CN120699170B

CN120699170B - A trispecific single-chain antibody and its uses

Info

Publication number: CN120699170B
Application number: CN202511178413.3A
Authority: CN
Inventors: 马晓敏
Original assignee: Zhongsheng Kangyuan Bio Tech Beijing Co ltd
Current assignee: Zhongsheng Kangyuan Bio Tech Beijing Co ltd
Priority date: 2025-08-22
Filing date: 2025-08-22
Publication date: 2025-11-25
Anticipated expiration: 2045-08-22
Also published as: CN120699170A

Abstract

本申请提供了一种三特异性单链抗体及其用途，其中所述三特异性单链抗体包含特异性结合CLEC9A的第一抗原结合部分、特异性结合PDL1的第二抗原结合部分、以及特异性结合CTLA4的第三抗原结合部分。本申请提供的三特异性单链抗体桥接DC细胞和T细胞，可实现DC细胞有效抗原的提呈，并能够激活T细胞，其在减少抗体介导的细胞毒性的同时，增强了对提呈pHLA抗原靶细胞的特异性杀伤，在癌症免疫治疗领域具有广阔的应用前景。This application provides a trispecific single-chain antibody and its uses. 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 in this application bridges dendritic cells (DCs) and T cells, enabling the presentation of effective antigens by DCs and activating T cells. It reduces antibody-mediated cytotoxicity while enhancing the specific killing of target cells presenting pHLA antigens, showing broad application prospects in the field of cancer immunotherapy.

Description

Trispecific single-chain antibody and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a trispecific single-chain antibody and application thereof.

Background

The only T cell that can activate among professional Antigen Presenting Cells (APC) is the Dendritic Cell (DC), which plays an important role in the immune process. DC cells activated under conditions of phagocytizing foreign protein antigens and the like can recruit other immune cells (such as macrophages (Mac), eosinophils, natural killer cells (NK), T cells and the like) and interact, thereby further activating the T cells and promoting the differentiation of the T cells into effector T cells or memory T cells, and realizing the regulation or killing of target cells.

Immune checkpoint inhibition and CAR-T and other immunotherapeutic approaches have made great advances in cancer treatment, but only a fraction of the population can benefit. Drug resistance of tumors to radiotherapy such as small molecule drugs and the like and immune escape of tumor cells to immunotherapy restrict the effectiveness of tumor therapy. T cells play an important role in inhibiting and killing tumor cells, but three signals are required for effective activation of T cells, including a first signal, a pHLA (peptide-HLA) tumor-specific or related antigen presented by DC cells interacts with T Cell Receptor (TCR), a second signal, co-stimulatory molecules CD80 and CD86 of DC cells interact with T cell surface CD28, a third signal, cytokines secreted by activated DC cells (e.g., IFN-I factor IFN alpha, IL-12, etc.), and IFN gamma secreted by T cells further activate DC cells, thereby increasing T cell activity and promoting expansion of T cells.

The immune escape mechanism of the tumor comprises that the tumor cells down regulate the expression of Human Leukocyte Antigens (HLA) or secrete immune suppression cytokines such as IL10, TGF-beta and the like, accelerate the immune cells to be in a depletion state, thereby promoting the immune cells to highly express proteins such as PD1, CTLA4, TIM3 and the like, and the other mechanism is that the tumor cells prevent the DC cells from recruiting the immune cells or damaging the functions of the DC cells through highly expressing immune checkpoint proteins such as PDL1, thereby avoiding the killing of the innate and adaptive immune cells.

Thus, promoting the efficient recruitment of DC cells to immune cells and activating DC cells and recruited immune cells to achieve effective control of tumor cells is an important point of current research.

Disclosure of Invention

The application aims to provide a trispecific single-chain antibody bridging DC cells and T cells and application thereof.

In particular, the application relates to the following:

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 CTLA 4.

2. The trispecific single-chain antibody of item 1, wherein the first antigen-binding portion comprises VHH _CLEC9A, preferably the amino acid sequence of VHH _CLEC9A is shown as SEQ ID No. 2.

3. The trispecific single-chain antibody of item 1 or 2, wherein the second antigen-binding portion comprises VHH _PDL1, preferably the amino acid sequence of VHH _PDL1 is shown as SEQ ID No. 3.

4. The trispecific single-chain antibody of any of claims 1-3, wherein the third antigen-binding portion comprises VHH _CTLA4, preferably the amino acid sequence of VHH _CTLA4 is shown as SEQ ID No. 4.

5. The trispecific single-chain antibody of any one of claims 1-4, wherein the first antigen-binding portion, the second antigen-binding portion, the third antigen-binding portion are linked to each other by a short linking peptide;

Preferably, the amino acid sequence of the short connecting peptide is (GGS) _n, and n is a natural number of 10-20;

further preferably, the amino acid sequence of the short connecting peptide is (GGS) ₁₀.

6. The trispecific single-chain antibody of item 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.

7. The trispecific single-chain antibody of any one of claims 1-6, wherein the trispecific single-chain antibody comprises a cytokine moiety.

8. The trispecific single chain antibody of item 7, wherein the cytokine moiety is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, ifnα, ifnβ, or mutants thereof;

preferably, the cytokine moiety is selected from ifnα or a mutant thereof;

Further preferably, the cytokine moiety is an IFN alpha mutant, the amino acid sequence of which is shown in SEQ ID NO. 5.

9. The trispecific single-chain antibody of item 8, wherein the cytokine moiety, the third antigen-binding moiety are linked to each other by a long linker peptide;

Preferably, the amino acid sequence of the long connecting peptide is (GGS) _n, and n is a natural number of 10-20;

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

10. The trispecific single-chain antibody of item 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 any one of claims 1-10, wherein the trispecific single-chain antibody comprises a signal peptide having the amino acid sequence shown in SEQ ID No. 1;

preferably, the signal peptide is located at the N-terminus of the trispecific single chain antibody.

12. The trispecific single-chain antibody of any one of claims 1-11, wherein the trispecific single-chain antibody comprises a His-tag peptide having the amino acid sequence shown in SEQ ID No. 8;

Preferably, the His-tag peptide is located at the C-terminus of the trispecific single chain antibody.

13. The trispecific single-chain antibody according to any of claims 1-12, 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 nucleic acid 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 of 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 1-13.

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

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

19. Use of the trispecific single-chain antibody of any one of claims 1-13 or the pharmaceutical composition of claim 17 or 18 in the manufacture of a medicament for the treatment and/or prevention of a tumor.

20. The use of claim 19, wherein the tumor is a tumor expressing pHLA tumor-specific or related antigen.

The beneficial effects are that:

The application provides a trispecific single-chain antibody capable of bridging DC cells and T cells, which effectively pulls in the distance between the DC cells and the T cells, inhibits the expression of immune checkpoints PDL1 and CTLA4 and realizes the activation of the functions of the DC cells and the T cells by targeting CTLA4, PDL1 and CTLA4 and connecting IFN alpha 2 ^Q124R with reduced affinity. The trispecific single-chain antibody reduces the cytotoxicity mediated by the antibody to a certain extent, enhances the specific killing of the antigen target cells presenting pHLA, and has wide application prospect in the field of cancer immunotherapy.

Drawings

FIG. 1 is a schematic diagram of the structure of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single-chain antibody;

FIG. 2 is a schematic diagram of the structure of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 tetra-specific single-chain antibody;

Fig. 3A to fig. 3B are graphs showing the results of antibody purity detection. Wherein FIG. 3A is a SDS-PAGE of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single-chain antibodies, and FIG. 3B is a SDS-PAGE of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 tetraspecific single-chain antibodies.

FIGS. 4A-4B are spatial block diagrams of the scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single chain antibodies. Wherein FIG. 4A is a 3D structure of the antibody, and FIG. 4B is a 3D structure of the interaction of the antibody with the corresponding antigen protein.

FIG. 5 is a 3D block diagram of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 tetra-specific single-chain antibodies.

FIG. 6 is a graph showing the results of CMV-induced expansion of CD8 ⁺ and CD4 ⁺ T cells.

FIGS. 7A-7C are graphs showing the results of the detection of affinity of the scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single chain antibody for mDC cells. Wherein FIG. 7A shows the Kd values of the antibody and the mDC cell surface CLEC9A and PDL1, FIG. 7B shows the K _D value of the antibody and the mDC cell surface CLEC9A, and FIG. 7C shows the K _D value of the antibody and the mDC cell surface PDL 1.

FIGS. 8A-8B are graphs showing the results of the detection of affinity of the trispecific single-chain antibodies scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R for T cells. Wherein FIG. 8A shows the Kd values of the antibodies and the T cell surface CTLA4, and FIG. 8B shows the K _D values of the antibodies and the T cell surface CTLA 4.

FIGS. 9A-9 and C are graphs showing the results of the detection of affinity of the four specific single chain antibodies of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 for mDC cells. Wherein, FIG. 9A is the Kd value of antibody and mDC cell surface CLEC9A and CD40, FIG. 9B is the K _D value of antibody and mDC cell surface CLEC9A, and FIG. 9C is the K _D value of antibody and mDC cell surface CD 40.

FIGS. 10A-10C are graphs showing the results of the detection of affinity of the scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 tetra-specific single chain antibody for T cells. Wherein, FIG. 10A shows the Kd values of antibodies and T cell surface CTLA4 and PD1, FIG. 10B shows the K _D values of antibodies and T cell surface CTLA4, and FIG. 10C shows the K _D values of antibodies and T cell surface PD 1.

FIGS. 11A-11B are graphs showing the results of the Elispot assay 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 tetraspecific single chain antibody mediated activation of T cells by DC cells. Fig. 11A is a graph of statistical results of the number of points of Elispot, and fig. 11B is a graph of the actual number of points of Elispot.

FIG. 12A-FIG. 12B are graphs showing the results of detection of T cell activation by the 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 tetraspecific single chain antibody. Wherein FIG. 12A is a graph showing the results of detection of CD69 expression in T cells, and FIG. 12-B is a graph showing the results of detection of TIM3 expression in T cell immune checkpoints.

FIGS. 13A-13F are graphs showing the results of the detection of the stability of the scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single chain antibody in mDC cell culture broth. Wherein, FIG. 13A is a graph of the detection result of the binding strength of the antibody to the mDC cells, FIG. 13B is a graph of the detection result of the proportion of the mDC cells bound to the antibody, FIG. 13C is a graph of the detection result of the expression of PDL1 on the surface of the mDC cells, FIG. 13D is a graph of the detection result of the proportion of the cells expressing PDL1, FIG. 13E is a graph of the detection result of the expression of CLEC9A on the surface of the mDC cells, and FIG. 13F is a graph of the detection result of the proportion of the cells expressing CLEC 9A.

FIG. 14A-FIG. 14C are graphs showing the results of detection of non-targeted toxicity of a tri-specific single chain antibody to mDC cells by scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R. Fig. 14A is a graph showing the detection result of the activity of the mDC cells, fig. 14 and B are a graph showing the detection result of the number of the mDC cells, and fig. 10 and C are a graph showing the detection result of the apoptosis rate of the mDC cells.

FIG. 15 is a graph showing the results of 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 tetraspecific single-chain antibody mediated killing of CMV-loaded T2 cells by T cells.

Detailed Description

The application will be further illustrated with reference to the following examples, which are to be understood as merely further illustrating and explaining the application and are not to be construed as limiting the application.

Unless defined otherwise, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the materials and methods are described herein below. In case of conflict, the present specification, including definitions therein, will control and materials, methods, and examples, will control and be in no way limiting. The application is further illustrated below in connection with specific examples, which are not intended to limit the scope of the application.

Definition of the definition

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 is a group V C lectin-like receptor (CLR) that functions as an activating receptor and is expressed on DC cells. CLEC9A can act as an endocytic receptor on a small fraction of DC cells dedicated to uptake and processing of material from dead cells. CLEC9A recognizes actin in a filiform form, is associated with actin binding proteins, can be exposed when the cell membrane is damaged, and can mediate cross-presentation of dead cell-associated antigens.

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

The term "CTLA4", cytotoxic T lymphocyte-associated protein 4, as used herein, inhibits an immune response by binding to the ligands CD80 (also known as B7-1) and CD86 (also known as B7-2). CTLA-4 inhibits immune responses in a variety of ways, such as 1) competes with the T cell costimulatory receptor CD28 for its ligands CD80 and CD86, thereby blocking costimulation, and 2) emits a negative signal that inhibits T cell activation. CTLA-4 inhibitors can allow T cells to proliferate and attack tumor cells by inhibiting CTLA-4 molecules.

The term "antibody" as used herein is used in the broadest sense and covers a variety of 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 an antibody moiety, 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 comprising an antigen recognition site of a desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

Typically, an intact or full length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second, and third constant regions (CH 1, CH2, CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL), the light chain constant region comprising either Kappa (Kappa) or Lambda (Lambda). The full length antibody may be any kind of antibody, such as IgD, igE, igG, igA or IgM (or subclasses thereof as described above), but the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant region of the heavy chain. Typically, immunoglobulins have five main classes IgA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

The term "binding" or "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g., binding of an antibody to an epitope.

The term "antigen binding portion" as used herein refers to an anti-polypeptide molecule that specifically binds to an epitope. Specific antigen binding portions may 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 antigen-binding antibody, or a combination thereof.

The term "nanobody" as used herein refers to a heavy chain antibody in which a naturally deleted light chain is present in a camelid or the like, whose variable region is cloned to give a single domain antibody consisting of only the heavy chain variable region, also known as VHH (Variable domain of HEAVY CHAIN of HEAVY CHAIN anti), which is the smallest functional antigen binding fragment.

The terms "VHH", "nanobody" and "single domain antibody" as used herein have the same meaning and are used interchangeably to refer to cloning the variable regions of a heavy chain antibody, constructing a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function. Typically, after a heavy chain antibody is obtained with naturally deleted light and heavy chain constant regions 1 (CH 1), the variable regions of the heavy chain of the antibody are cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

The term "trispecific" as used herein means that the antibody is capable of specifically binding at least three different antigenic determinants.

The term "tetraspecific" as used herein means that the antibody is capable of specifically binding at least four different antigenic determinants.

The terms "first," "second," "third," and the like, as used herein, are used merely for convenience in distinguishing when more than one of each type of section is present. No particular order or orientation is intended to be imparted unless explicitly stated.

The term "vector" as used herein 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 and 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".

The terms "host cell", "host cell line", "host cell culture" or related terms as used herein refer to a cell (or population thereof) into which a foreign (exogenous or transgenic) nucleic acid has been introduced. The external nucleic acid may comprise an expression vector operably linked to the transgene, and the host cell may be used to express the nucleic acid and/or polypeptide encoded by the external nucleic acid (transgene). The host cell (or population thereof) may be a cultured cell or may be extracted from a subject. Regardless of the number of pathways, the host cell (or population thereof) includes primary subject cells and their progeny. The progeny cells may or may not carry the same genetic material as the parent cells. Host cells encompass offspring cells. In one embodiment, a host cell describes any cell (including progeny thereof) that has been modified, transfected, transduced, transformed and/or manipulated in any manner to express an antibody as disclosed herein. In one embodiment, a host cell (or population thereof) may be introduced with an expression vector operably linked to a nucleic acid encoding a desired antibody described herein. The host cells and populations thereof can carry expression vectors stably integrated into the host genome or can carry extrachromosomal expression vectors. In one embodiment, the host cells and populations thereof may carry extrachromosomal vectors that exist after several cell divisions, or that exist temporarily and disappear after several cell divisions.

The term "K _D" as used herein refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen, obtained from the ratio of the dissociation rate constant (K _d) to 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 antigen. The specific binding properties between two molecules can be determined using methods well known in the art.

The term "pharmaceutical composition" as used herein means an article of manufacture which assumes a form that enables the biological activity of the active ingredient contained therein to be effected, the pharmaceutical composition being free of additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

The term "pharmaceutically acceptable carrier" as used herein means an ingredient in the pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The terms "tumor" and "cancer" are used interchangeably herein and generally refer to a neoplasm formed by local tissue cell proliferation under the influence of various tumorigenic factors. In one embodiment, the tumor is a tumor that expresses pHLA tumor-specific or related antigens.

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 that are expressed on the cell surface in complexes with peptide fragments (antigens) of self or non-self proteins. MHC class I molecules interact with CD8 ⁺ cytotoxic T cells and play an important role in destroying organ transplant rejection or infected cells.

The term "HLA class I molecule" or "HLA class I molecule" as used herein refers to a protein product of a wild-type or variant HLA class I gene encoding an MHC class I molecule. Thus, "HLA class I molecules" and "MHC class I molecules" are used interchangeably herein.

The term "pHLA" as used herein may refer to peptide-bound human leukocyte antigens.

Antibodies to

The present application provides 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 CTLA 4.

In a specific embodiment, the first antigen binding portion comprises VHH _CLEC9A. In a specific embodiment, the VHH _CLEC9A has the sequence

SEQ ID NO: 2:

QVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTWLQMNSLRPEDTAWYCKAFTRGDDYWGQGTQVTVSS Is a sequence of amino acids of (a).

In a specific embodiment, the second antigen binding portion comprises VHH _PDL1. In a specific embodiment, the VHH _PDL1 has the sequence

SEQ ID NO: 3:

QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS Is a sequence of amino acids of (a).

In a specific embodiment, the third antigen binding portion comprises VHH _CTLA4. In a specific embodiment, the VHH _CTLA4 has the sequence

SEQ ID NO: 4:

QVQLVESGGGLVQPGGSLRLSCAASGYIYSAYCMGWFRQAPGKGLEGVAAIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADVIPTETCLGGSWSGPFGYWGQGTLVTVSS Is a sequence of amino acids of (a).

In a specific embodiment, the first antigen binding portion, the second antigen binding portion, and the third antigen binding portion are linked to each other by a short linking peptide. In a specific embodiment, the amino acid sequence of the short connecting peptide is (GGS) _n, and n is a natural number from 10 to 20. In a specific embodiment, the amino acid sequence of the short connecting peptide is (GGS) ₁₀.

In a specific embodiment, 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.

In a specific embodiment, the trispecific single chain antibody comprises a cytokine moiety.

In a specific embodiment, the cytokine moiety is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN alpha, IFN beta, or mutants thereof. In a specific embodiment, the cytokine moiety is selected from ifnα or a mutant thereof. In a specific embodiment, the cytokine moiety is an IFN alpha mutant having the sequence

SEQ ID NO: 5:

CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE Is a sequence of amino acids of (a).

In a specific embodiment, the cytokine moiety, the third antigen binding moiety are linked to each other by a long linker peptide. In a specific embodiment, the amino acid sequence of the long connecting peptide is (GGS) _n, and n is a natural number from 10 to 20. In a specific embodiment, the amino acid sequence of the long connecting peptide is (GGS) ₂₀.

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

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

In a specific embodiment, the trispecific single chain antibody comprises a His tag peptide having the amino acid sequence of sequence 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.

In a specific embodiment, the trispecific single chain antibody has

SEQ ID NO: 9:

MYRMQLLSCIALSLALVTNSQVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTWLQMNSLRPEDTAWYCKAFTRGDDYWGQGTQVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSQVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSQVQLVESGGGLVQPGGSLRLSCAASGYIYSAYCMGWFRQAPGKGLEGVAAIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADVIPTETCLGGSWSGPFGYWGQGTLVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEHHHHHHHHHH Is a sequence of amino acids of (a).

Nucleic acids, host cells and methods of production

The present application provides a nucleic acid, wherein the nucleic acid comprises a nucleic acid encoding any of the foregoing trispecific single chain antibodies.

Wherein the nucleic acid may be obtained according to methods conventional in the art, for example, DNA sequences encoding trispecific single-chain antibodies may be obtained by PCR amplification, DNA recombination or chemical synthesis techniques.

The present application provides a host cell, wherein the host cell comprises any one of the nucleic acids described above.

Wherein the exogenous nucleic acid can be introduced into the host cell according to methods conventional in the art. The method of introducing the exogenous nucleic acid may vary depending on the host cell used, and the host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell, e.g., CHO, HEK293 cells. When the host cell is a higher eukaryotic cell, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electrotransfection or liposome packaging may be used.

The present application provides a method for producing a trispecific single-chain antibody, wherein the method comprises culturing any one of the host cells described above to produce any one of the trispecific single-chain antibodies described above.

Typically, the host cells are cultured under conditions suitable for antibody expression, and then purified using conventional immunoglobulin purification procedures, such as protein A sepharose purification resins, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, to give antibodies by conventional separation and purification means well known to those skilled in the art.

Pharmaceutical composition

The present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises any one of the foregoing trispecific single chain antibodies.

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

Pharmaceutically acceptable carriers may include, among others, nontoxic 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, hexamethyl diammonium chloride (hexamethonium chloride), benzalkonium chloride, benzethonium chloride, phenols, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, 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 (monosacchandes), disaccharides, glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt forming counterions such as sodium salt-formingcounter-ion complexes such as sodium, e.g., TWEEN-n-TWEEN-complexes, and non-surface active agents such as Zn-PEG(s), and the like.

Use of the same

The application also provides the use of any one of the foregoing trispecific single-chain antibodies or any one of the foregoing pharmaceutical compositions in the manufacture of a medicament for the treatment and/or prevention of a tumor.

In a specific embodiment, the tumor is a tumor that expresses pHLA tumor-specific or related antigens.

In a specific embodiment, the tumor is a tumor presenting tumor-specific or associated antigens via MHC class I molecules.

On the basis of a bispecific single chain antibody (bispecific single-chain diabody, scDB) structure, the application designs a trispecific single chain antibody capable of bridging DC cells and T cells by utilizing VHH and a factor for activating the T cells, and the trispecific single chain antibody has full advantages in anti-tumor effect.

According to the application, through detecting the activation of the T cells by the trispecific single-chain antibody-mediated DC cells, the activation of the T cells by the trispecific single-chain antibody can be obviously improved, and meanwhile, the IFN gamma secretion is increased, and compared with scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 four-specific single-chain antibodies which also bridge the DC cells and the T cells, the effect is better.

In the application, the activation of the T cells by the trispecific single-chain antibody is detected, so that the trispecific single-chain antibody can directly activate the T cells, and compared with the tetraspecific single-chain antibody, the expression of CD69 in the T cells is obviously improved, and the expression of T cell immune checkpoint TIM3 is reduced.

In the application, the non-targeted toxicity of the trispecific single-chain antibody to the mDC cells is detected, so that the activity rate, the number and the apoptosis rate of the mDC cells are not obviously changed, and the trispecific single-chain antibody has only slight toxicity to the mDC cells.

In the application, the killing effect of the T cells mediated by the trispecific single-chain antibody on the T2 cells loaded with CMV is detected, so that the killing effect of the trispecific single-chain antibody on the target cells is specific and better than that of the tetraspecific single-chain antibody.

In general, the trispecific single-chain antibody provided by the application can realize the presentation of effective antigens of DC cells and activate T cells, enhances the specific killing of antigen target cells presenting pHLA while reducing the cytotoxicity mediated by the antibody, has better safety, and is expected to be used for the immunotherapy of tumors.

Examples

The present application will be described with reference to specific examples, but the scope of the application is not limited thereto. Unless otherwise specified, reagents and equipment used in the following examples are all conventional in the art and are commercially available. The methods used are all routine experimental methods, which can be carried out without any doubt by a person skilled in the art on the basis of the examples and with corresponding results.

Example 1

1.1 ScDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single chain antibody structural design

The multifunctional scDB antibody (scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R) is formed by combining a nanometer antibody CLEC9A variable region (VHH _CLEC9A) targeting DC cells, a nanometer antibody PDL1 variable region (VHH _PDL1), a nanometer antibody CTLA4 variable region (VHH _CTLA4) targeting T cells and a cytokine IFN alpha mutant (IFN alpha 2 ^Q124R), and has the structure shown in figure 1, and the amino acid sequence is shown in the following table 1.

TABLE 1 scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R

Amino acid characteristic region composition

Wherein the VHH _CLEC9A sequence was derived from patent application CN201780021194.5, the VHH _PDL1 sequence and the VHH _CTLA4 sequence were derived from monoclonal antibody erfonrilimab, the IFN alpha 2 ^Q124R sequence was derived from UniProtKB database (P01563), the signal peptide sequence was deleted and glutamine at position 124 was mutated to arginine. These polypeptide sequences are joined according to SP-VHH_CLEC9A-(GGS)₁₀-VHH_PDL1-(GGS)₁₀-VHH_CTLA4-(GGS)₂₀-IFNα2^Q124R-H₁₀ assemblies. Through reverse translation into DNA sequence, adding 5 'UTR and 3' UTR sequences capable of improving mRNA translation efficiency at the 5 'end and the 3' end, and finally synthesizing scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R by a chemical synthesis mode, wherein the specific sequence information is as follows:

The amino acid sequence of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R is:

MYRMQLLSCIALSLALVTNSQVQLQESGGGLVQPGGSLRLSCAASGRIFSVNAMGWYRQAPGKQRELVAAITNQGAPTYADSVKGRFTISRDNAGNTWLQMNSLRPEDTAWYCKAFTRGDDYWGQGTQVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSQVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSQVQLVESGGGLVQPGGSLRLSCAASGYIYSAYCMGWFRQAPGKGLEGVAAIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADVIPTETCLGGSWSGPFGYWGQGTLVTVSSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFRRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEHHHHHHHHHH (SEQ ID NO: 9);

the nucleotide sequence of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R is:

GCCGCCACCATGTACAGGATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCCAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCCGCATCTTCAGCGTGAACGCCATGGGCTGGTACCGCCAGGCCCCCGGCAAGCAGCGCGAGCTGGTGGCCGCCATCACCAACCAGGGCGCCCCCACCTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCGGCAACACCTGGCTGCAGATGAACAGCCTGCGCCCCGAGGACACCGCCTGGTACTGCAAGGCCTTCACCCGCGGCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCAAGATGAGCAGCCGCCGCTGCATGGCCTGGTTCCGCCAGGCCCCCGGCAAGGAGCGCGAGCGCGTGGCCAAGCTGCTGACCACCAGCGGCAGCACCTACCTGGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACAGCTTCGAGGACCCCACCTGCACCCTGGTGACCAGCAGCGGCGCCTTCCAGTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTACATCTACAGCGCCTACTGCATGGGCTGGTTCCGCCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCGCCATCTACATCGGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGTGATCCCCACCGAGACCTGCCTGGGCGGCAGCTGGAGCGGCCCCTTCGGCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCTGCGACCTGCCCCAGACCCACAGCCTGGGCAGCCGCCGCACCCTGATGCTGCTGGCCCAGATGCGCCGCATCAGCCTGTTCAGCTGCCTGAAGGACCGCCACGACTTCGGCTTCCCCCAGGAGGAGTTCGGCAACCAGTTCCAGAAGGCCGAGACCATCCCCGTGCTGCACGAGATGATCCAGCAGATCTTCAACCTGTTCAGCACCAAGGACAGCAGCGCCGCCTGGGACGAGACCCTGCTGGACAAGTTCTACACCGAGCTGTACCAGCAGCTGAACGACCTGGAGGCCTGCGTGATCCAGGGCGTGGGCGTGACCGAGACCCCCCTGATGAAGGAGGACAGCATCCTGGCCGTGCGCAAGTACTTCCGCCGCATCACCCTGTACCTGAAGGAGAAGAAGTACAGCCCCTGCGCCTGGGAGGTGGTGCGCGCCGAGATCATGCGCAGCTTCAGCCTGAGCACCAACCTGCAGGAGAGCCTGCGCAGCAAGGAGCACCACCACCACCACCACCACCACCACCACTGAAACGGGCTGATGCTGCACCAACTGTATCCA (SEQ ID NO: 10).

1.2 scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 four-specific single-chain antibody structural design

The multifunctional scDB antibody (scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4) is designed by combining a nanometer antibody CLEC9A variable region (VHH _CLEC9A) targeting DC cells, a CD40 activating antibody heavy chain variable region (VH _CD40) and a CD40 activating antibody light chain variable region (VL _CD40), and a nanometer antibody CTLA4 (VHH _CTLA4) variable region, a PD1 heavy chain variable region (VH _PD1) and a PD1 light chain variable region (VL _PD1) targeting T cells, wherein the amino acid sequences of the multifunctional scDB antibody (scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4) are shown in the following table 2.

TABLE 2 scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4

Amino acid characteristic region composition

Wherein the VHH _CLEC9A sequence is derived from patent application CN201780021194.5, the variable region sequence of anti-CD 40 is derived from monoclonal antibody Sotigalimab, the variable region sequence of anti-PD 1 is derived from monoclonal antibody pembrolizumab, and the VHH _CTLA4 sequence is derived from monoclonal antibody erfonrilimab. These polypeptide sequences are joined according to SP-VHH_CLEC9A-L3-VL_CD40-L1-VH_PD1-L2-VL_PD1-L1-VH_CD40-L3-VHH_CTLA4-H₁₀ assemblies. Through reverse translation into DNA sequence, adding 5 'UTR and 3' UTR sequences capable of raising mRNA translation efficiency in 5 'end and 3' end, and synthesizing scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 in chemical synthesis mode, its amino acid sequence is shown as SEQ ID NO. 18, and its nucleotide sequence is shown as SEQ ID NO. 19.

1.3 Expression and purification of recombinant antibody fusion proteins

The synthetic scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R nucleotide and scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 nucleotide were respectively constructed into pcDNA3.4 expression vectors, transfected into Chinese hamster ovary Cells (CHO), cultured in serum-free medium for 6 days, centrifuged, and the cell supernatants were harvested, and the antibodies were purified with immobilized nickel metal affinity chromatography columns under low flow rate conditions, and protein purity was confirmed by SDS-PAGE.

As a result, the purity of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R was 70% and the purity was higher, and the purity of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 was 60%, as shown in FIGS. 3A-3B.

1.4 Prediction of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single chain antibody structure

AlphaFold 3 was used to predict the spatial structure of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R. The amino acid sequence of the antibody with the signal guide peptide removed is input into AlphaFold, 5 predicted structures can be obtained, the ranking score (ranking score) is selected to be higher, the processing is performed by ChimeraX software, and finally the predicted 3D structure (shown in figure 4 and A) is output, so that the spatial structure of different domains of the antibody can be obviously distinguished.

To further confirm the interaction of the antibodies with each antigen protein CLEC9A, PDL1, CLTA4, the antibodies and the interacted proteins were input into AlphaFold for prediction to obtain 5 prediction models, the higher ranking_score was selected and treated with ChimeraX software, and as a result, as shown in fig. 4B, the antibodies interacted with CLEC9A, PDL, CLTA4 proteins in close proximity to each other.

1.5 ScDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 four-specific single chain antibody structure prediction

AlphaFold 3 was used to predict the spatial structure of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4. The amino acid sequence of the antibody with the signal guide peptide removed is input into AlphaFold, 5 prediction models can be obtained, the higher ranking_score is selected, the processing is performed by ChimeraX software, and finally the predicted 3D structure (shown in fig. 5) is output, so that the result shows that the spatial structure of different domains of the antibody can be obviously distinguished.

Example 2

2.1 Preparation of imDC cells

Purifying human peripheral blood to obtain Peripheral Blood Mononuclear Cells (PBMC) by Ficoll density gradient centrifugation, separating with CD14 magnetic beads to obtain CD14 ⁺ mononuclear cells, counting cells, calculating activity, suspending CD14 ⁺ mononuclear cells (1-5×10 ⁶ cells/mL) with serum-free medium containing rhGM-CSF and rhIL-4, culturing in culture flask at 37deg.C in 5% CO ₂ incubator (denoted as D0), supplementing liquid after culturing for 2-3 days, and culturing for 2-3 days (D5-D6) to obtain immature dendritic cells (immature DENDRITIC CELL, imDC).

2.2 Preparation of mDC cells

The imDC cells are collected for maturation culture, the amount of the cytokines to be added is calculated according to the volume of the maturation culture medium, the maturation cytokines are added into a serum-free culture medium (the maturation cytokine combination comprises rhGM-CSF, rhIL-4, rhTNF-alpha and the like), and the mixture is placed in a 37 ℃ and 5% CO ₂ incubator for culture for 18 to 24 hours, and finally mature dendritic cells (texture DENDRITIC CELL, mDC) are obtained.

2.3 Expansion of antigen peptide specific T cells

PBMC from 2.1 above were resuspended in serum-free medium at 1X 10 ⁶ cells/mL, 1. Mu.g/mL CMV/pp65 _495-504 was added, the unbound small peptides were removed by centrifugation at 300g for 2 hours at 37℃and the medium washed once, followed by 2.5X10 ⁶ cells/mL in complete medium (50 IU/mL IL-2, 1% (v/v) GlutaMAXTM, 1% (v/v) autologous serum autologous serum were added to serum-free medium), plated in culture plates, placed in 37℃5% CO ₂ incubator for culture, 1.5 volumes of complete medium were supplemented at day 5, and cells were harvested at day 9, finally CMV-specific T cells were obtained.

Anti-CD 3 is coated on the culture plate, and anti-CD 28 is added to the culture medium to expand T cells, so that control T cells are finally obtained.

The composition of T cells was analyzed by flow, and as shown in fig. 6, a higher proportion of CD8 ⁺ cells could be obtained in the CMV-treated group, while the majority of CD4 ⁺ cells were obtained in the control group.

Example 3

3.1 Affinity of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single-chain antibodies with mDC cells

The mDC cells obtained in example 2.2 were resuspended in PBS+HSA buffer at 2X 10 ⁶ cells/mL, 200. Mu.L of suspension (4X 10 ⁵ cells) was taken for each reaction, different concentrations of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R (units. Mu.g/mL) were added, 0.5, 0.2, 0.78, 3.125, 12.5, 50, 200, incubated for 1 hour at room temperature, the cells were harvested and resuspended in PBS, 1000-fold dilutions of Alexa Fluor [ sic ] 647 anti-His were added, incubated at room temperature in the dark for 15-30 minutes, washed once with PBS, followed by resuspension of the cells with flow buffer, and the affinity of the antibodies to the mDC cells was examined.

As a result, as shown in FIGS. 7A to 7C, the dissociation rate constants (Kd) of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R with the mDC cell surface CLEC9A and PDL1 were 4.351. Mu.g/mL (FIG. 7A), the equilibrium dissociation constant with CLEC9A (K _D) was 0.03179 (FIG. 7B), and the equilibrium dissociation constant with PDL 1K _D was 0.4997 (FIG. 7C).

3.2 Affinity of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R trispecific single-chain antibodies to T cells

The method comprises the steps of enriching T cells by using anti-CD 3 magnetic beads, resuspending the T cells in serum-free culture medium added with 50 IU/mL IL-2, anti-CD 28 and autologous plasma at 1.5X10/mL, planting the T cells in anti-CD 3 coated culture plates, activating the T cells for 3 days, then supplementing serum-free culture medium added with IL-2 every 2 days, culturing for 10 days to obtain amplified T cells, resuspending the T cells in PBS+HSA buffer solution at 2X 10 ⁶ cells/mL, taking 200 mu L of suspension (4X 10 ⁵ cells) per reaction, adding scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R (unit mu g/mL) at 0.5, 0.2, 0.78, 3.125, 12.5, 50 and 200, harvesting the cells, resuspending the cells in PBS, adding 1000 times of Alexa Fluor for 64 anti-His, keeping away from light for 15-30 minutes, washing once, and detecting the affinity of the T cells with the PBS buffer solution.

As a result, as shown in FIGS. 8A-8B, kd of scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R to CTLA4 on T cell surface was 165.7. Mu.g/mL, affinity was low (FIG. 8A), and K _D to CTLA4 was 0.168 (FIG. 8B).

3.3 Affinity of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 tetraspecific single strand with mDC cells

The mDC cells obtained in example 2.2 were resuspended in PBS+HSA buffer at 2X 10 ⁶ cells/mL, 200. Mu.L of suspension (4X 10 ⁵ cells) was taken for each reaction, different concentrations of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 (units. Mu.g/mL) were added, 0.01, 0.04, 0.16, 0.64, 2.56, 10.24, 40.96, incubated for 1 hour at room temperature, the cells were harvested and resuspended in PBS, 1000-fold dilutions of Alexa Fluor 647-anti-His were added, incubated at room temperature in light-shielding for 15-30 minutes, PBS was washed once, and then the cells were resuspended with streaming buffer, and the affinity of antibodies to the mDC cells was detected.

As a result, FIG. 9A-FIG. 9C show that Kd of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 with mDC cell surface CLEC9A and CD40 is 7.117. Mu.g/mL (FIG. 9A), K _D with CLEC9A is 0.5705 (FIG. 9B), and K _D with CD40 is 0.104 (FIG. 9C).

3.4 ScDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 four-specific single chain affinity to T cells

The method comprises the steps of enriching T cells by using anti-CD 3 magnetic beads, re-suspending the T cells in serum-free medium added with 50 IU/mL IL-2, anti-CD 28 and autologous plasma at 1.5X10/mL, planting the T cells in a culture plate coated with anti-CD 3, activating the T cells for 3 days, then supplementing serum-free medium added with IL-2 every 2 days, culturing for 10 days to obtain amplified T cells, re-suspending the T cells in PBS+HSA buffer solution at 2X 10 ⁶ cells/mL, taking 200 mu L of suspension (4X 10 ⁵ cells) per reaction, adding scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 (unit mu g/mL) at different concentrations, incubating the obtained cells in PBS at room temperature for 1 hour, adding 1000 times diluted Alexa Fluor 64 anti-His, incubating the obtained cells in PBS at room temperature for 15-30 minutes, washing the obtained cells, and detecting the affinity of the T cells by using the PBS buffer solution after one time.

As a result, as shown in FIGS. 10A to 10C, kd of scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 with T cell surface CTLA4 and PD1 was 1.589. Mu.g/mL (FIG. 10A), K _D with CTLA4 was 0.6877 (FIG. 10B), and K _D with PD1 was 0.2043 (FIG. C).

Example 4

4.1 Elispot detection antibodies promote activation of T cells by DC cells

ImDC and mDC cells obtained in 2.1 and 2.2 of example 2 were resuspended in serum-free medium, CD14 ^- cells eluted from 2.1 magnetic beads of example 2 were resuspended in CTL medium, counted by AO/PI double staining and the viability was determined, the following experimental groupings were set (where Ab3 was scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R code; ab2 was scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 code):

A. CD ^- +pbs, negative control group;

B. CD14^-+Ab3;

C. CD14^-+Ab2;

D. CD ^- + PHA, positive control group;

E. imDC+CD14^-+PBS;

F. imDC+CD14^-+Ab3;

G. imDC+CD14^-+Ab2;

H. imDC+CD14^-+CMV

I. mDC+CD14^-+PBS;

J. mDC+CD14^-+Ab3;

K. mDC+CD14^-+Ab2。

DC cells were mixed with CD14 ^- cells at a ratio of 1:5, and the above-described groups were aliquoted into centrifuge tubes at 1X 10 ⁴ cells/well, with B, F and J groups added with 5. Mu.g/mL Ab3, C, G and K groups added with 5. Mu.g/mL Ab2, A, E and I groups added with PBS of corresponding volume, H groups added with 1. Mu.g/mL CMV/pp65 _495-504, the cells were plated in an Elispot plate, incubated in a 37℃ C, CO2 incubator for 18 hours, incubated with anti-IFNγ -biotin antibody (clone 4S.B3, BD) for 2 hours at room temperature, PBS washed four times, streptavidin alkaline phosphatase was added in PBS at a dilution of 1:1600, incubated for 2 hours at room temperature, PBS washed, colorimetric AP substrate was added, incubated for 10-30 minutes in the dark, and spots were quantified by the ImmunoSpot S6 system (CTL).

As shown in fig. 11A-11B, ab3 treatment group significantly increased the activation of T cells by imDC cells compared to PBS group, increased the number of the dots of Elispot from 94 to 297 by about 3-fold, the effect was comparable to the activation of T cells by imDC cells directly loaded with CMV/pp65 _495-504 (number of dots 332), while Ab2 treatment group number of dots 134 increased by about 1.4-fold, ab3 also increased the activation of T cells by mDC cells in the mDC cell group by 343, PBS group number of dots 250, by about 1.37-fold, while Ab2 treatment group number of dots 278 increased by about 1.1-fold, and in CD14 ^- cells PBS group number of dots 4, ab3 treatment group number of dots 42, and Ab2 treatment group number of dots 5. The results show that scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R can effectively promote the activation of T cells by DC cells, and can directly activate the T cells through IFN alpha 2 ^Q124R.

4.2 Detection of activation of T cells by antibodies

The CMV load obtained in example 2.3 was used to activate the expanded T cells, 1X 10 ⁶ cells were resuspended in 500. Mu.L of serum-free medium, scDB-VHH_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 equivalent amount of PBS was added, the cells were plated, and the cells were incubated in a 37℃ C, CO2 incubator for 24 hours, and the activation of the T cells was examined in a flow-through manner.

As shown in fig. 12A-12B, ab3 significantly increased CD69 expression in T cells compared to Ab2, i.e., increased activation of CD4 and CD8 cells (fig. 12A), and Ab3 decreased TIM3 expression at T cell immune checkpoints, whereas Ab2 did not significantly inhibit TIM3 (fig. 12B).

Example 5

5.1 Detection of antibody stability in mDC cell culture

The mDC cells obtained in example 2.2 were used, planted in 24-well plates, placed in a 37℃ C, CO 2-2 incubator for 5 hours of culture, 1. Mu.g/mL scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R (Ab 3, cultured for 0, 1, 3, 6, 12, 24, 48 hours) was added at various time points, and finally the cells were harvested and the antibody expression in the mDC cells and antibody-bound target were examined in a flow assay.

As shown in FIGS. 13A-13F, ab3 and mDC cells were gradually decreased with increasing culture time, the binding strength was highest after 1 hour of culture, the binding strength was decreased to 50% after 12 hours of culture, the binding strength was maintained at 30% after 24 hours of culture (FIG. 13A), positive mDC cells binding to Ab3 were maintained at a high ratio at different culture times (FIG. 13B), the expression strength and cell ratio of PDL1 were maintained at a low level at different culture times, indicating that Ab3 had a strong blocking effect on PDL1 (FIG. 13C-FIG. 13D), the expression strength and cell ratio of CLEC9A were maintained at a low level after 1-3 hours of culture, indicating that Ab3 could be strongly bound to CLEC9A, but the expression strength and cell ratio of CLEC9A were gradually increased after 3 hours, indicating that the binding capacity of Ab3 to CLEC9A was slightly decreased (FIG. E-FIG. 13F).

5.2 Detection of untargeted toxicity of antibodies to mDC cells

Using the mDC cells obtained in 2.2 of example 2, 5. Mu.g/mL scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R (Ab 3, incubation for 0, 1,3, 6, 12, 24, 48 hours) was added at various time points, and finally the cells were harvested, counted by AO/PI double staining, the number and viability of the mDC cells were recorded, and stained with Annexin V/PI and the apoptosis of the mDC cells was detected in a flow-through manner.

As a result, as shown in FIGS. 14A to 14C, the viability of mDC cells was decreased between 3 hours and 6 hours, but the viability was maintained at 90% after 6 hours (FIG. 14 and A), the number of mDC cells was decreased with the increase of the culture time, but the number of cells was maintained substantially unchanged after 6 hours (FIG. 14 and B), and the apoptosis rate of mDC cells was maintained at about 5% without significant difference between different culture times (FIG. 14 and C). The above results indicate that Ab3 is only slightly toxic to mDC cells.

Example 6

6.1 Antibody-mediated killing of T2 cells loaded with specific antigen

Polylysine was treated with 96-well E-PLATE PLATEs for 2 hours at room temperature, washed 3-5 times with PBS, resuspended cells with RPMI 1640+10% FBS+2mM glutamine (Glutamine) medium, AO/PI counted, 1X 10 ⁴ individual lymphocyte hybridoma cells (T2) were grown per well, incubated in an xCELLICE RTCA ESIGHT instrument for 24 hours, and the following groupings were set according to the experimental design:

A. tctrl+ab3+cmv, i.e. T cells expanded with anti-CD 3 and anti-CD 28, T2 cells loaded with CMV/pp65 _495-504, and scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124R (Ab 3) added;

B. Tctrl+ab2+cmv, i.e. T cells expanded with anti-CD 3 and anti-CD 28, T2 cells loaded with CMV/pp65 _495-504, and scDB-VHH _CLEC9A-VL_CD40-VH_PD1-VL_PD1-VH_CD40-VHH_CTLA4 (Ab 2) added;

C. tcmv +CMV, i.e., CMV loaded with activated expanded T cells, T2 loaded with CMV/pp65 _495-504, and PBS was added;

D. Tcmv +Ab3+CMV, i.e., CMV loaded with activated expanded T cells, T2 loaded with CMV/pp65 _495-504, and Ab3 added;

E. Tcmv +Ab2+CMV, i.e., CMV loaded with activated expanded T cells, T2 loaded with CMV/pp65 _495-504, and Ab2 added;

F. Tcmv +Ab3, i.e. CMV load activates expanded T cells, T2 cells do not load short peptides, and Ab3 is added;

G. Tcmv +Ab2, i.e. CMV load activates expanded T cells, T2 cells do not load short peptides, and Ab2 is added;

The T2 cells grown on the wall were washed once with PBS, the RPMI 1640+2mM Glutamine medium containing 1. Mu.g/mL CMV/pp65 _495-504 was added according to the group, the short peptide-free group was added only to the RPMI 1640+2mM Glutamine medium, and incubated in a 37℃ C, CO2 incubator for 1 hour, the PBS was washed once, the T cells obtained in 2.3 of example 2 were prepared during incubation of the short peptide with the T2 cells, resuspended in the RPMI 1640+10% FBS+2mM Glutamine medium, wherein the A, D, F group was added with 5. Mu.g/mL Ab3, the B, E, G group was added with 5. Mu.g/mL Ab2, and the C group was added with a corresponding volume of PBS, the T cells were seeded into the T2 cells at a ratio of effector cells: target cells (E: T) =1:1, and then the culture plate was placed in an xCELLIgene RTCA ESIGHT instrument, the resistance was measured every 15 minutes, and the effect of T cells on T2 cells was monitored in real time.

As shown in FIG. 15, the cell index of Tcmv +Ab3+CMV group was smaller than that of Tcmv +Ab2+CMV group, the cell index was increased continuously in the Tctrl+Ab3+CMV group and the Tctrl+Ab2+CMV group even when antibodies were added, the cell index was the largest in all the treated groups, tcmv had a certain killing effect on the CMV-loaded T2 cells, and Tcmv had a poor killing effect on the non-CMV-loaded T2 cells when antibodies were added. Thus scDB-VHH _CLEC9A-VHH_PDL1-VHH_CTLA4-IFNα2^Q124 mediates the killing of target cells by T cells is specific.

The above description is only a preferred embodiment of the present application, and is not intended to limit the application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. A trispecific single-chain antibody, wherein the trispecific single-chain antibody comprises VHH _CLEC9A that specifically binds to CLEC9A, VHH _PDL1 that specifically binds to PDL1, VHH _CTLA4 that specifically binds to CTLA4, and a cytokine portion;

The amino acid sequence of VHH _CLEC9A is shown in SEQ ID NO: 2, the amino acid sequence of VHH _PDL1 is shown in SEQ ID NO: 3, and the amino acid sequence of VHH _CTLA4 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 is attached to the N-terminus of the VHH _PDL1 , the VHH _CTLA4 is attached to the C-terminus of the VHH _PDL1 , and the cytokine portion is attached to the C-terminus of the VHH _CTLA4 .

2. The trispecific single-chain antibody according to claim 1, wherein VHH _CLEC9A , VHH _PDL1 , and VHH _CTLA4 are linked to each other by short linker 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 are linked to each other by a long linker peptide;

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.