Trispecific single-chain antibody and application thereof

Abstract

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

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to a trispecific single-chain antibody and uses thereof. Background Art

[0002] Dendritic cells (DCs) are the only professional antigen-presenting cells (APCs) that can activate naive T cells and play a crucial role in the immune process. Activated DCs, such as those that phagocytize foreign protein antigens, can recruit and interact with other immune cells (such as macrophages (MACs), eosinophils, natural killer (NK) cells, and T cells), further activating T cells and promoting their differentiation into effector or memory T cells, thereby regulating or killing target cells.

[0003] Immunotherapeutic approaches such as immune checkpoint inhibition and CAR-T have made significant progress in cancer treatment, but only a subset of the population can benefit. Tumor resistance to small molecule drugs and other radiotherapy and chemotherapy, as well as immune escape by tumor cells from immunotherapy, limit the effectiveness of cancer treatment. T cells play a crucial role in inhibiting and killing tumor cells, but effective T cell activation requires three signals: Signal 1, the interaction of pHLA (peptide-HLA) tumor-specific or associated antigens presented by dendritic cells (DCs) with the T cell receptor (TCR); Signal 2, the interaction of DC costimulatory molecules CD80 and CD86 with CD28 on the T cell surface; and Signal 3, the secretion of cytokines (such as IFN-I factors IFNα and IL-12) by activated DCs, as well as IFNγ secreted by T cells, further activate DCs, thereby increasing T cell activity and promoting T cell expansion.

[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, accelerating the exhaustion of immune cells, thereby prompting immune cells to highly express proteins such as PD1, CTLA4, and TIM3; another mechanism is that tumor cells highly express immune checkpoint proteins such as PDL1, preventing DC cells from recruiting immune cells or damaging the function of DC cells, thereby escaping the killing of innate and adaptive immune cells.

[0005] Therefore, promoting DC cells to effectively recruit immune cells and activate DC cells and recruited immune cells to achieve effective control of tumor cells has become the focus of current research. Summary of the Invention

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

[0007] Specifically, this application involves the following:

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

[0009] 2. The trispecific single-chain antibody of claim 1, wherein the first antigen-binding portion 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 portion 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 items 1 to 3, wherein the third antigen-binding portion 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 items 1 to 4, wherein the first antigen-binding portion, the second antigen-binding portion and the third antigen-binding portion are connected to each other via a short connecting peptide;

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

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

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

[0016] 7. The trispecific single-chain antibody of any one of items 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 a mutant thereof;

[0018] Preferably, the cytokine moiety is selected from IFNα or a mutant thereof;

[0019] Further 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 connected to each other via a long connecting peptide;

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

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

[0023] 10. The trispecific single-chain antibody of 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 items 1 to 10, wherein the trispecific single-chain antibody comprises a signal peptide, the amino acid sequence of which is 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 items 1 to 11, wherein the trispecific single-chain antibody comprises a His-tag peptide, the amino acid sequence of which is shown in SEQ ID NO: 8;

[0027] Preferably, the His-tag 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 items 1 to 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 coding sequence for the trispecific single-chain antibody of 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 cell of item 15 to produce the trispecific single-chain antibody of any one of items 1 to 13.

[0032] 17. A pharmaceutical composition, wherein the pharmaceutical composition comprises the trispecific single-chain antibody described in any one of items 1-13.

[0033] 18. A 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 to 13 or the pharmaceutical composition according to item 17 or 18 in the preparation of a drug for treating and / or preventing tumors.

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

[0036] Beneficial effects:

[0037] The present application provides a trispecific single-chain antibody capable of bridging DC cells and T cells, wherein the trispecific single-chain antibody targets CTLA4, PDL1 and CTLA4 and is linked to IFNα2 with reduced affinity. Q124R , effectively shortening the distance between DC cells and T cells, inhibiting the expression of immune checkpoints PDL1 and CTLA4, and achieving activation of DC cell and T cell functions. 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 antigens, and has broad application prospects in the field of cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0040] Figure 3 A-Figure 3 B are the results of antibody purity testing. Figure 3 A shows scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R SDS-PAGE of trispecific single-chain antibody; Figure 3 B is scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 SDS-PAGE image of the tetraspecific single-chain antibody.

[0041] Figure 4A-Figure 4B shows scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Figure 4A shows the spatial structure of a trispecific single-chain antibody. Figure 4A shows the 3D structure of the antibody, while Figure 4B shows the 3D structure of the antibody interacting with the corresponding antigen protein.

[0042] Figure 5 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 3D structure of the tetraspecific single-chain antibody.

[0043] Figure 6 CMV-induced CD8 + and CD4 + Figure 2 shows the detection results of T cell expansion.

[0044] Figure 7A-7C shows scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Figure 7 A shows the Kd values ​​of the trispecific single-chain antibody for CLEC9A and PDL1 on the surface of mDC cells; Figure 7 B shows the Kd values ​​of the antibody for CLEC9A on the surface of mDC cells. D Figure 7 C shows the K value of the antibody and PDL1 on the surface of mDC cells. D value.

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

[0046] Figures 9A-9C show scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The results of the affinity test of the tetraspecific single-chain antibody to mDC cells are shown in Figure 9A. Figure 9A shows the Kd value of the antibody to CLEC9A and CD40 on the surface of mDC cells; Figure 9B shows the Kd value of the antibody to CLEC9A on the surface of mDC cells. D Figure 9 C shows the K value of the antibody and CD40 on the surface of mDC cells. D value.

[0047] Figure 10A-Figure 10C are scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The results of the test of the affinity of the four-specific single-chain antibody to T cells. Figure 10 A shows the Kd value of the antibody to CTLA4 and PD1 on the surface of T cells; Figure 10 B shows the Kd value of the antibody to CTLA4 on the surface of T cells. D Figure 10 C shows the K value of the antibody and PD1 on the surface of T cells. D value.

[0048] Figure 11A-Figure 11B shows Elispot detection of scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Trispecific single-chain antibodies and scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Figure 11A shows the statistical results of the Elispot spot count, while Figure 11B shows the actual spot count results of the Elispot.

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

[0050] Figure 13A-13F shows scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Figures depicting the stability of trispecific single-chain antibodies in mDC cell culture medium. Figure 13A depicts the binding strength of the antibody to mDCs; Figure 13B depicts the proportion of mDCs that bind to the antibody; Figure 13C depicts the expression of PDL1 on the surface of mDCs; Figure 13D depicts the proportion of cells expressing PDL1; Figure 13E depicts the expression of CLEC9A on the surface of mDCs; and Figure 13F depicts the proportion of cells expressing CLEC9A.

[0051] Figures 14A-14C show scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Figure 14A shows the results of the non-targeted toxicity test of the trispecific single-chain antibody on mDC cells. Figure 14A shows the results of the mDC cell viability test; Figure 14B shows the results of the mDC cell number test; and Figure 10C shows the results of the mDC cell apoptosis test.

[0052] Figure 15 To detect scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Trispecific single-chain antibodies and scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4Figure 3 shows the results of tetraspecific single-chain antibody-mediated T cell killing of CMV-loaded T2 cells. DETAILED DESCRIPTION

[0053] The present application is further described below with reference to examples. It should be understood that the examples 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 those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0055] definition

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

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

[0058] The term "CTLA4," as used herein, stands for cytotoxic T lymphocyte-associated protein 4, which inhibits immune responses by binding to the ligands CD80 (also known as B7-1) and CD86 (also known as B7-2). CTLA-4 inhibits immune responses in various ways: for example, 1) competing with the T cell co-stimulatory receptor CD28 for its ligands CD80 and CD86, thereby blocking co-stimulation; and 2) sending negative signals that inhibit T cell activation. By inhibiting the CTLA-4 molecule, CTLA-4 inhibitors can enable T cells to proliferate and attack tumor cells.

[0059] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, 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 configuration of immunoglobulin molecules that contain an antigen recognition site of desired specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies.

[0060] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second, and third constant regions (CH1, CH2, CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL), with the light chain constant region comprising either kappa (kappa) or lambda (lambda). Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but antibodies need not belong to any specific class. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the heavy chain constant region. Generally, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are designated 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, for example, the binding of an antibody to an antigenic determinant.

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

[0063] The term "nanoantibody" used in this article refers to a heavy chain antibody that is naturally missing light chains and exists in camels and other organisms. Cloning its variable region can obtain 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] The terms "VHH," "nanobody," and "single-domain antibody" as used herein have the same meaning and are used interchangeably. They refer to cloning the variable regions of heavy chain antibodies to construct single-domain antibodies consisting solely of a single heavy chain variable region. These are the smallest fully functional antigen-binding fragments. Typically, a heavy chain antibody naturally lacking the light chain and 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 solely of a single heavy chain variable region.

[0065] The term "trispecific" as used herein means that the antibody is able to specifically bind to at least three different antigenic determinants.

[0066] The term "tetraspecific" as used herein means that the antibody is able to specifically bind to at least four different antigenic determinants.

[0067] The terms "first," "second," and "third" used herein are used only for the convenience of distinguishing when there are more than one part of each type. Unless explicitly stated, no particular order or orientation is intended.

[0068] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably 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) into which foreign (exogenous or transgenic) nucleic acids have been introduced. The external nucleic acid can include an expression vector operably linked to the transgene, and the host cell can be used to express nucleic acids and / or polypeptides encoded by the external nucleic acid (transgene). The host cell (or population thereof) can be a cultured cell or can be extracted from a subject. Without considering the number of pathways, the host cell (or population thereof) includes the primary subject cell and its progeny. Progeny cells may or may not carry the same genetic material as the parent cell. Host cells encompass progeny cells. In one embodiment, the host cell description is any cell (including progeny thereof) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express an antibody as disclosed herein. In one embodiment, the host cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding a desired antibody as described herein. The host cell and its population can carry an expression vector stably integrated into the host genome or can carry an extrachromosomal expression vector. In one embodiment, the host cells and populations thereof may carry an extrachromosomal vector that persists for several cell divisions or persists transiently and disappears after several cell divisions.

[0070] The term "K D ” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen and is obtained from the dissociation rate constant (K d ) than the binding rate constant (K a ) (i.e. K d / K a The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the 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 a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to exert its effect, and that contains no additional components that are unacceptably toxic to a subject to which the formulation is to be administered.

[0072] The term "pharmaceutically acceptable carrier" as used herein refers to an ingredient in 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" used herein are interchangeable and generally refer to neoplasms formed by the proliferation of local tissue cells under the action of various tumorigenic factors. In one embodiment, the tumor is a tumor that expresses pHLA tumor-specific or related antigens.

[0074] The term "HLA" as used herein refers to human leukocyte antigens. HLA genes encode major histocompatibility complex (MHC) proteins in humans. MHC proteins are expressed on the cell surface and are involved in the activation of immune responses. HLA class I genes encode MHC class I molecules, which are expressed on the cell surface in the form of complexes with peptide fragments (antigens) of self or non-self proteins. MHC class I molecules bind to CD8 + Cytotoxic T cells interact and play an important role in destroying organ transplant rejection or infected cells.

[0075] As used herein, the term "HLA class I molecule" or "HLA class I molecule" refers to the protein product of a wild-type or variant HLA class I gene encoding an MHC class I molecule. Thus, "HLA class I molecule" and "MHC class I molecule" are used interchangeably herein.

[0076] The term "pHLA" as used herein may refer to peptide-binding human leukocyte antigen.

[0077] Antibody

[0078] The present 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 a specific embodiment, the first antigen binding moiety comprises VHH CLEC9A In a specific embodiment, the VHH CLEC9A With sequence

[0080] SEQ ID NO: 2:

[0081] The amino acid sequence of QVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTWLQMNSLRPEDTAWYCKAFTRGDDYWGQGTQVTVSS.

[0082] In a specific embodiment, the second antigen binding moiety comprises VHH PDL1In a specific embodiment, the VHH PDL1 With sequence

[0083] SEQ ID NO: 3:

[0084] The amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS.

[0085] In a specific embodiment, the third antigen binding moiety comprises VHH CTLA4 In a specific embodiment, the VHH CTLA4 With sequence

[0086] SEQ ID NO: 4:

[0087] Amino acid sequence of QVQLVESGGGLVQPGGSLRLSCAASGYIYSAYCMGWFRQAPGKGLEGVAAIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADVIPTETCLGGSWSGPFGYWGQGTLVTVSS.

[0088] In a specific embodiment, the first antigen binding portion, the second antigen binding portion, and the third antigen binding portion are connected to each other via a short connecting peptide. In a specific embodiment, the amino acid sequence of the short connecting peptide is (GGS) n , wherein n is a natural number between 10 and 20. In a specific embodiment, the amino acid sequence of the short connecting peptide is (GGS) 10 .

[0089] In a specific embodiment, the first antigen binding moiety is linked to the N-terminus of the second antigen binding moiety, and the third antigen binding moiety is linked to the C-terminus of the second antigen binding moiety.

[0090] In a specific embodiment, said trispecific single chain antibody comprises a cytokine portion.

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

[0092] SEQ ID NO: 5:

[0093] The amino acid sequence of CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE.

[0094] In a specific embodiment, the cytokine portion and the third antigen binding portion are connected to each other via a long connecting peptide. In a specific embodiment, the amino acid sequence of the long connecting peptide is (GGS) n , wherein n is a natural number between 10 and 20. In a specific embodiment, the amino acid sequence of the long connecting peptide is (GGS) 20 .

[0095] In a specific embodiment, the cytokine moiety is linked to the C-terminus of the third antigen binding moiety.

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

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

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

[0099] SEQ ID NO: 9:

[0100] amino acid sequence.

[0101] Nucleic acids, host cells and production methods

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

[0103] The nucleic acid can be obtained according to conventional methods in the art, for example, a DNA sequence encoding a trispecific single-chain antibody can be obtained by PCR amplification, DNA recombination or chemical synthesis technology.

[0104] The present application provides a host cell, wherein the host cell comprises any of the aforementioned nucleic acids.

[0105] Exogenous nucleic acids can be introduced into the host cells according to conventional methods in the art. The method for introducing exogenous nucleic acids varies depending on the host cell used. The host cells can be prokaryotes, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, for example, CHO and HEK293 cells. When the host cells are higher eukaryotic cells, DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electrofection, or liposome packaging.

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

[0107] Typically, host cells are cultured under conditions suitable for antibody expression, and then the antibodies are 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, etc., which are conventional separation and purification methods well known to those skilled in the art.

[0108] Pharmaceutical composition

[0109] The present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises any one of the aforementioned trispecific single-chain antibodies.

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

[0111] The pharmaceutically acceptable carrier 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., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parahydroxybenzoates (alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (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 counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants such as Tween or polyethylene glycol (PEG).

[0112] use

[0113] The present application also provides use of any of the aforementioned trispecific single-chain antibodies or any of the aforementioned pharmaceutical compositions in the preparation of a drug for treating and / or preventing tumors.

[0114] In a specific embodiment, the tumor is a tumor that expresses a pHLA tumor-specific or -associated antigen.

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

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

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

[0118] In the present 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 the present application, the non-targeted toxicity of the trispecific single-chain antibody to mDC cells was detected, and it was found that there was no significant change in the viability, number and apoptosis rate of mDC cells, indicating that the trispecific single-chain antibody had only slight toxicity to mDC cells.

[0120] In the present 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 of target cells by T cells mediated by trispecific single-chain antibodies is specific, and the killing effect is better than that of tetraspecific single-chain antibodies.

[0121] In general, the trispecific single-chain antibody provided in this application can achieve effective antigen presentation by DC cells and activate T cells. While reducing antibody-mediated cytotoxicity, it enhances the specific killing of target cells presenting pHLA antigens, has good safety, and is expected to be used in tumor immunotherapy.

[0122] Example

[0123] The present application will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described scheme 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 structure design

[0126] Design of a nanobody CLEC9A variable region (VHH) targeting DC cells CLEC9A ), Nanobody PDL1 variable region (VHH PDL1 ); Nanoantibody CTLA4 variable region targeting T cells (VHH CTLA4); and cytokine IFNα mutant (IFNα2 Q124R ) to form a multifunctional scDB antibody (scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R ), structure such as Figure 1 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 is derived from patent application CN201780021194.5; VHH PDL1 Sequence and VHH CTLA4 Sequence derived from the monoclonal antibody erfonrilimab; IFNα2 Q124R The sequence was derived from the UniProtKB database (P01563), with the signal peptide sequence removed and the 124th glutamine mutated to arginine. CLEC9A -(GGS) 10 -VHH PDL1 -(GGS) 10 -VHH CTLA4 -(GGS) 20 -IFNα2 Q124R -H 10 Assembly and ligation. ScDB-VHH is synthesized by reverse translation into DNA sequence and adding 5' UTR and 3' UTR sequences at the 5' and 3' ends to improve the efficiency of mRNA translation. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R , its specific sequence information is as follows:

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

[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 Tetraspecific single-chain antibody structure design

[0136] Design of a nanobody CLEC9A variable region (VHH) targeting DC cells CLEC9A ), CD40 activating antibody heavy chain variable region (VH CD40 ), CD40 activating antibody light chain variable region (VL CD40 ); and nanoantibodies targeting T cells CTLA4 (VHH CTLA4 ) variable region, PD1 heavy chain variable region (VH PD1 ), PD1 light chain variable region (VL PD1 ) to form a multifunctional scDB antibody (scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 ), structure such as Figure 2 The 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 peptide sequences are arranged according to SP-VHH CLEC9A -L3-VL CD40 -L1-VH PD1 -L2-VLPD1 -L1-VH CD40 -L3-VHH CTLA4 -H 10 Assembly and ligation. ScDB-VHH is synthesized by reverse translation into DNA sequence and adding 5' UTR and 3' UTR sequences at the 5' and 3' ends to improve the efficiency of mRNA translation. 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 proteins

[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 The nucleotides were constructed into the pcDNA3.4 expression vector and transfected into Chinese hamster ovary (CHO) cells. The cells were cultured in serum-free medium for 6 days. The cell supernatant was harvested after centrifugation. The supernatant was purified by immobilized nickel metal affinity chromatography under low flow rate conditions, and the 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 trispecific single-chain antibody structures

[0146] Using AlphaFold 3 to predict scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The antibody amino acid sequence without the signal-guiding peptide was input into AlphaFold 3, resulting in five predicted structures. The structures with the highest ranking scores were selected and processed using ChimeraX software. Finally, the predicted 3D structure was output (as shown in Figure 4A). The results showed that the spatial structures of different antibody domains could be clearly distinguished.

[0147] To further confirm the interaction between the antibody and the antigenic proteins CLEC9A, PDL1, and CLTA4, the antibody and the interacting proteins were input into AlphaFold 3 for prediction, and 5 prediction models were obtained. The ones with higher ranking_scores were selected and processed using ChimeraX software. The results are shown in Figure 4B, showing that the antibody interacts closely with the CLEC9A, PDL1, and CLTA4 proteins.

[0148] 1.5 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Prediction of tetraspecific single-chain antibody structures

[0149] Using AlphaFold 3 to predict scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The spatial structure of the antibody was input into AlphaFold 3 after removing the signal-guiding peptide. Five prediction models were obtained. The ones with the highest ranking_score were selected and processed with ChimeraX software. Finally, the predicted 3D structure was output (e.g. Figure 5 The results show that the spatial structures of different antibody domains can be clearly distinguished.

[0150] Example 2

[0151] 2.1 Preparation of imDCs

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

[0153] 2.2 Preparation of mDCs

[0154] Collect imDC cells for maturation culture. Calculate the amount of cytokines required based on the volume of maturation culture medium. Add maturation cytokines to serum-free culture medium (maturation cytokine combinations include rhGM-CSF, rhIL-4, rhTNF-α, etc.) and culture in a 37°C, 5% CO2 incubator for 18-24 hours to obtain mature dendritic cells (mDCs).

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

[0156] The PBMCs prepared in 2.1 were cultured at a volume of 1 × 10 6 Cells / mL were resuspended in serum-free medium and 1 μg / mL CMV / pp65 was added. 495-504 , stimulated at 37°C for 2 hours, centrifuged at 300 g to remove unbound small peptides, and washed once with medium; then 2.5× 10 6 Cells / mL were resuspended in complete culture medium (serum-free medium supplemented with 50 IU / ml IL-2, 1% (v / v) GlutaMAX™, and 1% (v / v) autologous serum), 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 on the culture plate, and anti-CD28 was added to the culture medium to expand T cells and finally obtain control T cells.

[0158] The composition of T cells was analyzed by flow cytometry. Figure 6 As shown, the CMV-treated group could obtain a higher proportion of CD8 + cells, while the majority of cells obtained in the control group were CD4 + cell.

[0159] Example 3

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

[0161] The mDC cells obtained in 2.2 of Example 2 were cultured at 2×10 6 The cells / mL were resuspended in PBS+HSA buffer, and 200 μL of suspension (4× 10 5 cells), and different concentrations of scDB-VHH were added CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The concentrations of the antibodies (μg / mL) were as follows: 0.5, 0.2, 0.78, 3.125, 12.5, 50, and 200. The cells were incubated at room temperature for 1 hour. The cells were harvested and resuspended in PBS. A 1000-fold diluted Alexa Fluor® 647 anti-His was added and incubated at room temperature in the dark for 15-30 minutes. The cells were washed once with PBS and then resuspended in flow cytometry buffer. The affinity of the antibody to mDC cells was detected by flow cytometry.

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

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

[0164] T cells were enriched with anti-CD3 magnetic beads and cultured at a concentration of 1.5 × 10 6The cells / mL were resuspended in serum-free medium supplemented with 50 IU / mL IL-2, anti-CD28 and autologous plasma, and seeded on anti-CD3-coated culture plates to activate T cells for 3 days. Serum-free medium supplemented with IL-2 was then added every 2 days for 10 days to finally obtain expanded T cells. T cells were cultured at 2×10 6 The cells / mL were resuspended in PBS+HSA buffer, and 200 μL of suspension (4× 10 5 cells), and different concentrations of scDB-VHH were added CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R The concentrations of the antibodies (μg / mL) were as follows: 0.5, 0.2, 0.78, 3.125, 12.5, 50, and 200. The cells were incubated at room temperature for 1 hour. The cells were harvested and resuspended in PBS. 1000-fold diluted Alexa Fluor® 647 anti-His was added and incubated at room temperature in the dark for 15-30 minutes. The cells were washed once with PBS and then resuspended in flow cytometry buffer. The affinity of the antibody to T cells was detected by flow cytometry.

[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 a low affinity (Figure 8 A). D was 0.168 (Figure 8B).

[0166] 3.3 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Affinity of tetraspecific single chains for mDC cells

[0167] The mDC cells obtained in 2.2 of Example 2 were cultured at 2×10 6 The cells / mL were resuspended in PBS+HSA buffer, and 200 μL of suspension (4× 10 5 cells), and different concentrations of scDB-VHH were added CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 The concentrations of the antibodies (μg / mL) were 0.01, 0.04, 0.16, 0.64, 2.56, 10.24, and 40.96. The cells were incubated at room temperature for 1 hour. The cells were harvested and resuspended in PBS. 1000-fold diluted Alexa Fluor® 647 anti-His was added and incubated at room temperature in the dark for 15-30 minutes. The cells were washed once with PBS and then resuspended in flow cytometry buffer. The affinity of the antibody to mDC cells was detected by flow cytometry.

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

[0169] 3.4 scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 Affinity of tetraspecific single chains for T cells

[0170] T cells were enriched with anti-CD3 magnetic beads and cultured at a concentration of 1.5 × 10 6 The cells / mL were resuspended in serum-free medium supplemented with 50 IU / mL IL-2, anti-CD28 and autologous plasma, and seeded on anti-CD3-coated culture plates to activate T cells for 3 days. Serum-free medium supplemented with IL-2 was then added every 2 days for 10 days to finally obtain expanded T cells. T cells were cultured at 2×10 6 The cells / mL were resuspended in PBS+HSA buffer, and 200 μL of suspension (4× 10 5 cells), and different concentrations of scDB-VHH were added CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4The concentrations of the antibodies (μg / mL) were 0.01, 0.04, 0.16, 0.64, 2.56, 10.24, and 40.96. The cells were incubated at room temperature for 1 hour. The cells were harvested and resuspended in PBS. 1000-fold diluted AlexaFluor® 647 anti-His was added and incubated at room temperature in the dark for 15-30 minutes. The cells were washed once with PBS and then resuspended in flow cytometry buffer. The affinity of the antibody to T cells was detected by flow cytometry.

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

[0172] Example 4

[0173] 4.1 Elispot detection of antibodies promoting DC cell activation of T cells

[0174] The imDC cells and mDC cells obtained in 2.1 and 2.2 of Example 2 were resuspended in serum-free culture medium; the CD14 - The cells were resuspended in CTL culture medium, counted and viable by AO / PI double staining; the following experimental groups were set up (where Ab3 was scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R Synonym: Ab2 is scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 (alias):

[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 and CD14 - The cells were mixed at a ratio of 1:5 and grouped as above, with 1×10 4 Cells / well were divided into centrifuge tubes, in which 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 corresponding volumes of PBS, and group H was supplemented with 1 μg / mL CMV / pp65. 495-504 Cells were plated in Elispot plates and cultured in a 37°C, CO2 incubator for 18 hours. Plates were incubated with anti-IFNγ-biotin antibody (clone 4S.B3, BD Biosciences) for 2 hours at room temperature and washed four times with PBS. Streptavidin-alkaline phosphatase was added at a 1:1600 dilution in PBS and incubated for 2 hours at room temperature, followed by washing with PBS. A colorimetric AP substrate was added, the plates were incubated in the dark for 10-30 minutes, and the spots were quantified using the ImmunoSpot S6 system (CTL).

[0187] The results are shown in Figure 11 A-B. Compared with the PBS group, the Ab3-treated group can significantly improve the activation of imDC cells to T cells. The number of Elispot spots increased from 94 to 297, an increase of about 3 times, which is similar to the effect of directly loading CMV / pp65. 495-504The activation of T cells by imDC cells in the PBS group was comparable (number of spots was 332), while the number of spots in the Ab2-treated group was 134, with a smaller increase of about 1.4 times; in the mDC cell group, Ab3 could also enhance the activation of T cells by mDC cells, with a number of spots of 343, while the number of spots in the PBS group was 250, with an increase of about 1.37 times, and the number of spots in the Ab2-treated group was 278, with an increase of about 1.1 times; in CD14 - In the cells, the number of spots in the PBS group was 4, the number of spots in the Ab3-treated group was 42, and the number of spots in the Ab2-treated group was 5. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R It can effectively promote the activation of DC cells to T cells, and may be through IFNα2 Q124R Directly activate T cells.

[0188] 4.2 Detection of Antibody Activation of T Cells

[0189] The CMV-loaded activated and expanded T cells obtained in 2.3 of Example 2 were used to take 1×10 6 The cells were resuspended in 500 μL 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 (Ab2, 5 μg / mL) or an equal amount of PBS, the cells were seeded in culture plates, and cultured in a 37°C, CO2 incubator for 24 hours. The activation of T cells was detected by flow cytometry.

[0190] The results are shown in Figures 12A and 12B. Compared with Ab2, Ab3 can significantly increase the expression of CD69 in T cells, that is, 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 obvious inhibitory effect on TIM3 (Figure 12B).

[0191] Example 5

[0192] 5.1 Testing the stability of antibodies in mDC cell culture medium

[0193] The mDC cells obtained in 2.2 of Example 2 were seeded in a 24-well plate and cultured in a 37°C, CO2 incubator for 5 hours. 1 μg / mL scDB-VHH was added at different time points. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Ab3, cultured for 0 hour, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours). Finally, the cells were harvested and the expression of the antibody in mDC cells and the expression of the target bound by the antibody were detected by flow cytometry.

[0194] The results are shown in Figures 13A-13F. The binding of Ab3 to mDC cells gradually decreased with the increase of culture time. The binding strength was the highest after 1 hour of culture, decreased to 50% after 12 hours of culture, and maintained at 30% after 24 hours of culture (Figure 13A). After different culture times, the positive mDC cells binding to Ab3 maintained a high ratio ( Figure 13B ); at different culture times, the expression intensity and cell ratio of PDL1 remained at a low level, indicating that Ab3 had a strong blocking effect on PDL1 (Figure 13 C-Figure 13 D); from 1 to 3 hours of culture, the expression intensity and cell ratio of CLEC9A remained at a low level, indicating that Ab3 could strongly bind to CLEC9A, but after 3 hours, the expression intensity and cell ratio 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 non-targeted toxicity of antibodies on mDCs

[0196] The mDC cells obtained in 2.2 of Example 2 were used, and 5 μg / mL scDB-VHH was added at different time points. CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124R (Ab3, culture for 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h). Finally, the cells were harvested and counted by AO / PI double staining to record the number and viability of mDC cells. The cells were then stained with Annexin V / PI and the apoptosis of mDC cells was detected by flow cytometry.

[0197] The results, as shown in Figures 14A-14C, showed that mDC cell viability decreased after 3 and 6 hours of culture, but remained at 90% after 6 hours (Figure 14A). The number of mDC cells decreased with increasing culture time, but remained essentially unchanged after 6 hours (Figure 14B). The apoptosis rate of mDC cells did not differ significantly at different culture times, remaining at approximately 5% (Figure 14C). These results indicate that Ab3 is only mildly toxic to mDC cells.

[0198] Example 6

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

[0200] Poly-lysine was treated in 96-well E-PLATE plates at room temperature for 2 hours, washed 3-5 times with PBS, and cells were resuspended in RPMI 1640 + 10% FBS + 2mM glutamine (Glutamine) medium. AO / PI counting was performed, and 1×10 cells were seeded per well. 4 Human lymphocyte hybridoma cells (T2) were cultured in the xCELLigence RTCA eSight instrument for 24 hours and grouped as follows based on the experimental design:

[0201] A. Tctrl+Ab3+CMV, i.e., T cells and T2 cells expanded with anti-CD3 and anti-CD28 and 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., T cells expanded with anti-CD3 and anti-CD28, and T2 cells loaded with CMV / pp65 495-504 , and add scDB-VHH CLEC9A -VL CD40 -VH PD1 -VL PD1 -VH CD40 -VHH CTLA4 (Ab2);

[0203] C. Tcmv+CMV, i.e., CMV-loaded activated and expanded T cells, and T2 cells loaded with CMV / pp65 495-504 , and add PBS;

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

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

[0206] F. Tcmv+Ab3, i.e., CMV-loaded activated and expanded T cells, T2 cells not loaded with short peptides, and Ab3 added;

[0207] G. Tcmv+Ab2, i.e., CMV loaded activated and expanded T cells, T2 cells not loaded with short peptides, and Ab2 added;

[0208] The adherent T2 cells were washed once with PBS and then added with 1 μg / mL CMV / pp65 495-504 RPMI 1640 + 2mM Glutamine medium was added to the group without short peptide loading, and only RPMI 1640 + 2mM Glutamine medium was added. The cells were cultured in a 37°C, CO2 incubator for 1 hour and washed once with PBS. During the incubation of short peptides with T2 cells, the T cells obtained in 2.3 of Example 2 were prepared and resuspended in RPMI 1640 + 10% FBS + 2mM Glutamine medium, wherein groups A, D, and F were added with 5 μg / mL Ab3, groups B, E, and G were added with 5 μg / mL Ab2, and group C was added with the corresponding volume of PBS. T cells were seeded into T2 cells at a ratio of effector cells: target cells (E: T) = 1: 1, and the culture plate was then placed in the xCELLigence RTCAeSight instrument. The parameters were set to measure the resistance once 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 with the Tcmv+Ab2+CMV group, the cell index of the Tcmv+Ab3+CMV group was smaller; even with the addition of antibodies, the cell index of the Tctrl+Ab3+CMV group and the Tctrl+Ab2+CMV group continued to increase, and the index was the largest among all treatment groups; Tcmv also had a certain killing effect on T2 cells loaded with CMV; Tcmv had a poor killing effect on T2 cells not loaded with CMV when antibodies were added. Therefore, scDB-VHH CLEC9A -VHH PDL1 -VHH CTLA4 -IFNα2 Q124 The T cell-mediated killing of target cells is specific.

[0210] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

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.

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

2.

3. The trispecific single-chain antibody according to claim 1, wherein the second antigen-binding portion comprises VHH PDL1 , the VHH PDL1 The amino acid sequence is shown in SEQ ID NO:

3.

4. The trispecific single-chain antibody according to claim 1, wherein the third antigen-binding portion comprises VHH CTLA4 , the VHH CTLA4 The amino acid sequence is shown in SEQ ID NO:

4.

5. The trispecific single-chain antibody according to any one of claims 1 to 4, wherein the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion are connected to each other via a short linker peptide; The amino acid sequence of the short connecting peptide is (GGS) n , wherein n is a natural number between 10 and 20.

6. The trispecific single-chain antibody of claim 5, wherein the first antigen-binding moiety is linked to the N-terminus of the second antigen-binding moiety, and the third antigen-binding moiety is linked to the C-terminus of the second antigen-binding moiety. The trispecific single chain antibody according to claim 6 , wherein said trispecific single chain antibody comprises a cytokine portion.

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 a mutant thereof.

9. The trispecific single-chain antibody according to claim 8, wherein the cytokine portion and the third antigen-binding portion are connected to each other via a long linker peptide; The amino acid sequence of the long connecting peptide is (GGS) n , wherein n is a natural number between 10 and 20.

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

11. The trispecific single-chain antibody according to claim 10, 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.

12. The trispecific single-chain antibody according to claim 11, wherein the trispecific single-chain antibody comprises a His-tag peptide, the amino acid sequence of which is shown in SEQ ID NO: 8; The His-tag peptide is located at the C-terminus of the trispecific single-chain antibody.

13. 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.

14. A nucleic acid, wherein the nucleic acid comprises a protein encoding the trispecific single-chain antibody of any one of claims 1-13.

15. A host cell, wherein the host cell comprises the nucleic acid of claim 14. 16 . A method for producing a trispecific single-chain antibody, wherein the method comprises culturing the host cell of claim 15 to produce the trispecific single-chain antibody of any one of claims 1 to 13 .

17. A pharmaceutical composition, wherein the pharmaceutical composition comprises the trispecific single-chain antibody according to any one of claims 1 to 13.

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

19. Use of the trispecific single-chain antibody according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 17 or 18 in the preparation of a drug for treating and / or preventing tumors.

20. The use according to claim 19, wherein the tumor is a tumor expressing pHLA tumor-specific or -associated antigen.

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