CN118725136A

CN118725136A - Fusion protein of anti-PD-1/anti-TIGIT bispecific antibody and IL-2 and application thereof

Info

Publication number: CN118725136A
Application number: CN202310283485.9A
Authority: CN
Inventors: 陈羿; 蔡则玲; 何巧巧; 王雅玲; 方国波
Original assignee: Shanghai Celgen Biopharma Co ltd
Current assignee: Shanghai Celgen Biopharma Co ltd
Priority date: 2023-03-21
Filing date: 2023-03-21
Publication date: 2024-10-01
Also published as: WO2024193665A1

Abstract

Provided herein are fusion proteins of anti-PD-1/anti-TIGIT bispecific antibodies with IL-2 and uses thereof. In particular, provided herein is a fusion protein comprising a fused (I) first polypeptide and (II) second polypeptide, wherein the first polypeptide comprises an anti-TIGIT/anti-PD-1 bispecific antibody (BsAb) or antigen-binding portion thereof that is anti-TIGIT and anti-PD-1, wherein the BsAb or antigen-binding portion thereof comprises (a) TIGIT binding domain and (B) PD-1 binding domain; the second polypeptide comprises interleukin-2 (IL-2) or a variant thereof having lymphopoietic activity. Fusion protein coding sequences, vectors and cells, related products and uses thereof (e.g., for cancer diagnosis, prevention and/or treatment) are also provided.

Description

Fusion protein of anti-PD-1/anti-TIGIT bispecific antibody and IL-2 and application thereof

Technical Field

The present application belongs to the field of biotechnology and medicine. In particular, the application relates to antibody-cytokinin fusion proteins, in particular fusion proteins comprising a bispecific antibody capable of specifically binding PD-1 and TIGIT and interleukin-2 (IL-2) or various variants thereof. In addition, the application relates to polynucleotides encoding such fusion proteins, vectors and host cells expressing such fusion proteins. The application also relates to methods of producing, preparing such fusion proteins, and pharmaceutical compositions and methods of treatment for treating diseases.

Background

Interleukin-2 (IL-2), a T cell growth factor induced by antigen stimulation, can activate and promote proliferation and differentiation of T cells, and maintain growth and proliferation of various immune cells such as B cells, natural killer cells, macrophages. IL-2 thus plays an important role in the treatment of tumors and immunodeficiency diseases, and is one of the first immunotherapeutic drugs approved by the FDA for cancer. In recent years, although wild-type IL-2 has achieved remarkable results in the treatment of cancer, its short half-life in vivo may cause side effects such as fever, vomiting, diarrhea, dizziness and hypotension, and has led to the focus of research on novel mutant IL-2 proteins with greater specificity and better therapeutic effects.

IL-2 exerts biological activity by binding to IL-2 receptors (IL-2R) on cell membranes. The IL-2R receptor is a complex consisting of an alpha chain (55 kd), a beta chain (75 kd) and a gamma chain (64 kd). IL-2 binds IL-2R with high affinity only when 3 chains are present (Kd. Apprxeq.10 ^-11 M). Cells that only have β and γ chains expressed and lost α chains are able to perform signal transduction, but only bind to IL-2 with moderate affinity (Kd ≡10 ^-9 M). Cells expressing only the alpha chain bind IL-2 with low affinity (Kd. Apprxeq.10 ^-8 M) and are unable to signal transduction of the cells. The gamma subunit alone does not bind to IL-2. High affinity IL-2R is predominantly present in activated T, B lymphocytes and NK cells, most resting NK cells and macrophages express medium affinity IL-2R, while low affinity IL-2R is expressed in resting T cells (GAFFEN S L et al, cytokine,2004,28 (3): 109-123).

The research shows that the mutation of the IL-2 protein can enhance the affinity to IL-2 Rbeta, and compared with the wild IL-2, the novel IL-2 mutant protein can better induce the proliferation of cytotoxic T cells and reduce the expansion of Treg cells, thereby improving the anti-tumor effect (Levin et al, nature 484:529 (2012).

The Ig and ITIM domain-containing T cell immunoreceptor (T cell immunoreceptor with immunoglobulin and ITIM domains, TIGIT, also known as WUCAM, vstm3 or VSIG 9) is a checkpoint molecule expressed primarily on the surface of immune cells such as NK cells, T cells, treg cells, etc., which has an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the cytoplasmic tail, and is a typical inhibitory receptor protein (Yu et al, nat. Immunol.10:48-57,2009).

TIGIT can suppress the immune response of the body through a variety of mechanisms. TIGIT directly blocks the acquisition of primary T cell (Tn) activation, proliferation and effector functions by targeting signaling pathway molecules of T cell antigen receptor (TCR), and can inhibit cell proliferation and inflammatory cytokine production of cd4+ T cells (Fourcade J et al, JCI Insight,2018,3 (14): e 121157). TIGIT can indirectly inhibit T cells by modulating cytokine production by dendritic cells (Yu et al, nat. Immunol.10:48-57,2009). TIGIT can also enhance Tregs stability and its inhibitory function on IFN- γ producing T cell proliferation (Fourcade J et al, JCI Insight,2018,3 (14): e 121157). In a tumor environment, TIGIT is highly expressed on the surfaces of NK cells and effector T cells related to tumor infiltration or migration, CD155 is highly expressed on the surfaces of tumor cells, and tumor cells directly act on the NK cells and the effector T cells through the combination of CD155/TIGIT to inhibit the activity of the NK cells and the effector T cells (Li et al, J.biol. Chem.289:17647-17657,2014). TIGIT is a new target for immunotherapy with great potential for enhancing immune responses and preventing and/or treating tumors, infections or infectious diseases. CD155 blocking antibody of targeting TIGIT, especially anti-human TIGIT antibody, can release tumor killing activity of immune effector cells, and is expected to obtain good anti-tumor curative effect.

Immune checkpoint inhibition by PD-1 antibodies is one of the most widely used cancer immunotherapies at present, but this traditional therapy is not therapeutically effective enough, the population lacks response, and there is drug resistance and even serious adverse effects. Most of the immune checkpoint antibody drugs on the market at present are monoclonal antibodies, which are only aimed at one specific target. However, monoclonal antibody therapy may be resistant or unresponsive, and many diseases are affected by different signaling pathways, different cytokines, and mechanisms of receptor modulation, single-target immunotherapy may be less effective.

TIGIT is a potential new target for immunotherapy for enhancing immune response and preventing and/or treating tumor, infection or infectious disease, however, single drug therapy has limited efficacy, and effective combination therapy is still required to be developed.

Bispecific antibodies (Bispecific antibody, bsAb) are a class of genetically engineered antibodies, generally referred to as antibodies that specifically bind to two antigens or two different epitopes of the same antigen. At present, bsAb has become a hot spot in the field of antibody engineering and has wide application prospect in the treatment of autoimmune diseases, tumors and other diseases.

There remains an urgent need in the art to develop a highly potent dual-antibody drug capable of acting on TIGIT and PD-1 simultaneously and having improved IL-2 function.

Disclosure of Invention

The present invention provides a fusion protein comprising both a bispecific antibody moiety capable of specifically targeting both PD-1 and TIGIT targets and an IL-2 or variant moiety thereof. The in vivo and in vitro experiments prove that the high-efficiency biological activity of the fusion protein containing the bispecific antibody has great advantages in curative effect and cost compared with the combined administration, and has remarkable clinical significance and wide market space.

In some aspects of the invention, there is provided a fusion protein comprising:

(I) A first polypeptide comprising an anti-TIGIT/anti-PD-1 bispecific antibody (BsAb) against TIGIT and against PD-1, or an antigen-binding portion thereof, wherein the bispecific antibody or antigen-binding portion thereof comprises:

(A) TIGIT binding domain, and

(B) A PD-1 binding domain; and

(II) a second polypeptide comprising interleukin-2 (IL-2) or a variant thereof having lymphopoietic activity, wherein said second polypeptide is fused to said first polypeptide.

In some embodiments, the TIGIT binding domain in an anti-TIGIT/anti-PD-1 bispecific antibody employs an anti-TIGIT monoclonal antibody disclosed herein (e.g., P03489, P03479) or a functional fragment thereof (antigen binding fragment, e.g., see tables 2 and 3).

In some aspects of the invention, a nucleic acid molecule or combination thereof is provided that encodes a fusion protein of the invention.

In some aspects of the invention, there is provided a vector or cell comprising a fusion protein of the invention or a fragment thereof and/or a nucleic acid molecule or combination thereof, e.g., the cell is a Chimeric Antigen Receptor (CAR) cell.

In some aspects of the invention, there is provided a product comprising a fusion protein or fragment thereof, a nucleic acid molecule or combination thereof, a vector or a cell of the invention. The product may be selected from: pharmaceutical compositions, kits. Or the product may be used to: production of the fusion protein, production of derivatives (e.g., CAR cells) comprising the fusion protein, disease treatment, and/or disease detection.

In some aspects of the invention there is provided the use of a fusion protein as described herein or a fragment thereof, a nucleic acid molecule or combination thereof, a vector or cell or product for the manufacture of a medicament for the prevention and/or treatment of TIGIT and/or PD-1 overexpression-related diseases (e.g. cancer, infectious diseases).

Any combination of the technical solutions and features described above can be made by a person skilled in the art without departing from the inventive concept and the scope of protection of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present invention will be further described with reference to the accompanying drawings, wherein these drawings are provided only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.

Fig. 1: exemplary TIGIT/PD-1BsAb-IL-2 variant fusion proteins of the application are shown in schematic structural representations.

Fig. 2: flow cytometry analysis studies showing the binding of anti-human TIGIT monoclonal antibodies to membrane TIGIT are shown.

Fig. 2A: binding of chimeric antibody 7103-07 and humanized antibody P03479 to membrane TIGIT;

Fig. 2B: binding of anti-human TIGIT humanized antibody P03489 to membrane TIGIT.

Fig. 3: ELISA studies showing competitive binding of anti-human TIGIT monoclonal antibodies and CD155 to recombinant human TIGIT are shown.

Fig. 4: the promotion of ifnγ production by anti-human TIGIT monoclonal antibodies on PHA-activated PBMCs is shown.

Fig. 5: shows the effect of the humanized monoclonal antibody of the anti-human TIGIT in inhibiting the growth of tumor in mice:

fig. 5A: tumor volume versus time for each mouse of each group;

Fig. 5B: tumor weight of each mouse of each group at the end of the experiment (D17).

Fig. 6: SDS-PAGE method identifies the prepared TIGIT/PD-1BsAb-IL-2 (RLFA) fusion protein:

Fig. 6A: analysis of non-reducing and reducing proteins of TIGIT/PD-1BsAb-IL-2 (RLFA) fusion protein KD 5301A;

fig. 6B: analysis of non-reducing and reducing proteins of TIGIT/PD-1BsAb-IL-2 (RLFA) fusion protein KD 5301B.

Fig. 7: sandwich ELISA study of in vitro double binding of TIGIT/PD-1BsAb-IL-2 to antigen.

Fig. 8: studies of in vitro stimulation of lymphocyte CTLL-2 growth by TIGIT/PD-1 BsAb-IL-2.

In the above figures, p <0.05 and p <0.01.

Detailed Description

The invention provides an antibody-cytokinin fusion protein of an anti-PD-1/anti-TIGIT BsAb-IL-2 mutant, which comprises a bispecific antibody of TIGIT and PD-1 and a mutant IL-2 molecule. The mutant interleukin-2 molecule may increase affinity for IL-2rβ or decrease affinity for IL-2rβ, while decreasing affinity for IL-2rα, as compared to a wild-type IL-2 molecule. On the other hand, the PD-1/TIGIT bispecific antibody-IL-2 fusion protein can delay the half-life of IL-2 and has better biological activity than the single application of IL-2. The antibody part of the antibody is targeted to the tumor microenvironment with fusion, so that the depleted T lymphocytes and NK cells with high expression of TIGIT and PD-1 in the tumor microenvironment are stimulated, and the antitumor functions of the tumor microenvironment are activated. In addition, PD-1/TIGIT BsAb-IL-2 variants preferentially activate TIGIT and PD-1 highly expressed lymphocytes, and have weak activation on other lymphocytes, so that IL-2-induced side effects are reduced. The PD-1/TIGIT bispecific antibody can also release the tumor killing activity of immune effector cells, and synergistically kill tumor cells to obtain a stronger tumor inhibiting effect.

Thus, the present disclosure provides methods useful for preventing and/or treating TIGIT and PD-1 expression or over-expression related diseases, such as tumors, infections, or infectious diseases. Bispecific antibodies of the present disclosure are also capable of reducing or eliminating TIGIT and/or PD-1 expressing cells (e.g., treg cells) or their activity.

The present disclosure also provides a method of enhancing an immune response, preventing and/or treating a tumor, infection, or infectious disease comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a bispecific antibody described herein or a derivative thereof (e.g., a chimeric antigen receptor drug).

All numerical ranges provided herein are intended to expressly include all values and ranges of values between the endpoints of the range. The features mentioned in the description or the features mentioned in the examples can be combined. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

As used herein, "comprising," having, "or" including "includes" including, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "containing", "having" or "including".

Monoclonal antibodies and their preparation

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming a connecting loop, which in some cases may form part of a b-sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

As used herein, the term "target molecule" refers to a molecule capable of being specifically bound by an antibody or targeting domain thereof. In this context, the target molecule may be located on a cell that expresses TIGIT, PD-1, or both, either surface or highly, preferably the target molecule is exposed outside or enriched on the target cell. In some embodiments, the target molecule is expressed in a cancer cell, an infected cell. In some embodiments, the target molecule is TIGIT, PD-1, or both.

As used herein, the term "sequence identity" or "percent identity" refers to the percentage of identical residues (e.g., amino acids or nucleic acids) in a candidate sequence to a reference sequence after aligning the sequences and, if necessary, introducing gaps to obtain the maximum percentage of sequence identity. For example, as used herein, "at least 70% sequence identity" refers to a sequence identity between a candidate sequence and a reference sequence that is greater than 70%, such as 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any numerical point identity therein.

The present disclosure provides monoclonal antibodies, preferably humanized antibodies, that specifically bind to human TIGIT. In the present disclosure, "m" represents a murine portion and "h" represents a human portion unless otherwise specified. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising 3 Complementarity Determining Regions (CDRs), the antibody comprises a light chain variable region (VL) comprising 3 CDRs.

In some embodiments, the anti-TIGIT monoclonal antibody comprises an antibody of heavy chain complementarity determining regions (VH CDRs) 1-3 and/or light chain complementarity determining regions (VL CDRs) 1-3 selected from the group consisting of: (i) VH CDR1 selected from the group consisting of: 11 or 19; (ii) VH CDR2 selected from the group consisting of: SEQ ID NO 12 or 20; (iii) VH CDR3 selected from the group consisting of: 13 or 21; (iv) VL CDR1 selected from the group consisting of: 15 or 23; (v) VL CDR2 selected from the group consisting of: 16 or 24; (vi) VL CDR3 selected from the group consisting of: SEQ ID NO 17 or 25.

In some embodiments, the anti-TIGIT monoclonal antibody comprises a combination of VH CDRs 1-3 selected from the group consisting of: SEQ ID NOS 11, 12 and 13; or SEQ ID NOS 19, 20 and 21; and/or, a combination comprising VL CDRs 1-3 selected from the group consisting of: SEQ ID NOS 15, 16 and 17; or SEQ ID NOS.23, 24 and 25.

In some embodiments, the anti-TIGIT monoclonal antibody comprises a combination of VH CDRs 1-3 and VL CDRs 1-3 selected from the group consisting of: combinations of SEQ ID NOs 11, 12 and 13 with SEQ ID NOs 15, 16 and 17; or a combination of SEQ ID NOS: 19, 20 and 21 with SEQ ID NOS: 23, 24 and 25).

In some embodiments, an anti-TIGIT monoclonal antibody comprises an amino acid sequence comprising or having at least 80% sequence identity to a heavy chain variable region (VH) selected from the group consisting of seq id nos: SEQ ID NO. 10 or 18. In some embodiments, the anti-TIGIT monoclonal antibody comprises a light chain variable region (VL) selected from the group consisting of or an amino acid sequence having at least 80% sequence identity thereto: SEQ ID NO. 14 or 22.

In some embodiments, the anti-TIGIT monoclonal antibody comprises a combination of heavy chain variable regions (VH) and light chain variable regions selected from the group consisting of: 10 or a combination of an amino acid sequence having at least 80% sequence identity thereto and 14 or an amino acid sequence having at least 80% sequence identity thereto; or SEQ ID NO. 18 or a combination of an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO. 22 or an amino acid sequence having at least 80% sequence identity thereto.

The anti-TIGIT monoclonal antibodies provided herein are novel TIGIT monoclonal antibodies with high affinity, high specificity and superior effect. The antibody and the functional fragment thereof can effectively block the combination of TIGIT and ligand CD155 thereof, enhance the cytokine release of immune cells, enhance the activity of the immune cells and enhance the immune response, and are used for preventing and/or treating tumors, infections or infectious diseases. The anti-TIGIT antibodies of the present disclosure are also capable of reducing or eliminating TIGIT expressing cells (e.g., treg cells) or their activity. The anti-TIGIT monoclonal antibody and fragments thereof have even better effect than the three-phase clinical medicine tireli Li Youshan antibody (Tiragolumab) of roche, and further development can be carried out on the basis of the anti-TIGIT monoclonal antibody and fragments thereof to obtain a product more suitable for clinical application.

Various anti-PD-1 monoclonal antibodies can be used in the present application. In some embodiments, exemplary anti-PD-1 monoclonal antibodies comprise VL CDRs, VH and/or VL selected from pembrolizumab or nivolumab, or fragments having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity with the foregoing sequences and PD-1 binding activity, or a combination thereof.

Bispecific antibodies

The term "bispecific" as used herein refers to an antibody having the ability to specifically interact with 2 different ligands simultaneously.

After obtaining the high affinity, high specificity, high potency anti-TIGIT monoclonal antibodies and antigen-binding fragments thereof described herein (e.g., as described in the preceding section), known bispecific antibody construction methods and configurations may be employed to construct bispecific antibodies herein in combination with anti-PD-1 antibodies or antigen-binding fragments thereof.

The terms "antigen binding domain" of an antibody, and "antigen binding portion" are used interchangeably to refer to one or more portions of an antibody as used herein that have a specific binding affinity for TIGIT or PD-1. Portions of whole antibodies have been shown to be capable of performing the antigen binding function of antibodies. Examples of antigen binding moieties include, but are not limited to: (i) Fab portions, i.e., monovalent portions, comprising VL, VH, CL and CH1 domains; (ii) The F (ab') ₂ portion, a bivalent portion, comprises two Fab portions with hinge regions linked to each other by a disulfide bridge; (iii) an Fd moiety comprising VH and CH1 domains; (iv) Fv portion comprising FL and VH domains of a single arm of an antibody; (v) A dAb moiety consisting of a VH domain or of VH, CH1, CH2, DH3 or VH, CH2, CH3 (dAb, or single domain antibody, comprising only a VL domain that has also been shown to specifically bind to a target epitope).

In some embodiments, the TIGIT binding domain and/or the PD-1 binding domain is an antibody fragment selected from the group consisting of: fab fragments; monovalent fragments consisting of VL, VH, CL and CH1 domains; a F (ab) ₂ fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge of a hinge region; fd fragment consisting of VH and CH1 domains; fv fragments consisting of the VL and VH domains of the antibody single arm; a dAb fragment; an isolated CDR; and scFv.

Although the two domains of the Fv portion (i.e., VL and VH) are each encoded by a different gene, they can be joined to one another using synthetic linkers (e.g., poly-G ₄ S amino acid sequence (GGGGS) _n, n=any integer from 1 to 10) and recombinant methods, making it possible to prepare them as one protein chain, with the VL and VH domains combining to form monovalent molecules, known as single chain Fv (ScFv). The term "antigen binding portion" of an antibody also encompasses such single chain antibodies.

Other forms of single chain antibodies, such as "diabodies" are also included herein. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to combine the two domains on the same chain, forcing the domains to pair with complementary domains of different chains to form two antigen binding sites. Immunoglobulin constant region refers to either the heavy or light chain constant region. The human IgG heavy and light chain constant region amino acid sequences are known in the art.

In some embodiments, bispecific antibodies herein may be free of Fc fragments (non-IgG-like structures). The bispecific antibody has the advantages of simple and convenient production, high yield, low cost and the like. For example, a bispecific antibody herein may be formed from two single chain antibodies ScFv joined by a short peptide chain, an antibody dimer (diabody) consisting of two intersecting single chain antibodies ScFv. Bispecific antibodies of such structures can be further engineered to increase their stability, for example, by introducing cysteines at the C-terminus of their peptide chains to form disulfide bonds between VH-VL.

In some embodiments, the bispecific antibodies herein comprise an Fc fragment, i.e., are IgG-like structure bispecific antibodies. Such bispecific antibodies retain the corresponding function of the Fc fragment and have higher stability. Such bispecific antibodies may include, but are not limited to: pestle and socket structure ((Knob-into-hole, KIH)), scFv2-Fc, triomabs, double variable domain immunoglobulins (Dual-Variable Domain Immunoglobulin, DVD-Ig), and the like.

In some embodiments, bispecific antibodies herein employ a knob-and-socket structure, introducing mutations (e.g., S354C and T366W mutations) into the CH3 region of the heavy chain of one of the anti-TIGIT antibody and anti-PD-1 antibody, forming Knob structures by replacing a smaller size amino acid with a larger size amino acid, introducing mutations into the CH3 region of the heavy chain of the other antibody, forming a socket (Holes) structure by replacing a larger size amino acid with a smaller size amino acid, and utilizing the spatial structural complementarity characteristics of the sample socket structure to achieve proper assembly of the two antibodies.

In some embodiments, the bispecific antibodies herein have a configuration of a knob structure (see fig. 1), whose Fc can be engineered to incorporate a CrossMab. In particular, exemplary configuration 1 bispecific antibodies have the following structural features:

-introducing mutations S354C and T366W (EU numbering) in the constant region of one of the heavy chains, forming a knob heavy chain;

-introducing mutations Y349C, T366S, L a and Y407V (EU numbering) in the constant region of the other heavy chain to form the hole heavy chain;

-heterodimerization of the knob heavy chain and the hole heavy chain, thereby forming anti TIGIT/anti PD-1BsAbs.

In some embodiments, the bispecific antibodies herein have the configuration of an IgG-scFv structure (or scFv 2-IgG). In particular, exemplary IgG-scFv configuration bispecific antibodies have the following structural features:

-ligating scFv fragments of anti-TIGIT or PD-1 antibodies at the C-terminus of both heavy chains of anti-PD-1 or TIGIT antibodies;

-flexible peptide linkages between heavy chain and scFv, VH and VL of scFv, respectively, by n G ₄ S (i.e. GGGGS repeats, n greater than 3);

-these heavy chains form homodimeric bispecific antibodies with anti TIGIT antibody light chains or PD-1 antibody light chains, respectively.

In addition, in order to reduce antibody-mediated ADCC and CDC activity, 2 amino acids L234/L235 (EU numbering) of the two heavy chain CH1 regions of the antibody may be substituted with amino acids Ala (LALA variants) to eliminate the ADCC/CDC function of the antibody.

Bispecific antibodies herein can be identified by conventional means. For example, the binding specificity of a bispecific antibody can be determined using flow cytometry, immunoprecipitation, in vitro binding assays, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), biofilm layer optical interferometry (BLI).

Bispecific antibodies herein may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Fusion protein (polypeptide)

As used herein, the term "fusion protein (polypeptide)" refers to a fusion polypeptide molecule comprising an anti-PD-1/anti-TIGIT bispecific antibody polypeptide and an IL-2 polypeptide, wherein the components of the fusion protein are linked to each other directly by peptide bonds or by linkers. For clarity, the individual peptide chains of the antibody building blocks of the fusion protein may be non-covalently linked, e.g. by disulfide bonds. "fused" means that the components are linked by peptide bonds either directly or via one or more peptide linkers. As used herein, the term "element" or "building block" refers to an amino acid sequence that forms part of a fusion protein. The term "unit" or "monomer" refers to a basic fragment of an element's constituent function.

"Native IL-2", also referred to as "wild-type IL-2" (wtIL-2), means naturally occurring IL-2, as opposed to "modified IL-2", also referred to as "mutant IL-2" (mutIL-2), which has been mutated or modified relative to naturally occurring IL-2, e.g., in order to alter one or more properties of the native IL-2, such as stability, activity, etc. The modified IL-2 molecule may for example comprise modifications in the amino acid sequence, such as amino acid substitutions, deletions or insertions. As described in the background section, the structure and function of IL-2 has been studied and understood in the art. IL-2 polypeptides known in the art may be used in the present application, as may natural variants and fragments thereof (e.g., splice variants or allelic variants), as well as non-natural variants having IL-2 activity.

Wild-type human IL-2 polypeptides as used herein are known in the art. For example, the wild-type human IL-2 polypeptide may comprise the amino acid sequence of accession number UniProtKB-P60568, or the amino acid sequence as set forth in SEQ ID NO:9 (human IL-2), or may be a homologous sequence having the same or similar activity as the proteins (e.g., the homologous sequence may be obtained by databases or alignment software known in the art) or a functional/active fragment thereof.

In some embodiments, the mutant IL-2 molecule differs in the IL-2 region that binds to the IL-2Rα subunit from the IL-2 region that binds to the IL-2Rβ subunit. The introduction of amino acid mutations in these 2 regions of IL-2 can alter the binding affinity of IL-2 to the IL-2Rα subunit and to the IL-2Rβ subunit. For example, IL-2 mutants (L80F, R81D, L85V, I86V, I F) have increased affinity for binding to the IL-2Rβ subunit (Levin et al (2012) Nature 484, 529-533) and IL-2 mutants (F42A) have decreased affinity for binding to the IL-2Rα subunit. The IL-2 mutant gene (L80F, R81D, L85V, I86V, I F) can be artificially synthesized, and then an amino acid mutation (F42A) is introduced into the gene sequence by a gene point mutation method to construct the IL-2mut gene with reduced binding affinity for binding IL-2R alpha subunit and enhanced binding affinity for binding IL-2R beta subunit. In some embodiments, the IL-mutant comprises R38L and F42A mutations.

The mutant IL-2 polypeptides herein may be selected from: (a) A polypeptide having one or more additional amino acid residues in both stretches of the wild-type IL-2 amino acid sequence as described above; or (b) a protein or polypeptide derived from (a) which has wild-type IL-2 activity by substitution, deletion or addition of one or more amino acids within the wild-type IL-2 sequence. For example, the mutant IL-2 polypeptides herein can comprise a mutation that reduces IL-2Rα subunit binding affinity and/or increases IL-2Rβ subunit binding affinity as described previously.

The polypeptide elements and polypeptide elements in the fusion proteins of the present disclosure are preferably encoded by or humanized by a human gene or a homologous gene or family thereof. Variant forms of a protein or polypeptide in the present disclosure include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids having similar or similar properties typically does not alter the function of the protein or polypeptide. For another example, the addition of one or more amino acids at the C-terminal and/or N-terminal end will not normally alter the function of the protein or polypeptide, e.g., the fusion protein may or may not include the initial methionine residue while still having its desired viral infection control activity.

Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by sequences which hybridize under high or low stringency conditions to their protein coding sequences. Depending on the host used in the recombinant production protocol, the proteins or polypeptides of the invention may be glycosylated or may be non-glycosylated.

In the fusion proteins of the invention, the components (TIGIT binding domain, PD-1 binding domain and IL-2 or variant molecules thereof) are genetically fused to each other. Fusion proteins can be designed such that their components are fused directly to each other or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and efficacy can be tested. Additional sequences are included to incorporate cleavage sites to separate the individual components of the fusion protein (if desired), such as endopeptidase recognition sequences.

The individual polypeptide elements or peptide units within the elements of the fusion proteins herein may be linked by linkers. Flexible linkers are preferably employed in the present application to allow interactions between polypeptide elements or peptide units. Linkers useful in the fusion proteins of the application may comprise from 2 to 300 amino acid residues, for example from 5 to 100, from 10 to 50, from 15 to 3 amino acid residues. Exemplary linkers can be glycine linkers, such as (G) _n or glycine/serine linkers, such as the amino acid sequences of (GS) _n、(GGS)_n、(GGGS)_n、(GGGGS)_n or (GGGGGS) _n, where n is an integer of 1,2, 3, 4, 5, 6, 7, 8, 9, or 10.

IL-2 or variants thereof may be linked to the C-terminus, N-terminus, or in the middle of a bispecific polypeptide. In some embodiments, IL-2 is attached to the C-terminus of the PD-1 heavy chain.

The fusion proteins herein may also comprise a signal peptide, such as an amino acid sequence, typically 5-50 amino acids in length, that has the function of directing secretion, localization and/or transport of the fusion protein. In some embodiments, the signal peptide may be selected from, for example: tPA2 signal peptide, TFF2 signal peptide, IL-2 signal peptide, bPRL signal peptide CD33 protein signal peptide, and the like.

The fusion proteins herein may also comprise a label, e.g. for purification, detection, localization, such as a label selected from the group consisting of fluorescent labels, non-radionuclide labels, biotin-like labels, phosphorylation modification labels, peptide tags.

Coding molecule, expression vector containing the same and host cell

Nucleic acid molecules encoding the fusion proteins or fragments thereof are also provided herein. The sequences of these nucleic acid molecules can be obtained by conventional techniques, such as by PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody. Also disclosed herein are expression vectors containing the above nucleic acid molecules, e.g., pcdna3.4, pcdna3.1, etc.; also disclosed herein are host cells transformed with the above-described expression vectors, which may be, for example, CHO cells, COS cells, and the like.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

Currently, nucleic acid sequences encoding the fusion proteins herein (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The sequence can then be introduced into a variety of existing molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences herein by chemical synthesis.

The present invention also relates to vectors comprising the coding sequences described above and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells such as mammalian cells, e.g., CHO cells, COS cells, etc. Mammalian cell lines are commonly used as host cells for expression of eukaryotic cell-derived polypeptides. The propagation of mammalian cells in culture is well known in the art. See tissue culture, ACADEMIC PRESS, kruse and Patterson, eds. (1973), incorporated herein by reference. Preferred mammalian cells are a number of commercially available immortalized cell lines. These immortalized cell lines include, but are not limited to, chinese Hamster Ovary (CHO) cells, vero cells, hela sea-Law cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hep G2), and many other cell lines. They provide post-translational modifications to protein molecules, including proper folding, proper disulfide bond formation, and glycosylation at the correct site.

There are various methods for transforming host cells with expression vectors, and the transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene (1, 5-dimethyl-1, 5-diazaundecene polymethylbromide) -mediated transfection, protoplast fusion, electroporation, liposome-mediated transfection, and direct microinjection of DNA into the nucleus. Preferred methods herein are electroporation or liposome-mediated and the like. For example, invitrogen's liposome method kit can be used to transfect host cells such as COS, CHO cells, and the like.

Culturing the transformed host cell under conditions suitable for expression of the fusion protein herein. The antibodies herein are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, 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.

Conjugate, chimeric antigen receptor and immune cell expressing chimeric antigen receptor

Also provided in the application are conjugates comprising the fusion proteins herein, chimeric antigen receptors, and immune cells expressing the chimeric antigen receptors. Because of the specificity of the fusion proteins of the application or functional fragments thereof for TIGIT and PD-1, the conjugates, chimeric antigen receptors, and immune cells expressing the chimeric antigen receptors can target cells or tissues expressing or over-expressing TIGIT and/or PD-1, and preferably produce additive or synergistic effects. Furthermore, since the fusion protein of the present application itself has biological activity (e.g., has immunomodulatory activity), the conjugate thereof, the chimeric antigen receptor, and the antibody or antigen-binding fragment portion in immune cells expressing the chimeric antigen receptor can also exert corresponding effects.

In some embodiments, an antibody conjugate may comprise: a fusion protein or functional fragment thereof as described herein; a coupling moiety, such as a drug, toxin, cytokine, radionuclide or enzyme; and optionally, a joint. In some embodiments, the coupling is achieved by enzymatic coupling, chemical coupling, fusion, or the like. The linker may be a "cleavable linker" that facilitates release of the coupling moiety in the cell, such as an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker, and a disulfide-containing linker.

In some embodiments, the coupling moiety to which the antibody or fusion protein is attached may be, for example, a bioactive agent, which may be any synthetic, recombinantly produced, or naturally occurring substance that enhances and/or modulates a desired biological effect. In some embodiments, the bioactive agent can provide effects including, but not limited to: modulating, stimulating, and/or inhibiting a growth signal; modulating, stimulating, and/or inhibiting an anti-apoptotic signal; modulating, stimulating, and/or inhibiting an apoptosis or necrosis signal; modulating, stimulating, and/or inhibiting the ADCC cascade; modulating, stimulating, and/or inhibiting CDC cascades, and the like. For example, it may be a chemotherapeutic agent, toxin, cytokine, isotope, enzyme, etc.

Also provided in the application is a Chimeric Antigen Receptor (CAR) comprising an extracellular domain and an intracellular domain, wherein the extracellular domain comprises a fusion protein of the application or a functional fragment thereof. In some embodiments, the CARs of the application further comprise a transmembrane domain that connects the extracellular domain and the intracellular domain, such as the transmembrane portion of CD 28. The CAR of the present application may be a first, second, third or fourth generation CAR.

In some embodiments, the antibody or antigen-binding fragment thereof in a CAR of the application is, or consists of, an scFv or VH sdAb. In some embodiments, the intracellular domains of the CAR include those that mimic or approximate the signaling through natural antigen receptors, the signaling through such receptors in combination with co-stimulatory receptors, and/or the signaling through co-stimulatory receptors alone.

In some embodiments, the intracellular domain comprises one or more co-stimulatory domains and an activation domain, e.g., one or more selected from the group consisting of: ITAM domain, CD3 ζ, CD28, 4-1BB, OX40, CD27, ICOS or combinations thereof, e.g (CD28+CD3ζ)、(CD28+CD27+CD3ζ)、(CD28+OX40+CD3ζ)、(CD28+4-1BB+CD3ζ)、(CD28+CD27+OX40+CD3ζ)、(CD28+4-1BB+CD27+CD3ζ)、(CD28+4-1BB+OX40+CD3ζ)、(4-1BB+CD3ζ)、(4-1BB+OX40+CD3ζ)、(4-1BB+CD27+CD3ζ)、(CD27+CD3ζ)、(CD27+OX 40+CD3ζ)、(CD28Δ+CD3ζ)、(CD28Δ+CD27+CD3ζ)、(CD28Δ+OX40+CD3ζ)、(CD28Δ+4-1BB+CD3ζ)、(CD28Δ+4-1BB+OX40+CD3ζ)、(CD28Δ+CD27+OX40+CD3ζ)、(CD28Δ+4-1BB+CD27+CD3ζ))、(4-1BB+ICOS+CD3ζ)、(CD28+ICOS+CD3ζ)、(ICOS+CD3ζ).

In some embodiments, the extracellular domain of the CAR further comprises a hinge region, e.g., derived from an IgG hinge or a CD8 a/CD 28 extracellular region, such as selected from: igG4 FcΔEQ, igG4 FcΔQ, (t-12AA+t-20 AA), mKate, phiLov, dsRed, venus, eGFP, CH HA, (CD8α+t-20 AA), double t-20AA, (t-20AA+CD8α), (CD8α+leucine zipper Basep 1), (CD8α+leucine zipper Acid 1), 2D3, CD8α, or IgG4 Fc.

Also provided in the application is a transformed immune cell that expresses a chimeric antigen receptor of the application, e.g., the immune cell is selected from a T cell, such as an αβ T cell, γδ T cell, or NK T cell, or a T cell derived from a pluripotent cell. The immune cells may be from the individual to be treated or from other sources. The transformed (optionally expanded) immune cells may be administered to the individual to be treated, for example for adoptive therapy.

In addition, the application also includes a CAR construct, an expression vector, a preparation method and application thereof for transforming immune cells.

Pharmaceutical composition

Also provided herein is a pharmaceutical composition comprising a pharmaceutically effective amount of a fusion protein of the present disclosure or an immunoconjugate thereof, and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" as used herein means that the molecular entity and composition do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" shall be compatible with the active substances herein, i.e. capable of being blended therewith without substantially reducing the efficacy of the pharmaceutical composition in the usual manner. Such vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers is found in Remington pharmaceutical sciences (Remington's Pharmaceutical Sciences, mack Pub.Co., N.J.1991).

Such vectors include (but are not limited to): saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

The compositions herein may be administered orally, intravenously, intramuscularly or subcutaneously; oral or intravenous injection administration is preferred. The pharmaceutical compositions herein may be formulated into a variety of dosage forms as desired, and the dosage to be beneficial to the patient may be determined by the physician based on the type of patient, age, weight, general condition, mode of administration, and the like.

When a pharmaceutical composition is used, a safe and effective amount of the antibody or immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically about 0.1 μg to 5 mg per kilogram of body weight and in most cases no more than about 3 mg per kilogram of body weight, preferably the dose is about 1 to 10 μg per kilogram of body weight to about 1 mg per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

As used herein, the term "unit dosage form" refers to a dosage form that is required to prepare the compositions of the present disclosure for convenient administration in a single administration, including but not limited to various solid (e.g., tablets), liquid, capsules, sustained release formulations.

In another preferred embodiment of the present disclosure, the composition is in unit dosage form or multiple dosage form and wherein the active substance content is from 0.01 to 2000 mg/dose, preferably from 0.1 to 1500 mg/dose, more preferably from 1 to 1000 mg/dose. In another preferred embodiment of the present disclosure, 1 to 6 doses of the composition of the present disclosure, preferably 1 to 3 doses, are administered daily; most preferably, the daily dosage is 1 dose.

The fusion protein or the immunoconjugate can effectively block the combination of TIGIT and ligand CD155 thereof, can also effectively block the combination of PD-1 and PD-L1 ligand, enhance the cytokine release of immune cells, enhance the activity of the immune cells and enhance the immune response, and is used for preventing and/or treating tumors, infections or infectious diseases. The active substances herein are useful for the treatment of various types of tumors such as (including but not limited to): adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma; tumor tissue from adrenal gland, gall bladder, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary gland, spleen, testis, thymus, thyroid, or uterus; tumors of the central nervous system, such as glioblastoma, or astrocytoma; eye tumors (e.g., basal cell carcinoma, squamous cell carcinoma, or melanoma), endocrine gland tumors, neuroendocrine system tumors, gastrointestinal pancreatic endocrine system tumors, reproductive system tumors, or head and neck tumors.

Detection kit

Also provided herein is a kit for detecting TIGIT and/or PD-1, comprising the fusion protein described herein or an active fragment thereof, an immunoconjugate. The kit herein may be used to detect the presence or absence or presence or amount of TIGIT or PD-1 or both in a biological sample, the detection method comprising the steps of: (a) Contacting the sample with a fusion protein or immunoconjugate thereof in a kit herein; (b) Detecting whether a specific antigen-antibody complex (e.g., TIGIT antigen-antibody complex, PD-1 antigen-antibody complex, and/or TIGIT/PD-1 antigen-antibody dual complex) is formed, wherein the formation of a complex indicates the presence of TIGIT and/or PD-1 in the sample or quantitatively detecting the amount of antigen-antibody complex formed to reflect the amount of TIGIT and/or PD-1 in the sample. It is particularly preferred to use the kits herein to detect the presence of TIGIT and PD-1 simultaneously. The sample to be tested may or may not be pretreated, for example by extraction, purification or concentration.

The kit contains a container and the fusion protein herein, or a detection plate with the antibody, within the container, and instructions for use. Other reagents required for detection, such as buffers, indicators, etc., may also be included in the kit. The contents of the kit can be adapted by one skilled in the art according to specific needs.

Examples

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Appropriate modifications and variations of the invention may be made by those skilled in the art, and are within the scope of the invention.

The experimental methods described in the following examples, in which specific conditions are not specified, may be employed by conventional methods in the art, for example, with reference to the "molecular cloning Experimental guidelines" (third edition, new York, cold spring harbor laboratory Press, new York: cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. Methods for sequencing DNA are routine in the art and can also be provided for testing by commercial companies.

Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Example 1 acquisition of anti-TIGIT monoclonal antibody and its coding sequence, performance determination and construction of anti-TIGIT antibody-IL-2 fusion protein Gene

1.1 Anti-TIGIT monoclonal antibodies and the obtaining of the coding sequences thereof

The anti-TIGIT antibody is a plurality of anti-TIGIT humanized monoclonal antibodies invented by the inventor, the preparation method of the antibody and the amino acid sequence are shown in Chinese patent application (CN 202111105544.0, "monoclonal antibody targeting TIGIT", application day 2021, 9 months and 22 days).

Specifically, spleen cells of immunized mice (6-8 weeks old, about 18g in body weight, female, purchased from the laboratory animal manager of the family planning science research) were obtained by intraperitoneal and subcutaneous injection of recombinant human TIGIT (His tag, sino Biological,10917-H08H access # NP 776160.2.Met1-Phe138, the same shall apply hereinafter) according to the conventional method. Spleen cells were fused with mouse myeloma cells (available from the national academy of sciences cell bank under accession number TCM 18) and positive clones were screened by ELISA after fusion using TIGIT-Fc (Human TIGIT (Fc Tag): sino Biological, 10917-H02H), and multiple clones were selected, wherein clones numbered 7103-01 and 7103-07 each had an IgG1 heavy chain and a Kappa light chain.

The resulting cell line is cultured and the cells are expanded. After centrifugation, the supernatant was collected and the fusion Protein was purified from the supernatant using a Protein-A affinity column, run on an analytical column and each lane was loaded with 2. Mu.g of reduced and non-reduced samples, respectively. The voltage was 150V for 1 hour. The results show that: the molecular weight of the non-reduced antibody is about 150KD, the molecular weight of the reduced antibody is about 50KD and about 25KD respectively, and the molecular weight characteristics of the non-reduced antibody are met.

The binding of the resulting monoclonal antibodies to recombinant Human TIGIT antigen (Human TIGIT (His), sino Biological, 10917-H08H) was detected by ELISA, which showed binding affinities EC ₅₀ of the 7103-01 and 7103-07 monoclonal antibodies to TIGIT antigen, respectively, were: 0.0647nM and 0.075nM.

Total RNA from the hybridoma cells was extracted for reverse transcription. The obtained cDNA was used as a template, VH and VL genes of a hybridoma antibody were amplified with reference to von Boehmer, L. ,Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.Nat Protoc,2016.11(10):p.1908-1923.,, etc., and the TIGIT murine monoclonal antibody VH and VL sequences were sequenced.

Before humanizing the antibody, we constructed recombinant human mouse chimeric antibody expression plasmid, constructed the V region of hybridoma antibody to chimeric antibody expression vector (pcDNA3.1), i.e. the V region sequence was connected to the expression vector whose constant region was human IgG1 or human lambda/kappa, transiently expressed in HEK293F cells and purified. SDS-PAGE analysis of the purified product showed that: the molecular weight of the non-reduced antibody is about 150kDa, the molecular weight of the reduced antibody is 55kDa and 25kDa respectively, and the molecular weight characteristics of the antibody are met.

Humanized antibody sequences were designed based on murine maternal antibody sequences of numbers 7103-01 (VL: SEQ ID NO:26;VH:SEQ ID NO:27) and 7103-07 (VL: SEQ ID NO:28;VH:SEQ ID NO:29). The method comprises the following specific steps: first using Discovery Studio andAntibody Modeling, constructing a three-dimensional molecular model of the variable region by adopting a homologous modeling method. Next, structural simulations were performed on the parent antibody variable region and CDR, respectively, by comparing the existing antibody structures of the database. Meanwhile, cDNA derived human germline (Germline) sequences with high homology to murine maternal antibodies VH and VL, respectively, were selected for alignment. The IGHV1 with highest homology is selected as a humanized design template for heavy chain VH and sequence design. IGKV1 is selected as a humanized design template by a light chain VL, and a sequence is designed. A humanized anti-TIGIT monoclonal antibody was obtained with the following heavy and light chain sequence combinations:

TABLE 1 combination of different light and heavy chains of humanized antibodies and corresponding numbering

Humanized antibody sources	Light and heavy chain combination mode of humanized antibody	Antibody numbering
			7103-01	huVH4VL1	P03489
7103-07	huVH2VL1	P03479

TABLE 2P 03489 heavy and light chain sequences

TABLE 3P 03479 heavy and light chain sequences

According to the humanized design result, constructing the sequence on a pcDNA3.4 vector, and obtaining the plasmid after PCR, enzyme digestion, connection, transformation, identification, sequencing, comparison and extraction. According to the humanized expression combination, the above plasmid was expressed on demand in ExpiCHO-S cells for 7 days using ExpiCHO-S expression system (Thermo Fisher, A29133), and the expression amount was measured by HPLC on the 6 th day of expression. Finally, purifying by Protein A affinity chromatography to obtain antibody Protein.

To determine whether humanized TIGIT monoclonal antibodies can bind recombinant human TIGIT protein, ELISA tests were performed. The results show that: the humanized monoclonal antibodies P03489 and P03479 of the anti-human TIGIT can bind to the human TIGIT protein with EC ₅₀ of 0.5nM and 0.09nM respectively, which is obviously superior to the positive control Tirun Li Youshan against EC ₅₀ of 0.5nM, thus proving the excellent TIGIT binding performance of the monoclonal antibody of the application.

1.2 Biological film layer optical interferometry (BLI) study of antibody binding Activity to TIGIT

The dissociation constants (Kd) of human and mouse chimeric TIGIT monoclonal antibodies (7103-01 (hFc) and 7103-07 (hFc)) and humanized TIGIT monoclonal antibodies (P03489 and P03479) bound to recombinant human TIGIT were detected with a GATOR (ProbeLife) detection instrument and HFC (LOT#2007065Tray 2), human antibody Capture probes. The monoclonal antibodies were diluted to 30nM in binding buffer (Q buffer [ PBS (10 mM PH7.4) +0.02% Tween 20+0.2% BSA). Recombinant human TIGIT protein was diluted to different concentrations in a 2-fold gradient in Qbuffer, varying between 40nM and 10 nM.

Table 4 shows the BLI detection results. Humanized antibody #P03489 of 7103-01 has a binding affinity Kd to TIGIT comparable to that of its parent human murine chimeric antibody 7103-01 (hFc) of 1.19×10 ^-9 M and 1.73×10 ^-9 M, respectively. The binding affinity of humanized antibody #P03479 of 7103-07 to TIGIT was weaker than that of its parent murine chimeric antibody 7103-07 (hFc) to TIGIT, but its Kd also reached 8.43x10 ^-9 M and 2.96x10 ^-9 M, respectively. Experimental results show that the correlation coefficient R ² of all antibodies in the Global fitting mode is larger than 0.95, meets the requirement of system adaptability, and has reliable results.

TABLE 4 summary of the binding kinetics constants of TIGIT mAbs to recombinant human TIGIT

1.3 Flow cytometry (flow cytometry) studies of human murine chimeric and humanized monoclonal antibodies binding to TIGIT proteins on cell membranes

The CHO-K (CHO-K-TIGIT) cell line stably expressing full-length human TIGIT was constructed as follows: cDNA was cloned into mammalian cell expression vector pcDNA3.1 (Invitrogen) containing the rat glutamine synthetase gene using a plasmid (purchased from Sino Biological) containing full-length human TIGIT CDNA (NM-173799.2), and the above constructed plasmid was transfected into CHO-K cells by electrotransfection (Bio-Rad, gene Pulser Xcell) and after culturing the transfected cells in OptiCHO medium (Invitrogen) for 24-48 hours, the medium was changed to screening medium. The screening medium contained OptiCHO, 5. Mu.g/ml recombinant human insulin and 10. Mu.M methionine amino sulfoxide (MSX). Cells were cultured in an incubator at 37℃with 8% CO ₂. After 3 weeks, the screened cultured cells were flow-sorted (FACS) with anti-TIGIT antibody to give CHO-K monoclonal cell lines expressing human TIGIT on the cell membrane.

The flow cytometer research method for binding TIGIT antibody to cell membrane TIGIT is briefly described as follows: FACS buffer, i.e. 1×pbs+0.5% bsa solution, was first prepared. A desired number of CHO-K-TIGIT cells were obtained, a sample was obtained, the desired cell amount was about 1X 10-5 to 5X 10-5, TIGIT mAbs (7103-01 (hFc), 7103-07 (hFc), P03479 and P03489) and tireli Li Youshan antibody (Tiragolumab, roche, which had entered the three-phase clinical test; used as a positive control sample) were diluted in a predetermined ratio with FACS buffer, the cells were resuspended, the volume was about 100. Mu.L, incubated in a refrigerator at 4℃for 30 minutes, the cells were washed once with FACS buffer, FITC-anti-human IgG (Abcam: 6854) diluted with FACS buffer was incubated in a refrigerator at 4℃for 30 minutes, and finally, after washing the cells with FACS buffer, the cells were resuspended with 200. Mu.L of FACS buffer and examined on the machine.

FIG. 2 shows the results of a flow cytometry study. As shown in FIG. 2A, the TIGIT humanized monoclonal antibody P03479 bound well to CHO-TIGIT, and the EC ₅₀ binding to CHO-TIGIT cell membrane was 0.144 μg/mL (0.963 nM), with higher affinity than the EC ₅₀ 0.201.201 μg/mL (1.347 nM) of the female parent (7103-07 (hFc)) and the EC ₅₀ 0.574.574 μg/mL (3.844 nM) of the tireli Li Youshan antibody. As shown in FIG. 2B, the TIGIT humanized monoclonal antibody P03489 bound well to CHO-TIGIT, and EC ₅₀ bound to the CHO-TIGIT cell membrane was 0.114. Mu.g/mL (0.761 nM), and EC ₅₀ 0.202.202. Mu.g/mL (1.536 nM) of the high affinity Yu Tirui Li Youshan antibody.

1.4 Inhibition of recombinant human TIGIT-CD155 binding by human murine chimeric and humanized TIGIT monoclonal antibodies

To determine whether human murine chimeric and humanized TIGIT monoclonal antibodies can inhibit CD155 binding to TIGIT, an in vitro test was performed using ELISA. Tiarella Li Youshan antibody (Tiragolumab) was used as a positive control. Antigen TIGIT (Human TIGIT (His), sino Biological, 10917-H08H) was diluted with NaHCO ₃ solution at ph=9.6 to 1 μg/mL, 50 μl per well was added to 96-well elisa plates, refrigerator overnight at 4 ℃. After the next day, PBS was washed twice, blocked with 3% BSA, incubated at 37℃for 1.5h at 100. Mu.L per well, and washed 4 times with PBST. The gradient diluted TIGIT monoclonal antibody and CD155-mFC (6. Mu.g/mL) (Human CD155 (Fc Tag): sino Biological, 10109-H02H) were added to the enzyme label plate after the blocking wash, incubated at 37℃for 2H, washed with PBST for 4 times, added with a proportion of diluted secondary anti-HRP-antibody mFC (Jackson Immuno Research, 115-035-164), incubated at 37℃for 1.5H, finally washed with PBST for 4 times, added with TMB color development solution, incubated at 37℃for 10-15min, and the enzyme label read (wavelength 450nm and 655 nm).

Figure 3 shows the results of ELISA studies. As shown, both TIGIT monoclonal antibodies specifically block TIGIT binding to CD 155. The inhibitory activity of humanized mAb P03479, IC ₅₀, was 0.107 μg/mL (0.714 nM), blocking capacity was comparable to or better than the IC ₅₀ value of the human murine chimeric monoclonal antibody (7103-07 (hFc)) of 0.154 μg/mL (1.033 nM), and was significantly better than the tirelin Li Youshan anti-IC ₅₀ value of 0.378 μg/mL (2.534 nM).

1.5 In vitro enhancement of INFgamma production of human PBMC by humanized TIGIT monoclonal antibodies

After T lymphocyte activation, the lymphocytes secrete the cytokine ifnγ. To test the function of TIGIT monoclonal antibodies as positive modulators of T cell activation, we performed the following experiments demonstrating that blocking TIGIT signaling by humanized TIGIT monoclonal antibodies results in enhancement of ifnγ production by PBMC cells. The method is briefly described as follows: resuscitates frozen human peripheral blood mononuclear cells PBMC, adjusts the cell density to 5×10≡6/mL, dilutes the TIGIT antibody to be tested with RPMI 1640+10% FBS medium to a concentration of 20 μg/mL, PHA concentration of 6 μg/mL, adds 50 μl PHA dilution and 50 μl antibody dilution into 96-well plates, finally adds 100 μl cell suspension into 96-well plates, gently mixes them and cultures them in a 5% CO ₂ incubator for 4 days at 37 ℃. After 4 days, 100. Mu.L of the supernatant was aspirated to detect the content of IFNγ.

Fig. 4 shows the detection results. As shown, humanized TIGIT monoclonal antibodies P03479 and P03489 have a promoting effect on IFN gamma release from PHA-activated PBMC. The release amount of IFNγ added with the humanized TIGIT antibodies P03479 and P03489 is 81% and 36% higher than that of the blank (without the humanized TIGIT monoclonal antibody), and the effect is superior to that of tirelin Li Youshan antibody (only 16%).

1.6 Functional studies of humanized TIGIT monoclonal antibodies to inhibit tumor growth in mice in vivo

Since none of the antibodies of the present invention bound to mouse TIGIT, we studied the in vivo anti-tumor effect of the antibodies with human TIGIT gene knock-in mice (C57 BL/6). Human TIGIT gene knockout mice were kept in an SPF-grade environment. The mouse tumor model in this example is colon cancer MC38. The method is briefly described as follows: the mouse colon cancer MC38 tumor cells were from Shanghai, south mode Biotechnology Co., ltd. 24 human TIGIT gene knockout C57/B6 mice 7-9 weeks old were divided into 3 groups (4 male 4 female/group), and 1×10 ⁶ MC38 cells/mouse were injected under the armpit of the mice by subcutaneous inoculation. On the day of inoculation (D0), each group of mice was given PBS (control group), recombinant human IgG 110 mg/kg (control group) and TIGIT humanized antibody P03479 10mg/kg by intraperitoneal injection, respectively. The administration was 2 times per week for a total of 4 times. Tumor volume (i.e., major diameter x minor diameter ²/2) was measured at each dose and the mice were weighed. The test ends at D17. Mice were sacrificed by cervical dislocation and tumor was taken and weighed.

The results of the study are shown in FIG. 5. Fig. 5A shows tumor volume versus time for each mouse of each group, and fig. 5B shows tumor weight for each mouse of each group at the end of the experiment (D17). Experimental results show that TIGIT humanized antibody P03479 was effective in inhibiting tumor growth in mice (fig. 5A and 5B), and that at the end of the experiment (D17), the average weight of tumor in mice in group P03479 was significantly less than that in group hig 1 (P < 0.05).

Example 2 preparation of anti-PD-1/anti-TIGIT bispecific antibody-IL-2 fusion protein (BsAb-IL-2)

2.1. Antibody modification

In order to reduce antibody-mediated ADCC and CDC activities, 2 amino acids L234/L235 (EU numbering) in the CH1 domain of the two heavy chains of the antibody are substituted with amino acid Ala (LALA variant), eliminating the ADCC/CDC function of the antibody.

The anti-TIGIT/anti-PD-1 bispecific antibody was constructed in the form of "knob-in-hole," KIH for short (MERCHANT ET AL (1998) Nat Biotech 16, 677-681), in which mutations S354C and T366W (EU numbering) were introduced in one heavy chain constant region (knob heavy chain), mutations Y349C, T366S, L a and Y407V (EU numbering) were introduced in the other heavy chain constant region (hole heavy chain), and the knob heavy chain and hole heavy chain formed heterodimers, thereby forming an anti-TIGIT/anti-PD-1 BsAbs-IL-2 fusion protein.

To avoid 2 mismatched pairs of heavy and light chains of an antibody, 1 of the heavy and light chains of the antibody was engineered by replacing the CH1 sequence of the heavy chain of the antibody with the constant lambda domain of the light chain of the antibody, fusing the heavy chain variable domain to the constant lambda domain of the light chain of a human antibody, and fusing the light chain variable domain of the antibody to the CH1 sequence of human IgG1 (crossmab).

The genes are all obtained by artificial synthesis.

2.2. Wild-type IL-2 and mutant IL-2

The full-length gene (containing signal peptide sequence, SEQ ID NO: 9) of wild-type human IL-2 (WT) is synthesized artificially. The IL-2 region that binds to the IL-2Rα subunit differs from the IL-2 region that binds to the IL-2Rβ subunit by introducing amino acid mutations into these 2 regions of IL-2 that alter the binding affinity of IL-2 to the IL-2Rα subunit and to the IL-2Rβ subunit. IL-2 mutant (L80F, R81D, L85V, I86V, I F) has an increased affinity for binding to the IL-2Rβ subunit (Levin et al, (2012) Nature 484, 529-533), IL-2 mutant (R38L) has a decreased affinity for binding to the IL-2Rβ subunit (Chappel et al, (1991) Proc.Natl. Acad. Sci. U.S. A.88, 9036-9040), IL-2 mutant (F42A) has a decreased affinity for binding to the IL-2Rα subunit.

IL-2mut is characterized by the following mutations:

a) F42A-attenuation of IL-2/IL-2Rα interactions;

b) L80F, R81D, L85V, I86V, I F-enhancing IL-2/IL-2Rβ interactions;

c) R38L-attenuate IL-2/IL-2Rβ interactions.

2.3. Expression and purification of anti-TIGIT/PD-1 bispecific antibody-IL-2 (R38L, F a) fusion proteins

The IL-2 gene (R38L, F A) (RLFA for short) was ligated to the C-terminus of the heavy chain of PD-1 antibody (in this example, the PD-1 antibody is pembrolizumab) by a linker peptide (G ₄S)₃) by an overlap extension PCR method to construct the HC-IL-2 gene of the PD-1 antibody, the heavy chain of which may be the VH-CH 1-range-CH 2-CH3-linker-IL-2 structure (knob) (SEQ ID NO: 1) or the crossmab structure (knob) (SEQ ID NO: 2) of VH-CL-range-CH 2-CH 3-linker-IL-2. The remaining antibody heavy and light chains were obtained by artificial synthesis.

The heavy and light chain sequence numbers of the TIGIT/PD-1 bispecific antibody-IL-2 fusion protein are shown in Table 5

The heavy and light chain encoding DNA of each BsAb-IL-2 (RLFA) was cloned into expression vector pcdna3.4 (thermosfisher) to generate 4 independent vectors, and then the recombinant plasmids were extracted. The 4 plasmids were co-transfected into 293F cells with a transfection reagent of sinofection (SinoBiological). After cell culture for 7 days, the culture broth was purified from the supernatant by high-speed centrifugation through a Protein A affinity chromatography column to obtain TIGIT/PD-1BsAb-IL-2 (RLFA).

And (3) respectively adding the purified sample into a reduced protein electrophoresis loading buffer solution and a non-reduced protein electrophoresis loading buffer solution, boiling, and performing SDS-PAGE electrophoresis detection.

Experimental results and analysis

Electrophoresis patterns of KD5201A and KD5201B are shown in FIGS. 6A and 6B. Theoretical molecular weights of 2 heavy chains (HC-IL-2 and HC) of KD5301A are 65.7kDa and 49.0kDa, respectively, as indicated by the arrows (FIG. 6A); the theoretical molecular weights of the 2 light chains differ little, 23.4kDa and 23.7kDa, respectively, as indicated by the arrows (FIG. 6A). Theoretical molecular weights of 2 heavy chains (HC-IL-2 and HC) of KD5301B are 67.0kDa and 48.8kDa, respectively, as indicated by the arrows (FIG. 6B); the theoretical molecular weights of the 2 light chains differ by 883Da, and 2 bands are visible on SDS-PAGE gels as indicated by the arrows (FIG. 6B).

2.4SEC-HPLC analysis

Analysis was performed using the Acquity UPLC (Waters) system and protein BEH SEC, 200A, 1.7 μm, 4.6 mm. Times.150 mm, 10K-450K (Waters) was used as SEC column. SEC separation was performed uniformly at ambient temperature using a mobile phase consisting of PBS (ph 7.4 (day Hui Hua). The flow rate was 0.25mL min-1. The relative amounts of BiSpAb monomers, aggregates and fragments were quantified by calculating the peak area detected by an Ultraviolet (UV) detector at 280 nm.

Results display

The peak time of BsAbs-IL2 fusion protein was earlier than that of normal IgG, indicating that the molecular weight of the fusion protein was slightly greater than that of normal IgG, consistent with SDS-PAGE results.

2.5RPC-HPLC analysis

Analysis of the resulting antibody-IL-2 fusion protein by RPC-HPLC

Experimental results and analysis

IL-2 protein has strong hydrophobicity, and fusion protein fused with antibody becomes strong hydrophobic protein. RPC-HPLC results showed that the peak time of BsAbs-IL2 fusion protein was later than that of BsAbs without IL-2, indicating that the BsAbs-IL2 fusion protein became more hydrophobic, consistent with the expected results.

EXAMPLE 3 ELISA Studies of TIGIT/PD-1BsAb-IL-2 (RLFA) binding to antigen and IL-2R

In vitro binding activity of BsAb-IL-2 (RLFA) to its antigens TIGIT and PD-1 was examined by ELISA. The method is briefly described as follows: plates were incubated overnight at 4℃with 50. Mu.l of NaHCO ₃ (pH 9.6) buffer containing 1. Mu.l/mL of recombinant human TIGIT-His or PD-1-His or IL-2Rα subunit or IL-2Rβ subunit. After the next day, PBS was washed twice, blocked with 3% BSA, incubated at 37℃for 1.5h at 100. Mu.L per well, and washed 4 times with PBST. BsAb-IL-2 (RLFA) was diluted in a gradient with binding solution (TBST containing 1% BSA) and added to the blocked washed ELISA plate (50. Mu.l per well) and incubated for 2h at 37 ℃. Washing with PBST for 4 times, adding a certain proportion of diluted secondary alkaline phosphatase labeled antibody hFc (Jackson Immuno Research), incubating at 37 ℃ for 1.5h, finally washing with PBST for 4 times, adding PNPP color development solution, incubating at 37 ℃ for 10-15min, and reading by an enzyme-labeled instrument (wavelength 405nm and 490 nm).

The results show that TIGIT/PD-1BsAb-IL-2 (RLFA) can specifically bind TIGIT or PD-1, and has weak binding activity with IL-2Rα subunit or IL-2Rβ subunit. The above results indicate that fusion of the bispecific antibody to the IL-2 variant does not affect the binding of the bispecific antibody to its antigen, and that the IL-2 variant has the desired low IL-2rα subunit or IL-2rβ subunit binding activity.

EXAMPLE 4 TIGIT/PD-1BsAb-IL-2 (RLFA) binding kinetics study with antigen and IL-2R

The kinetic parameters of binding of bispecific antibodies to TIGIT, PD-1, IL-2 ra subunit and IL-2 ra subunit proteins were detected using biological membrane layer optical interferometry (BLI). The BLI assay instrument was GATOR (ProbeLife), the double antibody was captured with Human antibody Capture probe, and the antigens were HISTIDINE-tagged recombinant human TNFR2 and PD-1, respectively. The method is briefly described as follows: bsAb-IL-2 (RLFA) obtained by purification was diluted to 30nM in a binding buffer (Q buffer [ PBS (10 mM PH7.4) +0.02% Tween 20+0.2% BSA), bsAb-IL-2 was captured with Human antibody Capture probes, recombinant human TIGIT or PD-1 or IL-2Rα subunit or IL-2Rβ subunit proteins were diluted 2-fold in Q buffer to different concentrations, respectively, and varied in the range of 1nM to 100 nM. The captured BsAb-IL-2 (RLFA) sensor was placed in the above serially diluted antigen solution to start kinetic correlation assay.

Experimental results and analysis

The results show that BsAb-IL-2 (RLFA) has high affinity for its antigens TIGIT and PD-1, but has very weak affinity for IL-2Rα and β subunits, much weaker than the affinity of wild-type IL-2 for IL-2Rα and β subunits. These results are consistent with the expected results.

EXAMPLE 5 Sandwich ELISA Studies of TIGIT/PD-1BsAb-IL-2 (RLFA) double binding to antigen

A sandwich ELISA method is used to detect whether TIGIT/PD-1BsAb-IL-2 (RLFA) can bind to both TIGIT and PD-1. To prepare ELISA plates for analysis of bispecific antibody binding, the plates were plated overnight at 4℃with 50. Mu.l NaHCO ₃ (pH 9.6) containing 1. Mu.l/mL recombinant human TIGIT-His. After the next day, PBS was washed twice, blocked with 3% BSA, incubated at 37℃for 1.5h at 100. Mu.L per well, and washed 4 times with PBST. TIGIT/PD-1BsAb-IL-2 (RLFA) was diluted in a gradient with binding solution (TBST containing 1% BSA) and added to the blocked washed ELISA plate (50. Mu.l per well) and incubated for 2h at 37 ℃. After washing 4 times with PBST, recombinant human PD-1-mFc (mouse IgG1 Fc) diluted in binding solution was added to each well and incubated at 37℃for 2h. Washing with PBST for 4 times, adding alkaline phosphatase labeled anti-mouse IgG1 Fc antibody in a certain proportion, incubating at 37 ℃ for 1.5h, finally washing with PBST for 4 times, adding PNPP color development solution, incubating at 37 ℃ for 10-15min, and reading by an enzyme-labeled instrument (wavelength 405nm and 490 nm).

Experimental results and analysis

Sandwich ELISA results showed (fig. 7) that both TIGIT/PD-1BsAb-IL-2 (RLFA) fusion proteins KD5301A and KD5301B were able to bind both TIGIT and PD-1 specifically.

EXAMPLE 6 Sandwich BLI study of TIGIT/PD-1BsAb-IL-2 (RLFA) double binding to antigen

Whether TIGIT/PD-1BsAb-IL-2 (RLFA) can bind to TIGIT and PD-1 simultaneously was detected using biological membrane layer optical interferometry (BLI). The BLI test apparatus is GATOR (ProbeLife), and the method is briefly described as follows: recombinant human PD-1-mFc was captured with an antibody probe against mouse IgG1 Fc, TIGIT/PD-1BsAb-IL-2 (RLFA) (100 nM) was diluted in binding buffer (Q buffer [ PBS (10 mM PH7.4) +0.02%Tween 20+0.2% BSA ], incubated with the probe, the sensor that captured PD-1-mFc bound to the diabody, then recombinant TIGIT-His diluted in binding buffer was incubated with the probe, and TIGIT/PD-1BsAb-IL-2 (RLFA) binding to TIGIT was detected by the sensor.

The results showed that TIGIT/PD-1BsAb-IL-2 (RLFA) was able to bind both TIGIT and PD-1 specifically.

EXAMPLE 7 flow cytometry (flow cytometry) study of TIGIT/PD-1BsAb-IL-2 (RLFA) binding to TIGIT and PD-1 proteins on cell membranes

Stable cell lines expressed on the membrane were constructed using human TIGIT and PD-1 full-length cDNA. The cDNA of the antigen to be expressed is inserted into a mammalian cell expression vector pcDNA3.1 (ThermoFisher), an expression plasmid is transfected into Jurkat cells by an electrotransfection method, the cells are cultured in a medium containing G418 for 2 weeks, and then a flow separation instrument is used for separating single cell strains with high TIGIT or PD-1 expression. Single cell strain is cultivated in 96-well plate, amplified T25 square bottle is cultivated, then the expression of membrane protein is detected by flow, and the stable Jurkat cell strain with high expression is selected.

The flow cytometer research methods for the binding of TIGIT/PD-1BsAb-IL-2 (RLFA) to cell membrane TIGIT or PD-1 are briefly described as follows: FACS buffer, i.e. 1×pbs+0.5% bsa solution, was first prepared. Taking the required number of the constructed cells, diluting the needed diabody with a certain proportion by using a FACS buffer, uniformly mixing the diluted diabody with the cells, incubating the mixture for 30min at the temperature of 4 ℃ by using a FACS buffer, washing the cells once by using the FACS buffer, re-suspending the cells by using FITC-antihuman IgG (Abcam: 6854) diluted by using the FACS buffer, incubating the cells for 30min at the temperature of 4 ℃ by using the FACS buffer, washing the cells by using the FACS buffer, re-suspending the cells by using 200 mu L of the FACS buffer, and detecting the cells by taking the cells on a machine.

Experimental results show that TIGIT/PD-1BsAb-IL-2 (RLFA) can be combined with the specificity of TIGIT and PD-1 on cell membranes with high affinity.

Example 8 affinity detection of TIGIT/PD-1BsAb-IL-2 (RLFA) binding to IL-2Rα subunit and IL-2Rβ subunit on cell membranes in vitro

The binding affinity of TIGIT/PD-1BsAb-IL-2 (RLFA) to IL-2 ra and IL-2rβ subunits on cell membranes was examined using flow cytometry.

First we co-transfected human IL-2Rα, IL-2Rβ and IL-2Rγ expression plasmids (Sinobio) respectively in 293F cells transiently expressed the following IL-2R complex: IL-2Rαβγ (trimer), IL-2Rαγ (dimer), and IL-2Rβγ (dimer). The expression of the corresponding IL-2R subunit on the cell membrane was detected and confirmed with antibodies against IL-2R alpha, IL-2R beta and IL-2R gamma, respectively.

We then examined TIGIT/PD-1BsAb-IL-2 (RLFA) binding activity to the 3 293F cells expressing IL-2R complexes (αβγ, αγ and βγ) described above, respectively, using flow cytometry. The detection method is as described in example 1.3.

The results showed that TIGIT/PD-1BsAb-IL-2 (RLFA) has very low binding activity to IL-2Rαβγ, IL-2Rαγ and IL-2Rβγ.

EXAMPLE 9 inhibition of binding of TIGIT/PD-1BsAb-IL-2 (RLFA) to recombinant human TIGIT to CD155 or PD-1 to PD-L1

The inhibitory activity of TIGIT/PD-1BsAb-IL-2 (RLFA) binding to CD155 or PD-1 to PD-L1 was examined by ELISA. Antigen TIGIT or PD-1 (Sino Biological) was diluted to 1 μg/mL with NaHCO ₃ buffer (pH 9.6), 50 μl per well was added to 96-well elisa plates, refrigerator overnight at 4 ℃. After the next day, PBS was washed twice, blocked with 3% BSA, incubated at 37℃for 1.5h at 100. Mu.L per well, and washed 4 times with PBST. The gradient diluted TIGIT/PD-1BsAb-IL-2 (RLFA) and biotin-labeled recombinant human CD155 or PD-L1 (Sino Biological, the same applies below) with a fixed concentration are added into the enzyme label plate after closed washing after equal volume mixing, incubation is carried out for 2 hours at 37 ℃, the PBST is continuously used for washing for 4 times, alkaline phosphatase-labeled streptavidin with a certain proportion is added, incubation is carried out for 1.5 hours at 37 ℃, finally the PBST is used for washing for 4 times, PNPP color development liquid is added, and after incubation is carried out for 10-15 minutes at 37 ℃, the enzyme label instrument reads (wavelength 405nm and 490 nm).

Experimental results show that TIGIT/PD-1BsAb-IL-2 (RLFA) can effectively inhibit the binding of TIGIT to CD155 and PD-1 to PD-L1.

EXAMPLE 10 inhibition of TIGIT/PD-1BsAb-IL-2 (RLFA) binding of recombinant human CD155 or PD-L1 to TIGIT or PD-1 on cell membranes

The inhibition of the binding of recombinant human CD155 or PD-L1 to TIGIT or PD-1 on the cell membrane was studied by flow cytometry using TIGIT/PD-1BsAb-IL-2 (RLFA). The study procedure is briefly described as follows: FACS buffer, i.e. 1×pbs+0.5% bsa solution, was first prepared. A desired number of membrane TIGIT or PD-1 expressing Jurkat cells (described in example 7), a desired amount of cells ranging from about 1X 10-5 to 5X 10-5 cells were taken, the desired TIGIT/PD-1BsAb-IL-2 (RLFA) was diluted in a gradient with a FACS solution containing biotin-labeled CD155 or PD-L1, the cells were homogenized, the volume was about 100. Mu.L, incubated for 30min at 4℃in a refrigerator, after washing the cells once with FACS buffer, the cells were resuspended with FACS buffer-diluted PE-labeled streptavidin (Abcam: 6854) and incubated for 30min at 4℃in a refrigerator, and finally, after washing the cells with FACS buffer, the cells were resuspended with 200. Mu.L FACS buffer and examined on the machine.

Experimental results show that TIGIT/PD-1BsAb-IL-2 (RLFA) can effectively inhibit the binding of CD155 or PD-L1 with TIGIT or PD-1 on cell membranes.

EXAMPLE 11 functional Studies of in vitro stimulation of CTLL-2 cell proliferation by TIGIT/PD-1BsAb-IL-2 (RLFA)

IL-2 is one of the important growth factors for lymphocyte growth and stimulates the proliferation of a variety of lymphocytes, including mouse T-lymphocyte CTLL-2. CTLL-2 (China food and drug institute) was cultured in RPMI1640 medium (Gibco, 10% FBS (fetal bovine serum, gibco) and recombinant human (rh) IL-2 (Beijing Shuanglu pharmaceutical industry)). On the day before the experiment, 1X 10 ⁴ cells per well were added to 96-well cell culture plates containing 0.2mL of medium (10% FBS, without rhIL-2), then serial dilutions of TIGIT/PD-1BsAb-IL-2 (RLFA) to be assayed were added per well, and after two days of incubation, the cell growth status was examined using the CCK-8 cell growth assay kit. Wild-type recombinant human IL-2 (rhIL-2 (WT), sinoBiological, china) and the fusion protein of TIGIT antibody (P03479) with IL-2 (WT) (mAb-IL-2 (WT)) were used as control samples.

Experimental results show (FIG. 8), KD5301B has an activity EC ₅₀ of 1.7nM for stimulating CTLL-2 cells, whereas rhIL-2 (WT) and mAb-IL-2 (WT) have an activity EC ₅₀ of 0.07nM and 0.08nM, respectively, which is much stronger than KD5301B, indicating that the variant of KD5301 has very weak binding activity to IL-2R, and thus weak functional activity for stimulating lymphocyte growth.

EXAMPLE 12 in vitro Effect of TIGIT/PD-1BsAb-IL-2 (RLFA) on lymphocyte CD4+ and CD8+ growth

CD4+ and CD8+ T lymphocytes were isolated from peripheral blood or spleen of human TIGIT gene and PD-1 gene knockout mice and wild-type mice, respectively, co-cultured in vitro with TIGIT/PD-1BsAb-IL-2 (RLFA) for 3 days, and then examined for the numbers of TIGIT positive and negative, PD-1 positive and negative CD4+ and CD8+ lymphocytes in the gene knockout mice, and the numbers of CD4+ and CD8+ lymphocytes in the wild-type mice. Recombinant human IL-2 (WT) was used as a control.

Experimental results show that TIGIT/PD-1BsAb-IL-2 (RLFA) preferentially stimulated TIGIT-positive and PD-1-positive CD4+ and CD8+ lymphocyte proliferation compared to rhIL-2 (WT), whereas rhIL-2 (WT) stimulated all CD4+ and CD8+ lymphocyte proliferation.

EXAMPLE 13 investigation of in vitro activation of NK cells by TIGIT/PD-1BsAb-IL-2 (RLFA)

NK92 cells with high and low expression of TIGIT were selected from the NK92 cell population by FACS technology, and then were co-cultured with TIGIT/PD-1BsAb-IL-2 (RLFA) for 24 hours, to examine the expression of NK92 cell CD 107. CD107 is a marker of NK92 cell activation, CD107 is not expressed or is under expressed when NK92 cells are in a non-activated state. Upon activation, NK92 cells significantly increased expression of CD 107. Recombinant human IL-2 (WT) was used as a control.

Experimental results show that TIGIT/PD-1BsAb-IL-2 (RLFA) preferentially stimulated CD107 expression of NK92 for high TIGIT expression compared to rhIL-2 (WT), whereas rhIL-2 (WT) did not differentiate between activation of NK92 cells for high and low TIGIT expression.

Example 14 in vitro enhancement of INFγ production by human PBMC

After T lymphocyte activation, the lymphocytes secrete the cytokine ifnγ. To test whether TIGIT/PD-1BsAb-IL-2 could enhance the activity of activated T cells, we performed the following experiment. The method is briefly described as follows: the frozen human peripheral blood mononuclear cells PBMC are resuscitated, the cell density is adjusted to 5×10ζ6/mL, and the cells are cultured in PHA-containing (0.3-3 μg/mL) medium for 2 days to activate lymphocytes. PHA stimulated PBMC were washed with PBS, then incubated with medium containing TIGIT/PD-1BsAb-IL-2 (RLFA) for 4 days, and then the content of IFNγ in the supernatant was examined. Recombinant IL-2 (RLFA), TIGIT/PD-1BsAb and recombinant IL-2 (RLFA) plus TIGIT/PD-1BsAb were used as controls. Recombinant IL-2 (RLFA) and TIGIT/BsAbs preparation methods reference is made to the preparation of antibody-IL 2 fusion proteins of the invention.

Experimental results show that TIGIT/PD-1BsAb-IL-2 (RLFA) has stronger activity of activating T cell IFNγ expression, namely stronger capability of activating T cell functional activity compared with the single action of recombinant IL-2 (RLFA) or TIGIT/BsAbs or the combined action of the two.

EXAMPLE 15 study of the Effect of TIGIT/PD-1BsAb-IL-2 (RLFA) on lymphocyte CD4+ and CD8+ growth in vivo

We studied the stimulatory effects of the TIGIT/PD-1BsAb-IL-2 (RLFA) of the invention on T lymphocyte CD4+ and CD8+ growth in vivo in human TIGIT gene knock-in mice, human PD-1 gene knock-in mice and human TIGIT/PD-1 double gene knock-in mice.

The knock-in C57BL/6 mice (Baioerset) of 8-10 weeks old were randomly grouped according to body weight, 6 mice (male and female halves) of each group were injected with different doses of TIGIT/PD-1BsAb-IL-2 (RLFA), igG1-IL-2 (RLFA) and PBS (control group) of the present invention from the tail vein, respectively. Blood was taken from the eyes after 4 days, fluorescence-labeled anti-mouse CD4 or CD8 antibody and anti-human TIGIT and PD-1 antibody were added, after incubation for 1 hour, the cells were washed 3 times with PBS, and then the fluorescence intensity of each sample was detected with a flow cytometer, and the total cd4+ and cd8+ cells and NK cells content in the blood, and tigit+cd4+, tigit+cd8+, PD-1+cd4+, PD-1+cd8+ and tigit+nk cells content in the blood were calculated.

Experimental results show that TIGIT/PD-1BsAb-IL-2 (RLFA) preferentially stimulated proliferation of TIGIT and PD-1 positive lymphocytes compared to IgG1-IL-2 (RLFA).

EXAMPLE 16 in vivo inhibition of tumor growth Studies

We studied the function of the TIGIT/PD-1BsAb-IL-2 (RLFA) of the invention in inhibiting the growth of homologous tumors in human TIGIT/PD-1 double-gene knock-in mice.

Mice colon cancer cells MC38 were inoculated subcutaneously in 8-10C 57BL/6 mice (Baioser, male and female halves) and randomly grouped according to tumor size (8 mice/group, male and female halves) after about 7 days, and different doses of TIGIT/PD-1BsAb-IL-2 (RLFA), igG1-IL-2 (RLFA), TIGIT/PD-1BsAb or PBS were administered 2 times per week and 4 times in total, respectively, from the tail vein. Tumor volumes (length x width ²/2) were measured 2 times per week. The experiment was ended when the average tumor volume of the PBS group mice was greater than 2000mm ³, and spleen and tumor were weighed.

Experimental results show that the TIGIT/PD-1BsAb-IL-2 (RLFA) has a stronger ability to inhibit tumor growth than the IgG1-IL-2 (RLFA) and the TIGIT/PD-1 BsAb.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

The method comprises the following steps: sequence information

SEQ ID NO. 1: PD-1 antibody (pembrolizumab) heavy chain-linker-IL-2 (R38L, F42A), heavy chain containing LALA variation and S354C and T366W variation (knob heavy chain 1)

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG

GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT

TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST

YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKN

QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

SCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEHLLLDLQMI

LNGINNYKNPKLTLMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLI

SNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

SEQ ID NO. 2: PD-1 antibody (pembrolizumab) heavy chain-linker-IL-2 (R38L, F A), heavy chain variable domain fused to human antibody light chain constant lambda domain and human IgG1 hinge domain-CH 2-CH3 (knob heavy chain 2) containing LALA variation and S354C and T366W variation

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG

GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT

TVTVSSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV

TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAP

EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCR

EEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGGSGGGGSAPTSSSTKKTQLQLEH

LLLDLQMILNGINNYKNPKLTLMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF

HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

SEQ ID NO. 3: PD-1 antibody (pembrolizumab) light chain

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES

GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 4: PD-1 antibody (pembrolizumab) light chain, light chain variable domain fused to CH1 of human IgG1

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES

GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKSSASTKGPSVF

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 5: TIGIT antibody (P03489) heavy chain variable domain (SEQ ID NO: 84) fused to IgG1 Fc (hole heavy chain 1) containing LALA variants and Y349C, T366S, L368A and Y407V variants

QVQLVQSGAEVKKPGASVKVSCKASGYTFISYNIYWVRQAPGQGLEWMGGVNPSNGN

TNFNENFQGRVTMTVDTSISTAYMELSRLRSDDTAVYYCTRGNYYGYEFAYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 6: TIGIT antibody (P03479) heavy chain variable domain (SEQ ID NO: 88) fused to IgG1 Fc (hole heavy chain 2) containing LALA variants and Y349C, T366S, L a and Y407V variants

EVQVVESGGGLVKPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVAEISSGGSHTFYADTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARKTLDYYALDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 7: TIGIT antibody (P03489) light chain

DIQMTQSPSSMSASVGDRVTITCKASQHVSTAVAWYQQKPGKAPKLLIYSPSYRYTGVPSRFSGSGSGTDFTFTISSVQPEDIATYYCQQHYSTPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 8: TIGIT antibody (P03479) light chain

DVQITQSPSYLSASVGDRVTINCRASKSISKYLAWYQQKPGKAPKLLIYSGSRLQSGIPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 9: wild type IL-2 (WT)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

SEQ ID NO. 10-SEQ ID NO. 17: VH, VHCDR1-3, VL and VLCDR1-3 sequences of P03489

See Table 2

18 To 25 per cent of SEQ ID NO: VH, VHCDR1-3, VL and VLCDR1-3 sequences of P03479

See Table 3

SEQ ID NO. 26: VL of murine monoclonal antibody 7103-01

DIVMTQSHKFMSTSVGDRVSITCKASQHVSTAVAWYQQKPGQSPKLLIYSPSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIK

SEQ ID NO. 27: VH of murine monoclonal antibody 7103-01

QVQLQQPGAELVKPGASVKLSCKASGYTFISYNIYWVKQRPGQGLEWIGGVNPSNGNTNFNENFKSKATLTVDKSSSTAYMQLSSLTSDDSAVYFCTRGNYYGYEFAYWGQGTLVTVSA

SEQ ID NO. 28: VL of murine monoclonal antibody 7103-07

DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSRLQSGIPSKFSGSGYGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKVEIK

SEQ ID NO. 29: VH of murine monoclonal antibody 7103-07

EVQVVESGGGLVKPGGTLKLSCAASGFTFSTYAMSWVRQSPEQRLEWVAEISSGGSHTFYSDTVTGRFTISRDNAQNTLYLEMNSLRSEDTAMYYCARKTLDYYALDYWGQGTSVTVSS.

Claims

1. A fusion protein comprising:

(A) TIGIT binding domain comprising heavy chain complementarity determining regions (VH CDRs) 1-3 and/or light chain complementarity determining regions (VL CDRs) 1-3 selected from the group consisting of:

(i) VH CDR1 selected from the group consisting of: 11 or 19;

(ii) VH CDR2 selected from the group consisting of: SEQ ID NO 12 or 20;

(iii) VH CDR3 selected from the group consisting of: 13 or 21;

(iv) VL CDR1 selected from the group consisting of: 15 or 23;

(v) VL CDR2 selected from the group consisting of: 16 or 24;

(vi) VL CDR3 selected from the group consisting of: 17 or 25; and

(B) A PD-1 binding domain; and

(II) a second polypeptide comprising interleukin-2 (IL-2) or a variant thereof having lymphopoietic activity,

Wherein the second polypeptide is fused to the first polypeptide.

2. The fusion protein of claim 1, wherein,

(A) The TIGIT binding domain comprises a combination of VH CDRs 1-3 selected from the group consisting of: SEQ ID NOS 11, 12 and 13; or SEQ ID NOS 19, 20 and 21; and/or the number of the groups of groups,

The TIGIT binding domain comprises a combination of VL CDRs 1-3 selected from the group consisting of: SEQ ID NOS 15, 16 and 17; or SEQ ID NOS.23, 24 and 25

(E.g., the TIGIT binding domain comprises a combination of VH CDRs 1-3 and VL CDRs 1-3 selected from the group consisting of SEQ ID NOs 11, 12 and 13 in combination with SEQ ID NOs 15, 16 and 17;

Or a combination of SEQ ID NOS: 19, 20 and 21 with SEQ ID NOS: 23, 24 and 25);

And/or

(B) The PD-1 binding domain comprises an antigen binding domain of an anti-PD-1 antibody,

For example, the PD-1 binding domain comprises an antigen binding domain of an anti-PD-1 antibody;

For example, the PD-1 binding domain comprises a VL CDR, VH and/or VL selected from pembrolizumab or nivolumab, or a fragment having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity to the foregoing and having PD-1 binding activity, or a combination thereof; and/or

For example, the PD-1 binding domain comprises one or more PD-1 binding fragments selected from SEQ ID NO. 1,2, 3 or 4 or a fragment having at least 80% (e.g., 85%, 90%, 95%, 98%) sequence identity to the aforementioned sequences and having PD-1 binding activity or a combination thereof.

3. The fusion protein of claim 1, wherein the TIGIT binding domain comprises a heavy chain variable region (VH) selected from the group consisting of or an amino acid sequence having at least 80% sequence identity thereto: 10 or 18; and/or

The TIGIT binding domain comprises a light chain variable region (VL) selected from the group consisting of or an amino acid sequence having at least 80% sequence identity thereto: SEQ ID NO. 14 or 22,

(E.g., the TIGIT binding domain comprises a combination of a heavy chain variable region (VH) and a light chain variable region selected from the group consisting of SEQ ID No. 10 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID No. 14 or an amino acid sequence having at least 80% sequence identity thereto;

or a combination of the amino acid sequence of SEQ ID NO. 18 or an amino acid sequence having at least 80% sequence identity thereto and SEQ ID NO. 22 or an amino acid sequence having at least 80% sequence identity thereto.

4. The fusion protein of claim 1, wherein the TIGIT binding domain comprises one or more TIGIT binding fragments selected from the group consisting of: the fragment shown in SEQ ID NO 5,SEQ ID NO:6,SEQ ID NO:7,SEQ ID NO:8, or a fragment having at least 90% (e.g., 95%) sequence identity to the aforementioned sequence and having TIGIT binding activity.

5. The fusion protein of claim 1, wherein the TIGIT binding domain and/or PD-1 binding domain is an antibody fragment selected from the group consisting of: fab fragments; monovalent fragments consisting of VL, VH, CL and CH1 domains; a F (ab) ₂ fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge of a hinge region; fd fragment consisting of VH and CH1 domains; fv fragments consisting of the VL and VH domains of the antibody single arm; a dAb fragment; an isolated CDR; and scFv; and/or

The form of the bispecific antibody is selected from the group consisting of: 1) A fusion protein assembled from antigen binding regions (Fab); (2) symmetrical BsAb; (3) asymmetric BsAb; and/or

The bispecific antibody is a multivalent antibody, e.g., a 2-valent, 3-valent, 4-valent, 5-valent, 6-valent, or higher valent antibody; and/or

The fusion protein has a Knob (KIH) configuration (e.g., the bispecific antibody has a KIH configuration comprising a fragment comprising an antigen binding domain comprising a heavy chain selected from SEQ ID NOs 1,2, 5, or 6, a light chain selected from SEQ ID NOs 3,4, 7, or 8, or any combination thereof) or an IgG-scFv configuration with an scFv fragment linked at the C-terminus of the heavy chain; and/or

The bispecific antibody is a homodimer or multimer, or a heterodimer or multimer.

6. The fusion protein of claim 1, wherein the second polypeptide has one or more characteristics selected from the group consisting of:

(a) The second polypeptide further comprises a signal peptide;

(b) The IL-2 is wild-type IL-2 or a functional fragment thereof, e.g. derived from human, primate, rodent;

(c) The IL-2 variant has an increased binding affinity to the IL-2Rβ subunit and/or the IL-2 variant has a decreased binding affinity to the IL-2Rα subunit compared to wild-type IL-2;

(d) The lymphocyte growth promoting activity of said IL-2 variant is unchanged or enhanced compared to wild-type IL-2;

For example, the IL-2 variant comprises one or more mutations selected from the group consisting of: R38L, F42A, L F, R81D, L V, I86V, I F, for example comprising a combination of mutations: R38L and F42A, wherein the position of the mutation is according to EU numbering.

7. Fusion protein according to claim 1, wherein the first and second polypeptides are directly linked or linked by a linker, e.g. the linker is a glycine linker, such as Gn, or a glycine/serine linker, such as an amino acid sequence of (GS) n, (GGS) n, (GGGS) n, (GGGGS) n or (GGGGGS) n, wherein n is an integer of 1,2,3, 4, 5, 6, 7,8,9 or 10; and/or

The second polypeptide is linked to the N-terminus and/or C-terminus of the antibody heavy chain in the first polypeptide, e.g., at the C-terminus of the PD-1 binding domain antibody heavy chain; and/or

The fusion protein comprises: sequences shown in SEQ ID NOs 1, 3, 5 and 7; or the sequences shown in SEQ ID NOs 2, 4, 6 and 8; or a combination of fragments having at least 80% (e.g., 85%, 90%, 95%) sequence identity to the foregoing sequences and having the same or similar activity.

8. A nucleic acid molecule or combination thereof encoding the fusion protein of any one of claims 1-7.

9. A vector or cell comprising the fusion protein of any one of claims 1-7 and/or the nucleic acid molecule of claim 8 or a combination thereof, e.g., the cell is a Chimeric Antigen Receptor (CAR) cell.

10. A product comprising the fusion protein of any one of claims 1-7 or fragments thereof, the nucleic acid molecule of claim 8 or a combination thereof, the vector or cell of claim 9,

For example, the product is selected from: pharmaceutical compositions, kits, and kits; and/or the number of the groups of groups,

For example, the product is for: production of the bispecific antibody, production of a derivative (e.g. CAR cell) comprising the bispecific antibody, disease treatment and/or disease detection.

11. Use of a fusion protein according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8 or a combination thereof, a vector or cell according to claim 9 or a product according to claim 10 for the preparation of a medicament for the prophylaxis and/or treatment of TIGIT and/or PD-1 overexpression-related disorders,

For example, the TIGIT and/or PD-1 overexpression-related disorder is selected from:

Cancers (e.g., adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma; tumor tissue from adrenal gland, gall bladder, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary gland, spleen, testis, thymus, thyroid, or uterus; tumors of the central nervous system such as glioblastoma, or astrocytoma; eye tumors (e.g., basal cell carcinoma, squamous cell carcinoma, or melanoma), endocrine gland tumors, neuroendocrine system tumors, gastrointestinal pancreatic endocrine system tumors, reproductive system tumors, or head and neck tumors);

infection or infectious diseases, for example; HIV, viral infectious hepatitis, HTLV-1 virus infection, lymphocytic choriomeningitis virus infection, parasitic infection (metacercaria, schistosoma japonicum, plasmodium);

immunodeficiency diseases, such as: type i diabetes, multiple sclerosis, rheumatoid arthritis, celiac disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, idiopathic arthritis, crohn's disease, ulcerative colitis or systemic sclerosis, graft versus host disease, psoriasis, alopecia areata, atopic dermatitis, HCV-induced vasculitis, sjogren's syndrome, pemphigus, ankylosing spondylitis, behcet's disease, wegener's granulomatosis, autoimmune hepatitis, sclerosing cholangitis, gully-ston syndrome and macrophage activation syndrome, autoimmune thyroiditis, autoimmune uveitis, aplastic anemia.