed using the variable region (VHH) of the CLEC9A nanobody targeting DC cells CLEC9A ), nanobody PDL1 variable region (VHH) PDL1 ); CTLA4 variable region (VHH) of nanobodies targeting T cells CTLA4); and the cytokine IFNα mutant (IFNα2) Q124R The combination forms a multifunctional scDB antibody (scDB-VHH). CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R ), structure as Figure 1 As shown, its amino acid sequence is shown in Table 1 below.

[0127] Table 1 scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R

[0128] amino acid characteristic region composition

[0129]

[0130] Among them, VHH CLEC9A The sequence originates from patent application CN201780021194.5; VHH PDL1 Sequence and VHH CTLA4 The sequence is derived from the monoclonal antibody erfonrilimab; IFNα2 Q124R The sequences were derived from the UniProtKB database (P01563), with the signal peptide sequence removed and glutamine at position 124 mutated to arginine. These polypeptide sequences were then processed according to SP-VHH. CLEC9A -(GGS) 10 -VHH PDL1 -(GGS) 10 -VHH CTLA4 -(GGS) 20 -IFNα2 Q124R -H 10 Assembly and ligation. The mRNA was translated into DNA sequence via reverse translation, and 5' UTR and 3' UTR sequences to improve mRNA translation efficiency were added to the 5' and 3' ends. Finally, scDB-VHH was synthesized chemically. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The specific sequence information is as follows:

[0131] scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The amino acid sequence is as follows:

[0132] MYRMQLLSCIALSLALVTNSQVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTWLQMNSLRPEDTAWYCKAFTRGDDYWGQGTQVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSQVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSQVQLVESGGGLVQPGGSLRLSCAASGYIYSAYCMGWFRQAPGKGLEGVAAIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADVIPTETCLGGSWSGPFGYWGQGTLVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEHHHHHHHHHH (SEQ ID NO: 9);

[0133] scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The nucleotide sequence of:

[0134]

[0135] 1.2 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Design of four specific single-chain antibody structures

[0136] Designed using the variable region (VHH) of the CLEC9A nanobody targeting DC cells CLEC9A ), CD40 activates the antibody heavy chain variable region (VH) CD40 CD40-activated antibody light chain variable region (VL) CD40 ); and the T-cell-targeting nanobody CTLA4 (VHH CTLA4 ) Variable region, PD1 heavy chain variable region (VH PD1 ), PD1 light chain variable region (VL) PD1 The combination forms a multifunctional scDB antibody (scDB-VHH). CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 ), structure as Figure 2 As shown, its amino acid sequence is shown in Table 2 below.

[0137] Table 2 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4

[0138] amino acid characteristic region composition

[0139]

[0140]

[0141] Among them, VHH CLEC9A The sequence is derived from patent application CN201780021194.5; the variable region sequence of anti-CD40 is derived from the monoclonal antibody Sotigalimab; the variable region sequence of anti-PD1 is derived from the monoclonal antibody pembrolizumab; VHH CTLA4 The sequences are derived from the monoclonal antibody erfonrilimab. These polypeptide sequences are based on SP-VHH. CLEC9A -L3-VL CD40 -L1-VH PD1 -L2-VLPD1 -L1-VH CD40 -L3-VHH CTLA4 -H 10 Assembly and ligation. The mRNA was translated into DNA sequence via reverse translation, and 5' UTR and 3' UTR sequences to improve mRNA translation efficiency were added to the 5' and 3' ends. Finally, scDB-VHH was synthesized chemically. CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Its amino acid sequence is shown in SEQ ID NO: 18, and its nucleotide sequence is shown in SEQ ID NO: 19.

[0142] 1.3 Expression and purification of recombinant antibody fusion protein

[0143] The synthesized scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Nucleotides, and scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Nucleotides were constructed into the pcDNA3.4 expression vector and transfected into Chinese hamster ovary cells (CHO). The cells were cultured in serum-free medium for 6 days, and the cell supernatant was harvested after centrifugation. The supernatant was then purified into antibodies using an immobilized nickel affinity chromatography column under low flow conditions. Protein purity was confirmed by SDS-PAGE.

[0144] The results are shown in Figures 3A-3B, scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The purity is 70%, which is relatively high; scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The purity is 60%.

[0145] 1.4 scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Prediction of the structure of trispecific single-chain antibodies

[0146] AlphaFold 3 was used to predict scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The spatial structure of the antibody was determined. The amino acid sequence of the antibody with the signal guide peptide removed was input into AlphaFold 3, yielding five predicted structures. The structures with higher ranking scores were selected and processed using ChimeraX software. The final predicted 3D structure was output (as shown in Figure 4A). The results show that the spatial structures of different antibody domains can be clearly distinguished.

[0147] To further confirm the interaction between the antibody and each antigen protein CLEC9A, PDL1, and CLTA4, the antibody and the interacting proteins were input into AlphaFold 3 for prediction, resulting in 5 prediction models. The model with the higher ranking_score was selected and processed using ChimeraX software. The results are shown in Figure 4B, indicating that the antibody interacts closely with CLEC9A, PDL1, and CLTA4 proteins.

[0148] 1.5 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Prediction of the structure of four specific single-chain antibodies

[0149] AlphaFold 3 was used to predict scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The spatial structure was determined. The amino acid sequence of the antibody with the signal-guided peptide removed was input into AlphaFold 3, yielding five prediction models. The model with the highest ranking_score was selected, processed using ChimeraX software, and finally the predicted 3D structure was output (e.g., [example missing]). Figure 5 As shown in the figure, the results show that the spatial structures of different domains of the antibody can be clearly distinguished.

[0150] Example 2

[0151] 2.1 Preparation of imDC cells

[0152] Peripheral blood mononuclear cells (PBMCs) were purified from human peripheral blood using Ficoll density gradient centrifugation, and CD14 was then separated using CD14 magnetic beads.+ Mononuclear cells were counted and viability was calculated; CD14 cells were resuspended in serum-free medium supplemented with the differentiation-promoting cytokine combination rhGM-CSF and rhIL-4. + Monocytes (1-5×10) 6 Cells / mL were seeded into culture flasks and cultured in a 37°C, 5% CO2 incubator (denoted as D0). After 2-3 days of culture, the culture medium was added, and the culture was continued for another 2-3 days (D5-D6) to finally obtain immature dendritic cells (imDC).

[0153] 2.2 Preparation of mDC cells

[0154] imDC cells were collected and cultured to promote maturation. The amount of cytokines to be added was calculated based on the volume of the maturation culture medium. The maturation cytokines were added to the serum-free culture medium (the combination of maturation cytokines included rhGM-CSF, rhIL-4, rhTNF-α, etc.) and cultured in a 37°C, 5% CO2 incubator for 18-24 hours to finally obtain mature dendritic cells (mDC).

[0155] 2.3 Expansion of antigen peptide-specific T cells

[0156] The PBMC in 2.1 above is divided into 1 × 10 6 Cells / mL were resuspended in serum-free medium, and 1 µg / mL CMV / pp65 was added. 495-504 Stimulate at 37°C for 2 hours, centrifuge at 300g to remove unbound small peptides, and wash once with culture medium; then stir at 2.5 × 10⁻⁶. 6 Cells were resuspended in complete culture medium (50 IU / ml IL-2, 1% (v / v) GlutaMAX™, and 1% (v / v) autologous serum added to serum-free medium), seeded in culture plates, and cultured in a 37°C, 5% CO2 incubator. On day 5, 1.5 times the volume of complete culture medium was added, and cells were harvested on day 9 to obtain CMV-specific T cells.

[0157] Anti-CD3 was coated onto culture plates, and anti-CD28 was added to the culture medium to expand T cells, ultimately obtaining control T cells.

[0158] Flow cytometry analysis of T cell composition, results as follows Figure 6 As shown, the CMV processing group can obtain a higher proportion of CD8. + Cells, while the control group obtained mostly CD4 cells. + cell.

[0159] Example 3

[0160] 3.1 scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Affinity of trispecific single-chain antibodies to mDC cells

[0161] The mDC cells obtained in Example 2, 2.2 were used at a rate of 2 × 10⁻⁶. 6 Cells / mL were resuspended in PBS+HSA buffer, with 200 μL of suspension (4 × 10⁻⁶) for each reaction. 5 (cells), add different concentrations of scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Unit: μg / mL): 0.5, 0.2, 0.78, 3.125, 12.5, 50, 200. Incubate at room temperature for 1 hour, harvest cells and resuspend them in PBS. Add 1000-fold diluted Alexa Fluor® 647 anti-His and incubate at room temperature in the dark for 15-30 minutes. Wash once with PBS, then resuspend the cells with flow cytometry buffer. Flow cytometry is used to detect the affinity of the antibody for mDC cells.

[0162] The results are shown in Figures 7A-7C, scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The dissociation rate constant (Kd) with CLEC9A and PDL1 on the surface of mDC cells was 4.351 μg / mL (Figure 7A); the equilibrium dissociation constant (Kd) with CLEC9A was... D The value is 0.03179 (Figure 7B); compared with the K of PDL1 D It is 0.4997 (Figure 7 C).

[0163] 3.2 scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Affinity of trispecific single-chain antibodies to T cells

[0164] T cells were enriched using anti-CD3 magnetic beads, and the T cells were then arranged at a concentration of 1.5 × 10⁻⁶. 6Cells / mL were resuspended in serum-free medium supplemented with 50 IU / mL IL-2, anti-CD28, and autologous plasma, and seeded in anti-CD3 coated culture plates. T cells were activated for 3 days, followed by IL-2-supplemented serum-free medium every 2 days for 10 days to obtain expanded T cells. The T cells were then cultured at 2 × 10⁶ cells / mL. 6 Cells / mL were resuspended in PBS+HSA buffer, with 200 μL of suspension (4 × 10⁻⁶) for each reaction. 5 (cells), add different concentrations of scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Unit: μg / mL): 0.5, 0.2, 0.78, 3.125, 12.5, 50, 200. Incubate at room temperature for 1 hour, harvest cells and resuspend them in PBS. Add 1000-fold diluted Alexa Fluor® 647 anti-His and incubate at room temperature in the dark for 15-30 minutes. Wash once with PBS, then resuspend the cells with flow cytometry buffer. Flow cytometry is used to detect the affinity of the antibody for T cells.

[0165] The results are shown in Figures 8A-8B, scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The Kd of CTLA4 on the surface of T cells was 165.7 μg / mL, indicating low affinity (Figure 8A); the K of CTLA4 was... D It is 0.168 (Figure 8 B).

[0166] 3.3 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Affinity of four specific single-stranded molecules to mDC cells

[0167] The mDC cells obtained in Example 2, 2.2 were used at a rate of 2 × 10⁻⁶. 6 Cells / mL were resuspended in PBS+HSA buffer, with 200 μL of suspension (4 × 10⁻⁶) for each reaction. 5 (cells), add different concentrations of scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 (Unit: μg / mL): 0.01, 0.04, 0.16, 0.64, 2.56, 10.24, 40.96. Incubate at room temperature for 1 hour, harvest cells and resuspend them in PBS. Add 1000-fold diluted Alexa Fluor® 647 anti-His and incubate at room temperature in the dark for 15-30 minutes. Wash once with PBS, then resuspend the cells with flow cytometry buffer. Flow cytometry is used to detect the affinity of the antibody for mDC cells.

[0168] The results are shown in Figures 9A-9C, scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The Kd concentration of mDC cells with CLEC9A and CD40 on the cell surface was 7.117 μg / mL (Figure 9A); the Kd concentration of CLEC9A was... D It is 0.5705 (Figure 9 B); compared with the K of CD40 D It is 0.104 (Figure 9 C).

[0169] 3.4 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The affinity of four specific single-stranded molecules for T cells

[0170] T cells were enriched using anti-CD3 magnetic beads, and the T cells were then arranged at a concentration of 1.5 × 10⁻⁶. 6 Cells / mL were resuspended in serum-free medium supplemented with 50 IU / mL IL-2, anti-CD28, and autologous plasma, and seeded in anti-CD3 coated culture plates. T cells were activated for 3 days, followed by IL-2-supplemented serum-free medium every 2 days for 10 days to obtain expanded T cells. The T cells were then cultured at 2 × 10⁶ cells / mL. 6 Cells / mL were resuspended in PBS+HSA buffer, with 200 μL of suspension (4 × 10⁻⁶) for each reaction. 5 (cells), add different concentrations of scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4(Unit: μg / mL): 0.01, 0.04, 0.16, 0.64, 2.56, 10.24, 40.96. Incubate at room temperature for 1 hour, harvest cells and resuspend them in PBS. Add 1000-fold diluted AlexaFluor® 647 anti-His and incubate at room temperature in the dark for 15-30 minutes. Wash once with PBS, then resuspend the cells with flow cytometry buffer. Flow cytometry is used to detect the affinity of the antibody for T cells.

[0171] The results are shown in Figures 10A-10C, scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The Kd concentration of T cells with CTLA4 and PD1 was 1.589 μg / mL (Figure 10 A); the Kd concentration of CTLA4 was... D It is 0.6877 (Figure 10 B); compared with K of PD1 D It is 0.2043 (Figure 10 C).

[0172] Example 4

[0173] 4.1 Elispot assay shows that antibodies promote the activation of T cells by dendritic cells (DCs).

[0174] The imDC and mDC cells obtained in Examples 2.1 and 2.2 were resuspended in serum-free culture medium; the CD14 cells eluted from the magnetic beads in Example 2.1 were resuspended in serum-free culture medium. - Cells were resuspended in CTL medium, counted using AO / PI double staining, and viability was determined; the following experimental groups were set up (where Ab3 was scDB-VHH). CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Alternative name; Ab2 is scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 (Alternative name):

[0175] A. CD14 - +PBS, negative control group;

[0176] B. CD14 - +Ab3;

[0177] C. CD14 - +Ab2;

[0178] D. CD14 - +PHA, positive control group;

[0179] E. imDC+CD14 - +PBS;

[0180] F. imDC+CD14 - +Ab3;

[0181] G. imDC+CD14 - +Ab2;

[0182] H. imDC+CD14 - +CMV

[0183] I. mDC+CD14 - +PBS;

[0184] J. mDC+CD14 - +Ab3;

[0185] K. mDC+CD14 - +Ab2.

[0186] DC cells with CD14 - Cells were mixed at a ratio of 1:5, grouped as described above, and injected at a rate of 1 × 10⁻⁶. 4 Cells / well were aliquoted into centrifuge tubes. Groups B, F, and J were supplemented with 5 μg / mL Ab3; groups C, G, and K were supplemented with 5 μg / mL Ab2; groups A, E, and I were supplemented with the corresponding volume of PBS; and group H was supplemented with 1 μg / mL CMV / pp65. 495-504 Cells were seeded in Elispot culture plates and cultured at 37°C in a CO2 incubator for 18 hours. They were then incubated with anti-IFNγ-biotin antibody (clone 4S.B3, BD) at room temperature for 2 hours, followed by washing four times with PBS. Streptavidin alkaline phosphatase was added to PBS at a dilution of 1:1600 and incubated at room temperature for 2 hours, followed by washing with PBS. Colorimetric AP substrate was added and incubated in the dark for 10-30 minutes. Spots were then quantified using the ImmunoSpot S6 system (CTL).

[0187] The results are shown in Figures 11A-11B. Compared with the PBS group, the Ab3 treatment group significantly enhanced the activation of T cells by imDC cells, and the Elispot dot count increased from 94 to 297, an increase of approximately 3 times. The effect was comparable to that of directly loading CMV / pp65. 495-504The activation of T cells by imDC cells was comparable (332 dots), while the Ab2-treated group had 134 dots, a smaller increase of approximately 1.4 times. In the mDC cell group, Ab3 also increased the activation of T cells by mDC cells, with 343 dots, compared to 250 dots in the PBS group (an increase of approximately 1.37 times) and 278 dots in the Ab2-treated group (an increase of approximately 1.1 times). Regarding CD14… - In cells, the number of spots was 4 in the PBS group, 42 in the Ab3 treatment group, and 5 in the Ab2 treatment group. The results indicate that scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R It can effectively promote the activation of T cells by DC cells, and may do so through IFNα2. Q124R It directly activates T cells.

[0188] 4.2 Detection of antibody activation of T cells

[0189] Using the CMV-loaded T cells obtained in Example 2, section 2.3, 1×102 6 The cells were resuspended in 500 μL of serum-free medium and scDB-VHH was added. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Ab3, 5 μg / mL), scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Cells were seeded in culture plates with 5 μg / mL PBS or an equivalent amount of PBS and cultured at 37°C in a CO2 incubator for 24 hours. The activation of T cells was then detected by flow cytometry.

[0190] The results are shown in Figures 12A-12B. Compared with Ab2, Ab3 can significantly increase the expression of CD69 in T cells, which can increase the activation of CD4 and CD8 cells (Figure 12A). Ab3 can also reduce the expression of T cell immune checkpoint TIM3, while Ab2 has no significant inhibitory effect on TIM3 (Figure 12B).

[0191] Example 5

[0192] 5.1 Detection of antibody stability in mDC cell culture medium

[0193] The mDC cells obtained in Example 2, section 2.2 were seeded in 24-well plates and cultured at 37°C in a CO2 incubator for 5 hours. At different time points, 1 μg / mL scDB-VHH was added. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Ab3, cultured for 0 hours, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours), and finally harvested the cells. Flow cytometry was used to detect the expression of antibodies in mDC cells and the expression of the targets bound by the antibodies.

[0194] The results are shown in Figures 13A-13F. The binding of Ab3 to mDC cells gradually decreased with increasing culture time, reaching its highest strength after 1 hour of culture, decreasing to 50% after 12 hours, and remaining at 30% after 24 hours (Figure 13A). At different culture times, a high proportion of Ab3-binding positive mDC cells were maintained. Figure 13B At different culture times, the expression intensity and cell proportion of PDL1 remained at a low level, indicating that Ab3 had a strong blocking effect on PDL1 (Figure 13 C-Figure 13 D). After 1-3 hours of culture, the expression intensity and cell proportion of CLEC9A remained at a low level, indicating that Ab3 could bind strongly to CLEC9A. However, after 3 hours, the expression intensity and cell proportion of CLEC9A gradually increased, indicating that the binding ability of Ab3 to CLEC9A was slightly reduced (Figure 13 E-Figure 13 F).

[0195] 5.2 Detection of antibody non-targeted toxicity to mDC cells

[0196] Using the mDC cells obtained in Example 2, section 2.2, 5 μg / mL scDB-VHH was added at different time points. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Ab3, cultured for 0 hours, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours), and finally harvested the cells. The number and viability of mDC cells were recorded by counting using AO / PI double staining; and the apoptosis of mDC cells was detected by flow cytometry using Annexin V / PI staining.

[0197] The results are shown in Figures 14A-14C. After 3 and 6 hours of culture, the viability of mDC cells decreased, but after 6 hours, the viability remained at 90% (Figure 14A). The number of mDC cells decreased with increasing culture time, but after 6 hours, the number remained essentially unchanged (Figure 14B). The apoptosis rate of mDC cells showed no significant difference at different culture times, remaining at approximately 5% (Figure 14C). These results indicate that Ab3 has only mild toxicity to mDC cells.

[0198] Example 6

[0199] 6.1 Antibody-mediated killing effect of T cells on T2 cells loaded with specific antigens

[0200] Poly-L-lysine was used to treat 96-well E-PLATE plates at room temperature for 2 hours, followed by washing with PBS 3-5 times. Cells were then resuspended in RPMI 1640 + 10% FBS + 2mM glutamine medium, and AO / PI counts were performed. 1 × 10⁶ cells were seeded per well. 4 Personal lymphocyte hybridoma cells (T2) were cultured for 24 hours in an xCELLigence RTCA eSight instrument, and the following groups were set up according to the experimental design:

[0201] A. Tctrl+Ab3+CMV, i.e., using anti-CD3 and anti-CD28 expanded T cells and T2 cells loaded with CMV / pp65. 495-504 And add scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Ab3);

[0202] B. Tctrl+Ab2+CMV, i.e., using T cells and T2 cells loaded with CMV / pp65 that are anti-CD3 and anti-CD28 amplified. 495-504 And add scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 (Ab2);

[0203] C. Tcmv+CMV, i.e., T cells and T2 cells loaded with CMV to activate and expand, or CMV / pp65. 495-504 And add PBS;

[0204] D. Tcmv+Ab3+CMV, i.e., T cells and T2 cells loaded with CMV that have been activated and expanded, or CMV / pp65. 495-504And add Ab3;

[0205] E. Tcmv+Ab2+CMV, i.e., T cells and T2 cells loaded with CMV activated and expanded, with a CMV / pp65 ratio. 495-504 And add Ab2;

[0206] F. Tcmv+Ab3, which means T cells and T2 cells that are activated and expanded by CMV loading but not loaded with short peptides, and with the addition of Ab3;

[0207] G. Tcmv+Ab2, which means T cells and T2 cells that are activated and expanded by CMV loading but not loaded with short peptides, and with the addition of Ab2;

[0208] Adherent T2 cells were washed once with PBS, and then added with 1 µg / mL CMV / pp65 according to the grouping. 495-504 The T cells were cultured in RPMI 1640 + 2mM Glutamine medium, while the group without short peptide loading was cultured in RPMI 1640 + 2mM Glutamine medium only. The culture was then incubated at 37°C in a CO2 incubator for 1 hour, followed by washing with PBS once. During the incubation of the short peptide with T2 cells, the T cells obtained in Example 2, section 2.3 were prepared and resuspended in RPMI 1640 + 10% FBS + 2mM Glutamine medium. Groups A, D, and F received 5 µg / mL Ab3, groups B, E, and G received 5 µg / mL Ab2, and group C received the corresponding volume of PBS. T cells were seeded into T2 cells at an effector cell:target cell (E:T) ratio of 1:1. The culture plates were then placed in an xCELLigence RTCAeSight instrument, and resistance was measured every 15 minutes to monitor the killing effect of T cells on T2 cells in real time.

[0209] The results are as follows Figure 15 As shown, compared to the Tcmv+Ab2+CMV group, the cell index of the Tcmv+Ab3+CMV group was lower; the cell indexes of the Tctrl+Ab3+CMV and Tctrl+Ab2+CMV groups continued to increase even with the addition of antibodies, reaching the highest levels among all treatment groups; Tcmv also showed some killing effect on CMV-loaded T2 cells; however, Tcmv, with the addition of antibodies, had a poor killing effect on unloaded CMV T2 cells. Therefore, scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124 The killing of target cells by T cells is specific.

[0210] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A trispecific single-chain antibody, wherein the trispecific single-chain antibody comprises VHH that specifically binds to CLEC9A. CLEC9A VHH specifically binds to PDL1 PDL1 VHH specifically binds to CTLA4 CTLA4 and cytokines; The VHH CLEC9A The amino acid sequence of VHH is shown in SEQ ID NO:

2. PDL1 The amino acid sequence is shown in SEQ ID NO: 3, and the VHH CTLA4 The amino acid sequence is shown in SEQ ID NO: 4; The cytokine portion is an IFNα mutant, and the amino acid sequence of the IFNα mutant is shown in SEQ ID NO: 5; The VHH CLEC9A Connected to the VHH PDL1 The N-terminus of the VHH CTLA4 Connected to the VHH PDL1 The C-terminus of the cytokine is attached to the VHH. CTLA4 The C-terminus.

2. The trispecific single-chain antibody according to claim 1, wherein the VHH CLEC9A The VHH PDL1 The VHH CTLA4 They are linked together by short connecting peptides; The amino acid sequence of the short linker peptide is shown in SEQ ID NO:

6.

3. The trispecific single-chain antibody according to claim 1, wherein the cytokine portion and the VHH CTLA4 They are linked together by long linker peptides; The amino acid sequence of the long linker peptide is shown in SEQ ID NO:

7.

4. The trispecific single-chain antibody according to claim 1, wherein the trispecific single-chain antibody comprises a signal peptide, the amino acid sequence of which is shown in SEQ ID NO: 1; The signal peptide is located at the N-terminus of the trispecific single-chain antibody.

5. The trispecific single-chain antibody according to claim 1, wherein the trispecific single-chain antibody comprises a His-tagged peptide, the amino acid sequence of which is shown in SEQ ID NO: 8; The His-tagged peptide is located at the C-terminus of the trispecific single-chain antibody.

6. The trispecific single-chain antibody according to claim 1, wherein the amino acid sequence of the trispecific single-chain antibody is shown in SEQ ID NO:

9.

7. A nucleic acid, wherein the nucleic acid encodes a trispecific single-chain antibody according to any one of claims 1-6.

8. A host cell, wherein the host cell comprises the nucleic acid of claim 7.

9. A method for producing a trispecific single-chain antibody, wherein the method comprises culturing the host cell of claim 8 to produce the trispecific single-chain antibody of any one of claims 1-6.

10. A pharmaceutical composition comprising the trispecific single-chain antibody according to any one of claims 1-6.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

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