WO2022179004A1

WO2022179004A1 - Fusion protein with triple functions of tumor targeting, anti-cd3 and t-cell activation, and use thereof

Info

Publication number: WO2022179004A1
Application number: PCT/CN2021/099023
Authority: WO
Inventors: 屈向东; 潘琴
Original assignee: 启愈生物技术(上海)有限公司
Priority date: 2020-02-27
Filing date: 2021-06-08
Publication date: 2022-09-01
Also published as: CN113321738A

Abstract

A fusion protein with triple functions of tumor targeting, anti-CD3 and T-cell activation, and the use thereof. The trifunctional fusion protein comprises: an anti-tumor surface antigen (TAA) antibody part, an anti-CD3 antibody part, an IL15/IL15Rα complex part, and a heterodimer Fc part. The trifunctional antibody has a higher affinity and can solve the problem of mismatching of the light chain and the heavy chain of a bispecific antibody. The trifunctional antibody can bind to a T-cell surface antigen (CD3) and a tumor cell surface antigen (TAA) to form an immunological synapse, so that T-cells are directly activated, and cytotoxin or cytokine is accordingly released to kill tumor cells. In addition, the TAA antibody targets a IL15/IL15R complex to reach a tumor microenvironment, so that the proliferation of T-cells and NK cells is promoted, the problem of there being a low number of T-cells in the tumor microenvironment is overcome, the immune system is effectively activated, the tumor-killing effect is enhanced, and the treatment effect on solid tumors is greatly improved.

Description

Tumor targeting, anti-CD3 and T cell activation trifunctional fusion protein and its application

technical field

The invention relates to a tumor targeting, anti-CD3 antibody and T cell activation trifunctional fusion protein and its application, and belongs to the field of biomedical multifunctional antibodies.

Background technique

bispecific antibodies

Bispecific antibodies (BsAbs), also known as bifunctional antibodies, can simultaneously recognize and bind two different antigens and epitopes, and block two different signaling pathways to exert their effects. Compared with ordinary antibodies, BsAb adds a specific antigen binding site, which shows the following advantages in treatment:

Mediating immune cell killing of tumors: An important mechanism of action of bispecific antibodies is to mediate immune cell killing. Bispecific antibodies have two antigen-binding arms, one of which binds to the target antigen, and the other binds to effector cells. Tag antigen binding, which activates effector cells to target and kill tumor cells. The two bispecific antibody products that have been approved for marketing belong to this category. The catumaxomab developed by Trion Pharma can target the tumor surface antigen EpCAM and the T cell surface receptor CD3, while the blinatumomab developed by Micromet and Amgen can simultaneously bind CD19 and CD3. Both achieve the purpose of treating tumors by activating and recruiting killer T cells;

Dual-target signal blocking, exerting unique or overlapping functions, effectively preventing drug resistance: simultaneously combining dual targets and blocking dual signaling pathways is another important mechanism of action of bispecific antibodies. Receptor tyrosine kinases (RTKs) are the largest class of enzyme-linked receptors and play an important regulatory role in the process of cell proliferation, such as the Her family. RTKs are abnormally highly expressed on the surface of tumor cells, leading to malignant proliferation of tumor cells, so they are also important targets for tumor therapy. Single-target monoclonal antibodies against RTKs have been widely used in tumor therapy, however, tumor cells can activate intracellular activation by switching signaling pathways or by homologous or heterodimerization of HER family members themselves or between different members Signal for immune escape. Therefore, using bispecific antibody drugs to block two or more RTKs or their ligands at the same time can reduce the escape of tumor cells and improve the therapeutic effect;

With stronger specificity, targeting and reducing off-target toxicity: The two antigen-binding arms of bispecific antibodies can bind to different antigens. Binding specificity and targeting of cancer cells, reducing side effects such as off-target;

In terms of technology, in 1996, Ridgway et al. invented the "knobs into holes" (KiH) method of using CH3 domain mutation to generate bispecific humanized anti-CD3×CD4. A heterolog is finally formed by replacing a large amino acid (hole) with a small amino acid in one heavy chain of the bispecific antibody and a small amino acid (knob) with a large amino acid in the other heavy chain of the bispecific antibody dimer rather than homodimer. The CH3 mutants that form stable heterodimers were screened using phage display technology. In order to reduce monomer or homodimer by-products, the CH3 domain of the heavy chain was further designed, and interchain disulfide bonds were added to further increase the ratio of heterodimers. This technology can also fuse polypeptides, protein ligands or Ab fragments to both ends of the Fc chain (knob/hole) to generate various types of heterodimeric proteins. Roche Christian Klein Christian Klein Christian et al in 2010 published a new technique of CrossMab that reduces light chain mismatches (www.pnas.org/cgi/doi/10.1073/pnas.1019002108). Anti-angiopoietin-2 (Ang-2) and vascular endothelial growth factor were constructed with intact Fc and antigen binding domains by exchanging the heavy and light chain domains within the Fab region of one of the bispecific antibodies The CrossMab-bsAb of a(VEGF-A) has higher stability and affinity than its parent antibody. The CrossMab format can be further divided into three subtypes, CH1 with CL (CrossMabCH1-CL), VH with VL (CrossMabVH-VL), or VL-CL with VH-CH1 (CrossMabFab). CrossMabs do not require sequence optimization or additional linkers, making them an attractive method for designing novel bsAbs. Can be combined with (knob/hole) to ensure correct pairing of heavy chains.

IL15/IL15Ra

IL-15 is a 14–15kDa long cytokine that is important for NK cell, NKT cell and memory CD8 ⁺ T cell function. The content of IL-15 in the body is very small, but it is transduced and transported to the target cells by combining with its receptor IL-15Rα to produce a complex IL-15 superagonist (IL-15SA) with extremely high biological potency. IL-15SA strongly activates IL-15-responsive cells, especially NK cells, thereby promoting antitumor and antiviral functions.

Researchers first identified IL-15 as a T-lymphocyte growth factor in 1994, it shares approximately 19% sequence homology with IL-2, and many biological properties are very similar. The three-dimensional structure of IL-15 is similar to that of IL-2, consisting of four "up-down-down-down" helical bundles, and other cytokines such as IL-4, IL-7 and IL-9 also contain this conformation. IL-15 acts differently from other cytokines. IL-15 receptor alpha is expressed on IL-15-producing cells (such as macrophages and dendritic cells) and forms IL-15SA with IL-15 to deliver signals to express IL-15Rβ (also known as IL-2Rβ) and common γ chain (shared with IL-2, IL-4, IL-7, IL-9 and IL-21) NK, NKT and memory CD8 ⁺ T cells, very It is possible that this unique presentation confers IL-15 the ability to mediate its unique functions. Mouse IL-15 shares 70% amino acid sequence homology with human IL-15, and human IL-15 and mouse IL-15 also have similar trans-expression patterns, signaling pathways and biological activities. IL-15 is expressed in many cell types and tissues, including monocytes, macrophages, DCs, keratinocytes, fibroblasts, muscle cells, and neural cells. IL-15 plays an important role in innate and adaptive immunity as a pleiotropic cytokine.

Trans-expressed IL-15/IL-15Rα signaling induces the recruitment and activation of JAK1 and JAK3 in response to the β and γ chains expressed on cells, and the activated JAK1 and JAK3 further phosphorylate STAT3 and STAT5. STAT3 and STAT5 are phosphorylated to form homodimers that translocate to the nucleus and promote transcription of target genes. IL-15 signaling stimulates a series of downstream responses that induce cell growth, reduce apoptosis, and enhance activation and metastasis of immune cells. In the absence of high-affinity IL-15Rα, IL-15 alone can also bind to a moderate-affinity (Ka=1.10 ⁹ /M) β, γ receptor complex (IL-15Rβγ), induce other tyrosine kinases ( Such as Lck, Fyn, Lyn, Syk) phosphorylation activation, and interact with PI3K, MAPK pathway. It has been shown that the metabolic checkpoint kinase mTOR can also be activated by high concentrations of IL-15, which is associated with enhanced NK cell proliferation and activation: selective knockout of mTOR results in blocked bone marrow NK cell maturation. The ability of IL-15 to promote NK cell proliferation is partly due to IL-15-mediated aerobic glycolysis, the basal metabolism of NK cells in the absence of IL-15 is low, but by increasing IL-15 concentrations This physiological activity can be significantly enhanced.

Since IL-15 has similar immunological properties to IL-2: it induces the proliferation and survival of T cells, promotes the proliferation and differentiation of NK cells, and induces the production of cytotoxic T lymphocytes. But unlike IL-2, IL-15 has no obvious effect on Treg cells and does not cause capillary leak syndrome in mice or non-human primates (NHP), so compared with IL-2, IL-15 is a better choice for tumor immunotherapy. Rhesus monkey IL-15 (rIL-15) was the first form of IL-15 used in in vivo experiments, and researchers believe that rIL-15 can preferentially bind to cell surface IL-15Rα. Heterodimeric IL-15/IL-15Rα is the native form of IL-15 cleaved from cells and can respond independently of cell-stimulatory interactions, and Novartis is currently developing this form of molecule in solid tumors (NIZ985 ) clinical trials. The RLI developed by Cytune is a fusion protein composed of IL-15 linked to the Sushi domain of IL-15Rα, which acts as a soluble IL-15 agonist.

CD3 target related

T lymphocytes (T lymphocytes) are referred to as T cells, which are lymphoid stem cells derived from bone marrow. After differentiated and matured in the thymus, they are distributed to the immune organs and tissues of the whole body through lymph and blood circulation to exert immune function. T cells are highly efficient killer cells that rapidly destroy virus-infected cells and cancer cells. T cell killing requires the formation of immune synapses, and this process is highly dependent on TCR recognition of complexes formed by MHC molecules on the surface of antigen-presenting cells and the presented antigenic peptides. Activated immune synapses will release cytotoxins and cytokines to kill. In the process of immune synapse formation, limited by the distance between T cells and target cells, bispecific antibody-directed T cells must simulate the formation of immune synapses, and one end can bridge the T cell TCR receptor by targeting CD3. , the other end bridges the target cell by targeting the target cell surface antigen.

It is estimated that the majority of bispecific antibodies (67%) currently in clinical trials are aimed at combating hematological malignancies. In contrast, bispecific or multifunctional antibodies targeting solid tumors deserve further study because of their ability to target normal tissues or other complex factors (including immune-tolerant cancer stroma, neovascular disturbances, penetration of bispecific antibodies) deficiencies) have inevitable adverse effects. (Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics. Cell Mol Immunol. 2020 May;17(5):451-461)

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a new structural form of TAA/CD3/IL15 trifunctional fusion protein, the trifunctional fusion protein comprises: anti-tumor surface antigen (TAA) antibody part, anti-CD3 antibody part, IL15/ IL15Rα complex part, heterodimeric Fc part. The trifunctional fusion protein of the present invention simultaneously realizes the three functions of tumor targeting, T cell activation, and T cell and NK cell proliferation; the cytokine IL15 and IL15Ra have an ultra-high affinity (KD is about 30-100pM), and the use of IL15 and IL15Ra Replacing the CL and CH1 domains in one of the antibody structures respectively solves the mismatch of the light chain of the bispecific antibody, and at the same time resolves the mismatch of the heavy chain through the Fc heterodimer form. Biologically, the TAA/CD3/IL15 trifunctional fusion protein of the present invention, on the one hand, combines with T cell surface antigen (CD3) and tumor cell surface antigen (TAA) to form an immune synapse, thereby directly activating and proliferating T cells, and then Release cytotoxins or cytokines to kill tumor cells. The activation process of this T cell is simple and straightforward, and does not require the presentation of tumor cell antigens to generate specific T lymphocyte clones, so it is not restricted by MHC or HLA. On the other hand, through TAA antibodies targeting IL15/IL15R to the complex to the tumor microenvironment, the IL15/IL15Ra complex promotes the proliferation of T cells and NK cells, overcomes the low number of T cells in the tumor microenvironment, effectively activates the immune system, enhances The killing effect on tumors will greatly improve the therapeutic effect on solid tumors.

The present invention adopts following technical scheme:

A first aspect of the present invention provides a trifunctional fusion protein, characterized in that the trifunctional fusion protein comprises: an anti-tumor surface antigen (TAA) antibody part, an anti-CD3 antibody part, an IL15/IL15Rα complex part, a heterologous two a polymeric Fc portion; wherein the anti-tumor surface antigen antibody portion comprises an antibody that binds at least one TAA antigen.

In some embodiments, the anti-tumor surface antigen (TAA) antibody comprises a variant form thereof, or an antibody derivative, an antigen-binding fragment of TAA.

In some embodiments, the anti-CD3 antibody portion comprises a derivative or polypeptide fragment of an anti-CD3 antibody.

In some embodiments, the IL15 portion of the IL15/IL15Rα complex comprises mutations, truncations and various derivatives capable of binding IL15Ra comprising mutations, truncations and various derivatives capable of binding IL15.

In some embodiments, the trifunctional fusion protein of the present invention, the IL15/IL15Ra complex includes but is not limited to: 1) IL15 and its mutants, truncations and various derivatives that can bind IL15Ra; 2) IL15Ra and its derivatives Mutations, truncations and various derivatives that can bind to IL15;

In some embodiments, IL15/IL15Ra includes the mutations shown in the following table, and the counting method is based on the first amino acid of the IL15 sequence shown in SEQ ID NO: 1 and counted as position 1; IL15Ra shown in SEQ ID NO: 3 The first amino acid in the sequence starts at position 1:

组合 combination	IL15IL15		IL15RaIL15Ra
11	wt wt		D96D96
22	wtwt	D96/P97D96/P97
33	wtwt	D96/P97/A98D96/P97/A98
44	E87CE87C	D96/C97D96/C97
55	E87CE87C	D96/P97/C98D96/P97/C98
66	E87CE87C	D96/C97/A98D96/C97/A98
77	V49CV49C	S40CS40C
88	L52CL52C	S40CS40C
99	E89C E89C		K34CK34C
1010	Q48CQ48C	G38CG38C
1111	E53CE53C	L42CL42C
1212	C42S C42S		A37CA37C
1313	L45CL45C	G38CG38C
1414	L45CL45C	A37CA37C

In some embodiments, the IL15-related mutations shown in the following table are included (counting is based on the first amino acid of IL15 shown in SEQ ID NO: 1 starting at position 1):

组合 combination		IL15突变IL15 mutation
11	N1D N1D
22	N4D N4D
33	D8N D8N
44	D30N D30N
55	D61ND61N
66	E64QE64Q
77	N65DN65D
88	Q108EQ108E
99	N1D/D61NN1D/D61N
1010	N1D/E64QN1D/E64Q
1111	N4D/D61NN4D/D61N
1212	N4D/E64QN4D/E64Q
1313	D8N/D61ND8N/D61N
1414	D8N/E64QD8N/E64Q
1515	D61N/E64QD61N/E64Q
1616	E64Q/Q108EE64Q/Q108E
1717	N1D/N4D/D8NN1D/N4D/D8N
1818	D61N/E64Q/N65DD61N/E64Q/N65D
1919	N1D/D61N/E64Q/Q108EN1D/D61N/E64Q/Q108E
2020	N4D/D61N/E64Q/Q108EN4D/D61N/E64Q/Q108E

In some embodiments, the anti-TAA antibody and the anti-CD3 antibody comprise variable regions VL and VH, respectively, and a pair of disulfide bonds exists between the VL and VH of the anti-TAA antibody and/or anti-CD3 antibody, comprising the following Mutation combinations, according to EU counts.

In some embodiments, the heterodimeric Fc comprises differently mutated A, B chains; the A, B chains have the following combinations of mutations, counted according to EU:

In some embodiments, the Fc segment comprises Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc and mutants thereof; the A and B chains of the Fc segment, one of which is capable of binding protein A, The other chain is a mutant unable to bind protein A, which contains H435R or H435R/Y436F, according to EU counts.

In some embodiments, the Fc segment is in a form that eliminates the immune effect, comprising a combination of the following mutations:

In some embodiments, the anti-CD3 antibody comprises OKT3, SP34, UCTH1 and derivatives thereof or other CD3 binding antibodies, antibody fragments.

In some embodiments, the anti-TAA antibody is a monovalent or multivalent molecule, and the antigen or antigen-specific mutation to which the anti-TAA antibody binds comprises CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2 , CD24, CD47, CD133, CD217, CD239, CD274, CD276, PD-1, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, IL-6R, IL-2R, CCR4, VEGFR-2, CD6, integration α4, PDGFRα, NeuGcGM3, IL-4Rα, IL-6Rα.

In some embodiments, the anti-TAA antibody or anti-CD3 antibody is a chimeric, humanized or fully human antibody.

In some embodiments, the anti-TAA antibody, anti-CD3 antibody, IL15/IL15Rα, heterodimeric Fc are linked by a linker sequence, which is a low immunogenic amino acid sequence.

The anti-TAA antibody and the variable region VL and VH of the anti-CD3 antibody of the present invention and IL15 or IL15Rα have various permutations and combinations.

In some embodiments, the trifunctional fusion protein has the structure shown in formula I:

Chain 1: [VL1A/VH1A], [antiCD3VH/VL], [IL15/IL15Ra] fusion;

Chain 2: [antiCD3VL/VH], [VH1A/VL1A], [IL15Ra/IL15] fusion, C-terminal fusion [Fc (heterodimer A chain or B chain)]

Chain 3: [VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[CL]

(Formula I)

The chain 1: TAA-1 binding antibody variable region VL1A or VH1A and CD3 binding antibody variable region VL or VH and IL15 or IL15Ra fusion; the chain 2: CD3 binding antibody variable region VH or VL and The antibody variable region VH1A or VL1A that binds to TAA-1 is fused with IL15Ra or IL15, and the C-terminal is fused to the Fc of the heterodimer; the chain 3: from the N-terminal to the C-terminal, the antibody variable that binds to TAA-2 Region VH1B, CH1 fragment, Fc that can form heterodimers; said chain 4: C-terminal fusion CL fragment of antibody variable region VL1B that binds TAA-2.

In some embodiments, the trifunctional fusion protein has the structure shown in formula II:

Strand 1: [VL1A/VH1A], [IL15/IL15Ra] fusion

Chain 2: [VH1A/VL1A], [IL15Ra/IL15] fusion, C-terminal fusion [Fc (heterodimeric A chain or B chain)]

Chain 3: [VL1B/VH1B], [antiCD3VH/VL] fusion, C-terminal fusion [CH1]-[Fc (heterodimeric A chain or B chain)]

Strand 4: [antiCD3VL/VH], [VH1B/VL1B] fusion, C-terminal fusion [CL]

(Formula II)

The chain 1: TAA-1 binding antibody variable region VL1A or VH1A fused to IL15 or IL15Ra; the chain 2: TAA-1 binding antibody variable region VH1A or VL1A fused to IL15Ra or IL15; at its C-terminus Fusion can form a heterodimer Fc; the chain 3: TAA-2 binding antibody variable region VH1B or VL1B and CD3 binding antibody variable region VL or VH fusion, fused at its C-terminus [CH1]- [Fc (heterodimeric A chain or B chain)]; chain 4: fusion of TAA-2-binding antibody variable region VL1B or VL1B and CD3-binding antibody variable region VH or VL, C-terminal fusion of CL fragment.

In some embodiments, the trifunctional fusion protein has the structure shown in formula III:

Strand 1: [antiCD3VH/VL], [IL15/IL15Ra] fusion

Chain 2: [antiCD3VH/VL], [IL15Ra/IL15] fusion, C-terminal fusion [Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1]-[CL]

(Formula III)

Said chain 1: fusion of CD3-binding antibody VL or VH with IL15 or IL15Ra; said chain 2: fusion of CD3-binding antibody VH or VL with IL15Ra or IL15, and fusion of heterodimeric Fc at its C-terminus; The chain 3: from the N-terminus to the C-terminus, the TAA-binding antibody variable region VH1 is fused to the CH1 fragment, and the C-terminus is fused with an Fc that can form a heterodimer to form an antibody heavy chain; the chain 4: binds TAA The C-terminus of the antibody variable region VL1 is fused to the CL fragment to form the antibody light chain.

In some embodiments, the trifunctional fusion protein has the structure shown in formula IV:

Chain 1: [VL1/VH1], [antiCD3VH/VL] fusion, C-terminal link [IL15/IL15Ra];

Chain 2: [antiCD3VL/VH], [VH1/VL1] fusion, C-terminal link [IL15Ra/IL15]-[Fc (heterodimer A chain or B chain)]

Chain 3: [Fc (heterodimeric A chain or B chain)]

(Formula IV)

The chain 1: TAA-binding antibody variable region VL1 or VH1 and CD3-binding antibody variable region VL or VH fusion, C-terminal is connected to IL15 or IL15Ra; the chain 2: CD3-binding antibody variable region VH or VL It is fused to the variable region VH1 or VL1 of the antibody that binds to TAA, and the C-terminus is connected to [IL15Ra/IL15]-[Fc (heterodimer A chain or B chain)]; the chain 3: the Fc of the heterodimer chain A or chain B.

In some embodiments, the trifunctional fusion protein of the present invention includes the following forms:

Form one:

Contains the following 4 chains:

Chain 1: [VL1A/VH1A]-[antiCD3VH/VL]-[IL15/IL15Ra] or

[antiCD3VH/VL]-[VL1A/VH1A]-[IL15/IL15Ra],

Chain 2: is [VH1A/VL1A]-[antiCD3VL/VH]-[IL15Ra/IL15]-[Fc (heterodimeric A chain or B chain)] or

[antiCD3VL/VH]-[VH1A/VL1A]-[IL15Ra/IL15]-[Fc (heterodimeric A chain or B chain)]

Wherein VH1A and VL1A are antibody variable regions that bind antigen-1,

The three domains at the N-terminus of chain 2 are sequentially combined with the corresponding domains of chain 1, and fused at the C-terminus of chain 2 to form a heterodimeric Fc;

Chain 3: [VH1B]-[CH1]-[Fc (heterodimer B chain or A chain)], the antibody variable region VH1B that binds to antigen-2 is fused to the CH1 fragment, and the C-terminal fusion can form a heterologous The Fc of the source dimer forms the antibody heavy chain;

Chain 4: is [VL1B]-[CL], and the C-terminal of VL1B of the antibody variable region of antigen-2 is fused to the CL fragment to form an antibody light chain.

Form two:

Contains the following 4 chains:

Chain 1: [VL1A/VH1A]-[IL15/IL15Ra]-[antiCD3VH/VL] or

[antiCD3VH/VL]-[IL15/IL15Ra]-[VL1A/VH1A]

Chain 2: is [VH1A/VL1A]-[IL15Ra/IL15]-[antiCD3VL/VH]-[Fc (heterodimeric A chain or B chain)] or

[antiCD3VL/VH]-[IL15Ra/IL15]-[VH1A/VL1A]-[Fc (heterodimeric A chain or B chain)]

Wherein VH1A and VL1A are antibody variable regions that bind antigen-1,

The three domains at the N-terminus of chain 2 are sequentially combined with the corresponding domains on chain 1; re-fusion at the C-terminus of chain 2 can form a heterodimeric Fc;

Chain 4: is [VL1B]-[CL], and the C-terminal of VL1B of the antibody variable region of antigen-2 is fused to the CL fragment to form an antibody light chain. Form three:

Includes the following 4 chains:

Chain 1 is [IL15/IL15Ra]-[VL1A/VH1A]-[antiCD3VH/VL] or

[IL15/IL15Ra]-[antiCD3VH/VL]-[VL1A/VH1A]

Chain 2 is [IL15Ra/IL15]-[VH1A/VL1A]-[antiCD3VL/VH]-[Fc (heterodimeric A chain or B chain)] or

[IL15Ra/IL15]-[antiCD3VL/VH]-[VH1A/VL1A]-[Fc (heterodimeric A chain or B chain)]

Wherein VH1A and VL1A are antibody variable regions that bind antigen-1,

The three domains at the N-terminus of chain 2 are sequentially combined with the corresponding three domains on chain 1; re-fusion at the C-terminus of chain 2 can form a heterodimeric Fc;

Chain 4: is [VL1B]-[CL], and the C-terminal of VL1B of the antibody variable region of antigen-2 is fused to the CL fragment to form an antibody light chain. Form four:

Contains the following 3 chains:

Chain 1: [VL1A/VH1A]-[antiCD3VH/VL]-[IL15/IL15Ra] or

[antiCD3VH/VL]-[VL1A/VH1A]-[IL15/IL15Ra],

Wherein VH1A and VL1A are antibody variable regions that bind antigen-1,

Chain 3: [Fc (heterodimeric B chain or A chain)], forms a heterodimer with the Fc chain of chain 2.

Form five:

Includes the following 3 chains:

Chain 1: [VL1A/VH1A]-[IL15/IL15Ra]-[antiCD3VH/VL] or

[antiCD3VH/VL]-[IL15/IL15Ra]-[VL1A/VH1A]

Wherein VH1A and VL1A are antibody variable regions that bind antigen-1,

Form six:

Contains the following 3 chains:

Chain 1: [IL15/IL15Ra]-[VL1A/VH1A]-[antiCD3VH/VL] or

[IL15/IL15Ra]-[antiCD3VH/VL]-[VL1A/VH1A]

Chain 2: is [IL15Ra/IL15]-[VH1A/VL1A]-[antiCD3VL/VH]-[Fc (heterodimeric A chain or B chain)] or

Wherein VH1A and VL1A are antibody variable regions that bind antigen-1;

Form seven:

Includes the following 4 chains:

Strand 1: [antiCD3VH/VL]-[IL15/IL15Ra]

Chain 2: [antiCD3VL/VH]-[IL15Ra/IL15]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[CL]

in:

Chain 1: formed by the fusion of the variable region VL or VH of the CD3-binding antibody to the N-terminus of IL15 or IL15Ra;

Chain 2: The VL or VH of the antibody variable region that binds to CD3 is fused to the N-terminus of IL15 or IL15Ra, and the C-terminus of IL15 or IL15Ra is fused to form a heterodimeric Fc;

Chain 3: The antibody variable region VH1B that binds to the antigen is fused to the CH1 fragment, and the C-terminus of the CH1 fragment is fused to form a heterodimeric Fc to form the antibody heavy chain;

Chain 4: C-terminal fusion of the CL fragment of the antibody variable region VL1B that binds the antigen to form an antibody light chain,

Form eight:

Contains the following 4 chains:

Strand 1: [IL15/IL15Ra]-[antiCD3VH/VL]

Chain 2: [IL15Ra/IL15]-[antiCD3VL/VH]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[CL]

in:

Chain 1: IL15 or IL15Ra is fused to the N-terminus of the variable region VL or VH of the CD3-binding antibody;

Chain 2: IL15 or IL15Ra is fused to the N-terminus of the variable region VL or VH of the CD3-binding antibody, and fused to the C-terminus of the variable region VL or VH of the CD3-binding antibody to form a heterodimeric Fc;

Chain 3: The antibody variable region VH1B that binds the antigen is fused to the N-terminus of the CH1 fragment, and the C-terminus of the CH1 fragment is fused to form a heterodimeric Fc;

Chain 4: C-terminal fusion CL fragment of antigen-binding antibody variable region VL1B,

Chains

3 and 4 of form nine to form twelve are fused with antibody light chain constant region CL or heavy chain constant region CH1- The composition of CH2-CH3 is as follows:

Form nine:

Contains the following 4 chains:

Strand 1: [VL1A/VH1A]-[IL15/IL15Ra];

Chain 2: [VH1A/VL1A]-[IL15Ra/IL15]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B/VL1B]-[antiCD3VL/VH]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [antiCD3VH/VL]-[VL1B/VH1B]-[CL]

Chain 1: IL15 or IL15Ra is fused to the C-terminus of the variable region VL1A or VH1A of an antibody that binds antigen-1;

Chain 2: IL15Ra or IL15 is fused to the C-terminus of the antibody variable region VH1A or VL1A that binds antigen-1; Fc that can form a heterodimer is re-fused at its C-terminus;

Chain 3: The C-terminus of the antibody variable region VH1B or VL1B that binds to antigen-2 is fused to the antibody variable region VL or VH that binds to CD3, and the C-terminus is fused to the CH1 fragment and the Fc that can form a heterodimer;

Chain 4: The C-terminus of VH or VL of the antibody variable region that binds to CD3 is fused to the variable region VL1B or VH1B of the antibody that binds antigen-2, and the CL fragment is fused to the C-terminus of the variable region VL1B or VH1B of the antibody that binds antigen-2 ,

Form ten:

Contains the following 4 chains:

Strand 1: [IL15/IL15Ra]-[VL1A/VH1A];

Chain 2: [IL15Ra/IL15]-[VH1A/VL1A]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1A/VL1B]-[antiCD3VL/VH]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [antiCD3VH/VL]-[VL1B/VH1B]-[CL]

Chain 1: The C-terminal fusion of IL15 or IL15Ra binds the antibody variable region VL1A or VH1A of antigen-1;

Chain 2: The C-terminus of IL15Ra or IL15 is fused to the antibody variable region VH1A or VL1A of antigen-1; the C-terminus of IL15Ra or IL15 is fused to form a heterodimer Fc;

Chain 4: The C-terminus of VH or VL of the antibody variable region that binds to CD3 is fused to the variable region VL1B or VH1B of the antibody that binds antigen-2, and the CL fragment is fused to the C-terminus of the variable region VL1B or VH1B of the antibody that binds antigen-2 ;

Form Eleven:

Contains the following 4 chains:

Strand 1: [VL1A/VH1A]-[IL15/IL15Ra];

Chain 2: [VH1A/VL1A]-[IL15Ra/IL15]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [antiCD3VL/VH]-[VH1B/VL1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B/VH1B]-[antiCD3VH/VL]-[CL]

Chain 3: The C-terminus of the variable region VL or VH of the antibody that binds to CD3 is fused to the variable region VH1B or VL1B of the antibody that binds to antigen-2, and the C-terminus is fused to the CH1 fragment and the Fc that can form a heterodimer;

Chain 4: The C-terminus of the variable region VL1B or VH1B of the antibody that binds to antigen-2 is fused to the variable region VH or VL of the antibody that binds to CD3, and the CL fragment is fused to the C-terminus of the variable region VH or VL of the antibody that binds to CD3,

Form twelve:

Contains the following 4 chains:

Strand 1: [IL15/IL15Ra]-[VL1A/VH1A];

Chain 2: [IL15Ra/IL15]-[VH1A/VL1A]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [antiCD3VL/VH]-[VH1A/VL1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B/VH1B]-[antiCD3VH/VL]-[CL]

Chain 4: The C-terminus of the variable region VL1B or VH1B of the antibody that binds to antigen-2 is fused to the variable region VH or VL of the antibody that binds to CD3, and the CL fragment is fused to the C-terminus of the variable region VH or VL of the antibody that binds CD3.

Chains

3 and 4 of Form XIII to Form XVI consist of a CD3-binding antibody variable region and an antigen-2-binding antibody variable region fused in DVD-Ig format with antibody light chain constant region CL or heavy chain constant region CH1- The composition of CH2-CH3 is as follows:

Form Thirteen:

Contains the following 4 chains:

Strand 1: [VL1A/VH1A]-[IL15/IL15Ra];

Chain 2: [VH1A/VL1A]-[IL15Ra/IL15]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[antiCD3VH]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[antiCD3VL]-[CL]

Chain 3: The C-terminus of the antibody variable region VH1B that binds to antigen-2 is fused to the antibody variable region VH that binds to CD3, and the CH1 fragment and the Fc that can form a heterodimer are fused to its C-terminus;

Chain 4: The C-terminus of the antibody variable region VL1B that binds to antigen-2 is fused to the CD3-binding antibody variable region VL, and the CL fragment is fused to the C-terminus of the CD3-binding antibody variable region VL,

Form Fourteen:

Contains the following 4 chains:

Strand 1: [IL15/IL15Ra]-[VL1A/VH1A];

Chain 2: [IL15Ra/IL15]-[VH1A/VL1A]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[antiCD3VH]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[antiCD3VL]-[CL]

Form fifteen:

Contains the following 4 chains:

Strand 1: [VL1A/VH1A]-[IL15/IL15Ra];

Chain 2: [VH1A/VL1A]-[IL15Ra/IL15]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [antiCD3VH]-[VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Strand 4: [antiCD3VL]-[VL1B]-[CL]

Chain 3: The C-terminus of the antibody variable region VH that binds to CD3 is fused to the antibody variable region VH1B that binds to antigen-2, and the CH1 fragment and the Fc that can form a heterodimer are fused to its C-terminus;

Chain 4: The C-terminus of the variable region VL of the antibody that binds to CD3 is fused to the variable region VL1B of the antibody that binds to antigen-2, and the CL fragment is fused to the C-terminus of the variable region VL1B of the antibody that binds antigen-2,

Form sixteen:

Contains the following 4 chains:

Strand 1: [IL15/IL15Ra]-[VL1A/VH1A];

Chain 2: [IL15Ra/IL15]-[VH1A/VL1A]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [antiCD3VH]-[VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Strand 4: [antiCD3VL]-[VL1B]-[CL]

Chain 4: The C-terminus of the variable region VL of the antibody that binds to CD3 is fused to the variable region VL1B of the antibody that binds to antigen-2, and the CL fragment is fused to the C-terminus of the variable region VL1B of the antibody that binds to the antigen-2.

In some embodiments, chain 1 and chain 2 of the trifunctional fusion protein comprise the combinations described in the following table:

In some embodiments, the trifunctional fusion protein has the form:

Strand 1: [VL1A]-[antiCD3VH]-[IL15]

Chain 2: [VH1A]-[antiCD3VL]-[IL15Ra]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[CL]

in:

Chain 1: The antigen-binding antibody variable region VL1A and the CD3-binding antibody variable region VH are sequentially fused to the N-terminus of IL15;

Chain 2: The antigen-binding antibody variable region VH1A and the CD3-binding antibody variable region VL are integrated at the N-terminus of IL15Ra, and fused at the C-terminus of IL15Ra to form a heterodimeric Fc;

Chain 4: C-terminal fusion of the CL fragment of the variable region VL1B of the antigen-binding antibody to form an antibody light chain.

In some embodiments, the trifunctional fusion protein of the present invention also has the following forms:

Strand 1: [VL1A]-[antiCD3VH]-[IL15]

Chain 2: [VH1A]-[antiCD3VL]-[IL15Ra]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [Fc (heterodimeric A chain or B chain)]

in:

Chain 1: The antigen-binding antibody variable region VL1A and the CD3-binding antibody variable region VH are fused at the N-terminus of IL15;

Chain 2: The N-terminus of the variable region VL of the antibody that binds to CD3 is fused to the variable region VH1A of the antibody that binds to the antigen, and the C-terminus of the variable region VL of the antibody that binds to CD3 is fused to IL15Ra, which can form a heterologous two. Aggregate Fc;

Chain 3: Fc that forms a heterodimer.

In some embodiments, the trifunctional fusion protein comprises the following 4 chains:

Strand 1: [antiCD3VL]-[IL15]

Chain 2: [antiCD3VH]-[IL15Ra]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[CL]

in:

Chain 1: The VL of the antibody variable region that binds to CD3 is fused to the N-terminus of IL15;

Chain 2: The N-terminus of IL15Ra is fused to the CD3-binding antibody variable region VH, and its C-terminus is fused to an Fc that can form a heterodimer;

Chain 3: The antibody variable region VH1B that binds to the antigen is fused to the CH1 fragment, and the C-terminus is fused to the Fc that can form a heterodimer to form the antibody heavy chain;

In some embodiments, the trifunctional fusion protein also has the following characteristics:

Chain 3 and chain 4 consist of antibody variable regions that bind CD3 and antibody variable regions that bind antigen-2 fused in the form of diabody diabodies with antibody light chain constant regions CL or heavy chain constant regions CH1-CH2-CH3, including but not limited to Not limited to the following forms:

chain 1: [VL1A]-[IL15];

Chain 2: [VH1A]-[IL15Ra]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VL1B/VH1B]-[antiCD3VH/VL]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [antiCD3VH/VL]-[VH1B/VL1B]-[CL]

Chain 1: IL15 is fused to the C-terminus of the variable region VL1A of the antibody that binds Antigen-1;

Chain 2: IL15Ra is fused to the C-terminus of the antibody variable region VH1A that binds antigen-1; Fc that can form a heterodimer is re-fused at its C-terminus;

Chain 4: The C-terminus of the VH or VL antibody variable region that binds to CD3 is fused to the antigen-2-binding antibody variable region VL1B or VH1B, and the CL fragment is fused to its C-terminus.

In some embodiments, chain 3 and chain 4 of the trifunctional fusion protein are fused to an antibody light chain constant region CL or heavy antibody variable region from a CD3-binding antibody variable region and an antigen-2-binding antibody variable region in a DVD-Ig format Chain constant region CH1-CH2-CH3 composition, including but not limited to the following forms:

Contains the following 4 chains,

chain 1: [VL1A]-[IL15];

Chain 2: [VH1A]-[IL15Ra]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[antiCD3VH]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[antiCD3VL]-[CL]

Chain 3: The C-terminus of the antibody variable region VH1B that binds to antigen-2 is fused to the antibody variable region VH that binds to CD3, and the C-terminus is fused to the CH1 fragment and the Fc that can form a heterodimer;

Chain 4: Antigen-2-binding antibody variable region VL1B is fused to the C-terminus of the CD3-binding antibody variable region VL, and a CL fragment is fused to its C-terminus.

In some embodiments, the TAA-binding antibodies include, but are not limited to, anti-CD20, CD19, CD38, CD40, CD30, CD33, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, PD-1, etc. surfaces Antibodies to antigens, or CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, Antibodies against targets such as 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, glypican-3, etc.

In some embodiments, the TAA of the trifunctional fusion protein is CLDN18.2.

In some embodiments, the TAA includes EGFR and folate receptor alpha.

In some embodiments, TAA-1 and TAA-2 of the trifunctional fusion protein are both CLDN18.2; the Fc is IgG4 subtype; the IL15 and IL15Ra complex fusion proteins of the chain 1 and chain 2 There are disulfide bonds formed.

In some embodiments, the 4 chains of the trifunctional fusion protein comprise the sequences SEQ ID NO:36, SEQ ID NO:35, SEQ ID NO:34, SEQ ID NO:33; or SEQ ID NO:38, SEQ ID NO:37, SEQ ID NO:34, SEQ ID NO:33; or SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:34, SEQ ID NO:33; or SEQ ID NO:42, SEQ ID NO:41, SEQ ID NO:34, SEQ ID NO:33.

In some embodiments, the TAA-1 and TAA-2 are one of EGFR and folate receptor alpha, respectively; Fc is an IgG4 heterodimer; the IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have two Sulfur bond formation.

In some embodiments, the 4 chains of the trifunctional fusion protein comprise the sequences SEQ ID NO:46, SEQ ID NO:45, SEQ ID NO:44, SEQ ID NO:43; or SEQ ID NO:48, SEQ ID NO:47, SEQ ID NO:44, SEQ ID NO:43; or SEQ ID NO:50, SEQ ID NO:49, SEQ ID NO:44, SEQ ID NO:43; or SEQ ID NO:52, SEQ ID NO:51, SEQ ID NO:44, SEQ ID NO:43; or SEQ ID NO:56, SEQ ID NO:55, SEQ ID NO:54, SEQ ID NO:53; or SEQ ID NO:58, SEQ ID NO:57, SEQ ID NO:54, SEQ ID NO:53; or SEQ ID NO:60, SEQ ID NO:59, SEQ ID NO:54, SEQ ID NO:53; or SEQ ID NO:62, SEQ ID NO:61, SEQ ID NO:54, SEQ ID NO:53.

In some embodiments, the TAA of the trifunctional fusion protein is CLDN18.2; the Fc is of the IgG4 subtype; and the IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bond formation.

In some embodiments, the three chains of the trifunctional fusion protein comprise the sequences of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:63; or SEQ ID NO:67, SEQ ID NO:66, SEQ ID NO:63 ID NO:63; or SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:63; or SEQ ID NO:71, SEQ ID NO:70, SEQ ID NO:63.

In some embodiments, the trifunctional fusion protein is arranged from the N-terminus to the C-terminus, and it has the structure shown in formula V:

Strand 1: [antiCD3VL]-[IL15]

Chain 2: [antiCD3VH]-[IL15Ra]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1]-[CL]

(Formula V)

Wherein the VH1 and VL1 are variable regions of anti-TAA antibodies.

In some embodiments, the TAA is CLDN18.2; the Fc is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bond formation, and the 4 chains comprise the sequence SEQ ID NO. :72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75.

In some embodiments, the trifunctional fusion protein chain 3 and chain 4 are composed of a CD3-binding antibody variable region and a TAA-2-binding antibody variable region fused to heavy chain constant regions CH1-CH2 in the form of a diabody diabody -CH3 or antibody light chain constant region CL composition.

In some embodiments, the TAA-1 and TAA-2 of the trifunctional fusion protein are both CLDN18.2; the Fc is IgG4 subtype; the IL15 and IL15Ra complex fusion proteins of the chain 1 and chain 2 There is disulfide bond formation; the trifunctional fusion protein comprises the sequence SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:77, SEQ ID NO:76; or SEQ ID NO:79, SEQ ID NO: 78, SEQ ID NO:81, SEQ ID NO:80; or SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:83, SEQ ID NO:82; or SEQ ID NO:79, SEQ ID NO: 78. SEQ ID NO:85, SEQ ID NO:84.

In some embodiments, the 3 and chain 4 of the trifunctional fusion protein are composed of a CD3-binding antibody variable region and a TAA-2-binding antibody variable region fused to the heavy chain constant regions CH1-CH2- CH3 or antibody light chain constant region CL is composed of the structure shown in formula VI:

Strand 1: [VL1A/VH1A]-[IL15/IL15Ra];

Chain 2: [VH1A/VL1A]-[IL15Ra/IL15/]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[antiCD3VH]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[antiCD3VL]-[CL]

(Form VI)

Wherein, the chain 1: the C-terminus of the antibody variable region VL1A or VH1A that binds to TAA-1 is fused to IL15 or IL15Ra; the chain 2: the C-terminus of the antibody variable region VH1A or VL1A that binds to TAA-1 is fused to IL15Ra or IL15, whose C-terminus can be re-fused to form a heterodimeric Fc; the chain 3: the C-terminus of the antibody variable region VH1B that binds to TAA-2 is fused to the antibody variable region VH that binds to CD3, and the C-terminus is re-assembled. The CH1 fragment and the Fc that can form a heterodimer are fused; the chain 4: the C-terminus of the antibody variable region VL1B that binds TAA-2 is fused to the variable region VL of the CD3-binding antibody, and the CL fragment is fused to its C-terminus.

In some embodiments, TAA-1 and TAA-2 of the trifunctional fusion protein are both CLDN18.2; the Fc is an IgG4 heterodimer; the IL15 and IL15Ra of the chain 1 and chain 2 are complexed The fusion protein has disulfide bond formation; the trifunctional fusion protein comprises the sequences SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:87, SEQ ID NO:86.

In some embodiments, the IL15 sequence is shown in SEQ ID No. 1 or SEQ ID No. 2; the IL15Rα sequence is shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 2 No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9.

In some embodiments, the sequence of the anti-CD3 antibody comprises SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

In some embodiments, the human Fc fragment sequence comprises SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.

The second aspect of the present invention provides the application of the trifunctional fusion protein according to the first aspect of the present invention in the preparation of medicines for cancer, infection or immune regulation diseases.

In some embodiments, the present invention provides the use of the trifunctional fusion protein in the preparation of a medicament for inhibiting tumor growth.

In some embodiments, the cancer or tumor comprises: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer , neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, bone marrow cancer, lymphoma, leukemia, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

Beneficial effects of the invention: The present invention provides a new structural form of TAA/CD3/IL15 trifunctional fusion protein: the cytokine IL15 and IL15Ra have an ultra-high affinity (KD is about 30-100pM), and IL15 and IL15Ra are used separately. Substitute the CL and CH1 domains in one of the antibody structures to resolve the light chain mismatch of the bispecific antibody, while resolving the heavy chain mismatch through the Fc heterodimer form. Biologically, the TAA/CD3/IL15 trifunctional fusion protein of the present invention, on the one hand, combines with T cell surface antigen (CD3) and tumor cell surface antigen (TAA) to form an immune synapse, thereby directly activating and proliferating T cells, and then Release cytotoxins or cytokines to kill tumor cells. The activation process of this T cell is simple and straightforward, and does not require the presentation of tumor cell antigens to generate specific T lymphocyte clones, so it is not restricted by MHC or HLA. On the other hand, through TAA antibodies targeting IL15/IL15R to the complex to the tumor microenvironment, the IL15/IL15Ra complex promotes the proliferation of T cells and NK cells, overcomes the low number of T cells in the tumor microenvironment, effectively activates the immune system, enhances The killing effect on tumors will greatly improve the therapeutic effect on solid tumors.

Description of drawings

Figure 1 is a schematic representation of Forms IA and IB of trifunctional fusion proteins.

Figure 2 is a schematic representation of the forms IC and ID of the trifunctional fusion protein.

Figure 3 is a schematic representation of Form IE of the trifunctional fusion protein.

Figure 4 is a schematic representation of the formal IF of the trifunctional fusion protein.

Figure 5 is a schematic representation of the form IG of the trifunctional fusion protein.

Figure 6 is a schematic representation of Form IH of the trifunctional fusion protein.

Figure 7 is a schematic representation of Form IIA of the trifunctional fusion protein.

Figure 8 is a schematic representation of Form IIB of the trifunctional fusion protein.

Figure 9 is a schematic representation of Form IIC of the trifunctional fusion protein.

Figure 10 is a schematic representation of Form IID of the trifunctional fusion protein.

Figure 11 is a schematic representation of Form IIE of the trifunctional fusion protein.

Figure 12 is a schematic representation of Form IIF of the trifunctional fusion protein.

Figure 13 is a schematic representation of Form IIG of the trifunctional fusion protein.

Figure 14 is a schematic representation of Form IIH of the trifunctional fusion protein.

Figure 15 is a schematic representation of Form IIIA of the trifunctional fusion protein.

Figure 16 is a schematic representation of Form IIIB of the trifunctional fusion protein.

Figure 17 is a schematic representation of Forms IVA and IVB of trifunctional fusion proteins.

Figure 18 is a schematic representation of forms IVC and IVD of trifunctional fusion proteins.

Figure 19 is the plasmid structure of QD3208.

Figure 20 is the plasmid structure of QD3209.

Figure 21 is a reducing SDS-PAGE electropherogram.

Figure 22 is a non-reducing SDS-PAGE electropherogram.

Figure 23 is a graph showing the experimental results of FACS detecting the binding of the CLDN18.2/CD3/IL15 trifunctional fusion protein of the present invention to CHOS-CLDN18.2 cells. FACS实验结果表明CLDN18.2/CD3/IL15三功能融合蛋白QP32083209,QP331633193320,QP34683469,QP34683475,QP34683478,QP36503651,QP36633651, QP34683472,QP36543651,QP36573651,QP36603651均结合稳定表达CLDN18.2的CHOS细胞，即结合人CLDN18.2 protein. At the same time, the positive control CLDN18.2 mAb QP14611463 binds to CHOS-CLDN18.2, while the negative control human IgG does not bind to CHOS-CLDN18.2.

Figure 24 is a graph showing the experimental results of FACS detecting the binding of the CLDN18.2/CD3/IL15 trifunctional fusion protein of the present invention to Jurkat cells. FACS实验结果表明CLDN18.2/CD3/IL15三功能融合蛋白QP331633173318,QP331633193320,QP331633213322,QP331633233324,QP32083209,QP34683469,QP34683475,QP36503651,QP36633651,QP34683472,QP36543651,QP36573651,QP36603651均结合天然表达CD3的Jurkat细胞，即Binds to human CD3 protein, while CLDN18.2 mAb QP14611463, IL15/IL15Ra fusion protein QP33123313 and human IgG do not bind to Jurkat cells.

[Corrected 23.06.2021 under Rule 91]
Figure 25 is a graph showing the results of the Mo7e cell proliferation assay to detect the biological activity of IL15/IL15Ra in the CLDN18.2/CD3/IL15 trifunctional fusion protein of the present invention. The results of the Mo7e cell proliferation assay showed that the CLDN18.2/CD3/IL15 trifunctional fusion proteins QP34683469, QP36503651, QP36633651, QP32083209, QP33123313, QP36603651, QP36573651, and QP36543651 could promote the proliferation of Mo7e cells, indicating that there are IL15/IL15Ra in the trifunctional fusion proteins. The biological activity was significantly weaker than that of the IL15/IL15Ra fusion protein QP33123313, which was consistent with our designed goal of reducing the toxicity of IL15/IL15Ra. Figure 26 is a graph showing the experimental results of the PBMC killing assay to detect the biological function activity of antiCLDN18.2/antiCD3 in the CLDN18.2/CD3/IL15 trifunctional fusion protein of the present invention. The results of PBMC killing experiments showed that CLDN18.2/CD3/IL15 trifunctional fusion proteins QP34683478, QP36503651, QP36633651, QP32083209, QP34683469, QP34683475 could induce T cells in PBMC to kill human gastric cancer cells NUGC4-CLDN18.2 that stably express CLDN18.2 Cells, and CD3 monoclonal antibody OKT3 and human IgG did not kill.

Figure 27 is a graph of the experimental results of FACS detecting the binding of EGFR/folate receptor/CD3/IL15 multifunctional fusion protein to A431 cells naturally expressing EGFR. The results of FACS experiments showed that EGFR/folate receptor/CD3/IL15 multifunctional fusion proteins QP34843487, QP35293530, QP35293644, and QP35293645 all bound to A431 cells that naturally express EGFR, that is, they all bound to EGFR protein.

Figure 28 is a graph showing the results of FACS detection of EGFR/folate receptor/CD3/IL15 multifunctional fusion protein combined with SK-OV-3 cells naturally expressing folate receptor protein. The results of FACS experiments showed that EGFR/folate receptor/CD3/IL15 multifunctional fusion proteins QP34843487, QP35293530, QP35293644 and QP35293645 all bound to SK-OV-3 cells naturally expressing folate receptor protein, that is, they all bound to folate receptor protein.

Figure 29 is a graph showing the experimental results of FACS detecting the binding of the EGFR/folate receptor/CD3/IL15 multifunctional fusion protein of the present invention to Jurkat cells. The results of FACS experiments showed that EGFR/folate receptor/CD3/IL15 multifunctional fusion proteins QP34843487, QP35293530, QP35293644, and QP35293645 all bound to Jurkat cells that naturally express CD3, that is, binding to human CD3 protein, that is, binding to human CD3 protein.

Figure 30 is a graph of the experimental results of the Mo7e cell proliferation assay detecting the biological activity of IL15/IL15Ra in the EGFR/folate receptor/CD3/IL15 multifunctional fusion protein of the present invention. The results of the Mo7e cell proliferation experiment showed that the EGFR/folate receptor/CD3/IL15 multifunctional fusion proteins QP35293533, QP35293644, and QP35293645 could promote the proliferation of Mo7e cells, indicating that their IL15/IL15Ra had biological activity, and were significantly weaker than the IL15/IL15Ra fusion proteins. QP33123313, which is consistent with our designed target to reduce IL15/IL15Ra toxicity.

Figure 31 is a graph showing the experimental results of the PBMC killing assay to detect the biological function activity of antiEGFR/antiCD3 in the fusion protein QP35293530 of the present invention. The results of PBMC killing experiments showed that EGFR/folate receptor/CD3/IL15 multifunctional fusion protein QP35293530 could induce T cells in PBMCs to kill human epidermal cancer cell line A431 that naturally expresses EGFR, and CD3 mAb OKT3 and human IgG did not kill.

Figure 32 is a graph of the experimental results of the PBMC killing assay to detect the biological function activity of anti-folate receptor/antiCD3 in fusion protein QP35293530. The results of the PBMC killing experiment showed that the EGFR/folate receptor/CD3/IL15 multifunctional fusion protein QP35293530 could induce T cells in PBMC to kill the human epidermal cancer cell line SK-OV-3, which naturally expresses the folate receptor. No kills.

Detailed ways

1. Terminology

For easier understanding of the present invention, some technical and scientific terms of the present invention are explained before describing the embodiments.

Unless otherwise expressly defined elsewhere in the present application documents, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter and one-letter codes for amino acids used in the present invention are known to those skilled in the art, or described in J. biol. Chem., 1968, 243:3557.

The "IL-15" of the present invention can be any IL-15 or a mutant capable of binding IL15Rα, such as human IL-15 or non-human mammalian or non-mammalian IL-15. Exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, etc.; preferably human IL-15. The term "mutant capable of binding to IL15Rα" refers to an increased or decreased affinity for IL-15 and its receptor obtained by one or more amino acid substitutions, additions or deletions mutations, or an increase or decrease in the activity of stimulating T cells or NK cells. mutant molecules. The "IL-15" of the present invention is preferably its variant form, more preferably the amino acid sequence is SEQ ID No.1 or SEQ ID No.2.

The "IL-15Rα" described in the present invention can be any species of IL-15Rα or a mutant capable of binding IL15Rα, such as human IL-15Rα or non-human mammalian IL-15Rα or non-mammalian IL-15Rα. Exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, and the like. Preferably human IL-15Rα, more preferably human IL-15Rα extracellular domain fragment, abbreviated as IL-15Rα ECD (see database UniProtKB, accession number Q13261, 31-205aa). The term "mutant capable of binding IL15Rα" refers to a functional mutant formed by one or more amino acid deletion, insertion or substitution mutations in IL-15Rα, which has the ability to bind to its ligand molecule such as IL-15, preferably human IL The -15Rα molecule is more preferably a shortened form of the human IL-15Rα extracellular domain fragment, that is, a molecule with human IL-15 receptor α activity obtained by one or more amino acid deletion mutations from the C-terminal of the extracellular domain fragment, preferably retaining 65 - Deletion mutant forms of 178 amino acids, such as IL-15Rα (SEQ ID NOs: 3-9).

Fc heterodimer mutation refers to a change in Fc structure or function by the presence of one or more amino acid substitution, insertion or deletion mutations at appropriate sites in the Fc. Space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions, etc. can be formed between the mutant-designed Fc variants. Interaction between Fc mutants contributes to the formation of stable heterodimers. Preferred mutagenesis designs are those in the "Knob-in-hole" format. In addition, the Fc of the present invention may also have other mutations that lead to changes in its function, such as glycosylation mutations, FcγR binding region mutations (to adjust ADCC activity), and amino acid mutations to improve antibody stability. In the present invention, Fc includes Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc and mutations thereof, wherein one chain can bind proteinA, and the other chain is a mutant that cannot bind proteinA, including mutation H435R or H435R/Y436F , according to EU counts.

The term "linker sequence" refers to insertion into an immunoglobulin domain of one or more amino acid residues that provide sufficient mobility for the domains of the light and heavy chains to fold into an exchange dual variable region immunoglobulin. In the present invention, the variable regions VH, VL, IL-15, IL-15Rα, CH1, CL and FC of CD3 antibody and TAA antibody are linked to ensure the correct folding and peptide stability of the protein. The "linker peptide" of the present invention is preferably a low immunogenic amino acid disability.

"Modification" as used herein means amino acid substitutions, insertions and/or deletions in a polypeptide sequence or changes in the portion chemically linked to the protein. For example, the modification can be an altered carbohydrate or PEG structure attached to the protein. "Amino acid modification" as used herein means amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications are always amino acids encoded by DNA, such as the 20 amino acids with codons in DNA and RNA.

"Amino acid substitution" or "substitution" as used herein means replacing an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, a substitution refers to an amino acid at a particular position that is not naturally occurring, either in an organism or in any organism. For example, substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced by a tyrosine. For clarity, a protein that has been engineered to alter the nucleic acid coding sequence without altering the starting amino acid (eg, replacing CGG (which encodes arginine) with CGA (which still encodes arginine) to increase expression levels in the host organism) is not an "amino acid" Substitution"; that is, a protein is not an amino acid substitution if it has the same amino acid at the specific position where it starts, despite the creation of a new gene encoding the same protein.

As used herein, an "amino acid insertion" or "insertion" means an addition of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, -233E or 233E specifies a glutamate insertion after position 233 and before position 234. Additionally, -233ADE or A233ADE specifies an Ala Asp Glu insertion after position 233 and before position 234.

As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid sequence at a specified position in the parent polypeptide sequence. For example, E233- or E233#, E233( ) or E233del specify a glutamate deletion at position 233. Additionally, EDA233- or EDA233# specifies a deletion of the sequence Glu Asp Ala starting at position 233.

As used herein, "variant protein" or "protein variant" or "variant" means a protein that differs from that of the parent protein by at least one amino acid modification. A protein variant can refer to the protein itself, a composition comprising the protein, or the amino acid sequence that encodes it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, eg, about one to about seventy amino acid modifications, and preferably about one to about five amino acid modifications, compared to the parent. As described below, in some embodiments, the parent polypeptide, eg, an Fc parent polypeptide, is a human wild-type sequence, eg, an Fc region from IgGl, IgG2, IgG3, or IgG4, although human sequences with variants can also serve as "parental" sequences. "Polypeptide", for example, may include IgG1/2 hybrids. The protein variant sequences herein are preferably at least about 80% identical to the parent protein sequence, and most preferably at least about 90% identical, more preferably at least about 95-98-99% identical. A variant protein can refer to the variant protein itself, a composition comprising the variant protein, or the DNA sequence encoding it.

As used herein, "protein" means herein at least two covalently attached amino acids, which include proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. "analogs", such as peptoids (see Simon et al, PNAS USA 89(20):9367 (1992), incorporated by reference in its entirety incorporated). Amino acids can be naturally occurring or synthetic (e.g., amino acids not encoded by DNA); as understood by those of skill in the art. For example, for the purposes of the present invention, homophenylalanine, citrulline, ornithine, and norleucine are considered synthetic amino acids, and both D- and L- (R or S) configurations of amino acids can be utilized . Variants of the invention may comprise modifications, including synthetic amino acids incorporated using, for example, techniques developed by Schultz and colleagues, including but not limited to by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al. , 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3 and the method described by Chin et al., 2003, Science 301(5635):964-7 , all of which are incorporated by reference in their entirety. Additionally, polypeptides can include synthetic derivatization of one or more side chains or ends, glycosylation, PEGylation, cyclic arrangement, cyclization, linkers to other molecules, fusions to proteins or protein domains, and peptide tags or Marked additions.

Accordingly, as used herein, an "antibody variant" or "variant antibody" means an antibody that differs from a parent antibody by at least one amino acid modification. As used herein, "IgG variant" or "variant IgG" means an antibody that differs from a parent IgG (again, in many cases, the human IgG sequence) by at least one amino acid modification, and as used herein, " "Variant immunoglobulin" or "variant immunoglobulin" means an immunoglobulin sequence that differs from that of a parental immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" means a protein comprising amino acid modifications in the Fc domain. The Fc variants of the present invention are defined in terms of the amino acid modifications that constitute them. Thus, for example, N434S or 434S is an Fc variant having the substitution serine at position 434 relative to the parent Fc polypeptide, wherein numbering is according to the EU index. Likewise, M428L/N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 428L/434S. It should be noted that the order in which the substitutions are provided is arbitrary, that is, for example, 428L/434S is the same Fc variant as M428L/N434S, etc. For all positions discussed in the present invention in relation to antibodies, unless otherwise stated, amino acid position numbering is according to the EU index. The EU index, or the EU index as in the Kabat or EU numbering scheme, refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby incorporated by reference in its entirety). Modifications can be additions, deletions or substitutions. Substitutions can include naturally occurring amino acids and, in some cases, synthetic amino acids. Examples include US Patent No. 6,586,207; WO98/48032; WO03/073238; US2004-0214988A1; WO05/35727A2; WO05/74524A2; & P.G. Schultz, (2002), ChemBioChem 11:1135-1137; J.W. Chin, et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang, & P.G. Schultz, (2002) , Chem.1-10, all of which are incorporated by reference in their entirety.

As used herein, "protein" means herein at least two covalently attached amino acids, which include proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. "analogs", such as peptoids (see Simon et al, PNAS USA 89(20):9367 (1992), incorporated by reference in its entirety incorporated). Amino acids can be naturally occurring or synthetic (eg, amino acids not encoded by DNA); as understood by those of skill in the art. For example, for the purposes of the present invention, homophenylalanine, citrulline, ornithine, and norleucine are considered synthetic amino acids, and both D- and L- (R or S) configurations of amino acids can be utilized . Variants of the invention may comprise modifications, including synthetic amino acids incorporated using, for example, techniques developed by Schultz and colleagues, including but not limited to by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al. , 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3 and the method described by Chin et al., 2003, Science 301(5635):964-7 , all of which are incorporated by reference in their entirety. Additionally, polypeptides can include synthetic derivatization of one or more side chains or ends, glycosylation, PEGylation, cyclic arrangement, cyclization, linkers to other molecules, fusions to proteins or protein domains, and peptide tags or Marked additions.

As used herein, "residue" means a position in a protein and its associated amino acid identity. For example, asparagine 297 (also known as Asn297 or N297) is the residue at position 297 in human antibody IgGl.

The present invention includes antibodies or fragments of such antibodies so long as they exhibit the desired biological activity. Also included in the present invention are chimeric antibodies, eg, humanized antibodies. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. For example, humanization can be performed by substituting at least a portion of a rodent complementarity determining region for a corresponding region of a human antibody using methods described in the art.

Antibody: As used herein, "antibody" or "immunoglobulin" refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that is capable of non-covalent, reversible, and specificity bind antigen. For example, a naturally occurring "antibody" of the IgG class is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, bispecific or multispecific antibodies and anti-idiotype (anti-Id) Antibodies (including, for example, anti-Id antibodies to antibodies of the invention). These antibodies can belong to any isotype/type (eg, IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the light (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation and the like. By convention, the further the constant region domain is from the antigen binding site or amino terminus of the antibody, the higher it is numbered. The N-terminus is the variable region and the C-terminus is the constant region; the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

As used herein, "antibody fragment" refers to one or more portions of an antibody. In some embodiments, these moieties are part of one or more contact domains of an antibody. In some other embodiments, these moieties are antigen-binding fragments (which retain the ability to bind antigen non-covalently, reversibly, and specifically), sometimes referred to herein as "antigen-binding fragments," "which Antigen-binding fragment", "antigen-binding portion" and the like. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, comprising disulfide-linked fragments at the hinge region A bivalent fragment of two Fab fragments; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of the one-armed antibody; a dAb fragment consisting of the VH domain (Ward et al., ( 1989) Nature 341:544-546); and isolated complementarity determining regions (CDRs). Thus, the term "antibody fragment" encompasses proteolytic fragments of antibodies (eg, Fab and F(ab)2 fragments) and engineered proteins comprising one or more portions of an antibody (eg, scFv).

Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, tribodies, tetrabodies, v-NAR and bis-scFv (see For example, Hollinger and Hudson, 2005 Nature Biotechnology 23:1126-1136).

Antibody fragments can be incorporated into a single-chain molecule comprising a pair of tandem Fv fragments (eg, VH-CH1-VH-CH1), together with a complementary light chain polypeptide (eg, VL-VC-VL-VC) to form a pair Antigen binding regions (Zapata et al., 1995, Protein Eng. [Protein Engineering] 8:1057-1062; and US Pat. No. 5,641,870).

Antigen binding domain: The term "antigen binding domain" refers to the portion of a molecule that has the ability to bind non-covalently, reversibly and specifically to an antigen. Exemplary antigen-binding domains include antigen-binding fragments and portions of immunoglobulin-based scaffolds and non-immunoglobulin-based scaffolds that retain the ability to non-covalently, reversibly, and specifically bind antigen. As used herein, the term "antigen binding domain" encompasses antibody fragments that retain the ability to bind antigen non-covalently, reversibly and specifically.

Complementarity Determining Region: As used herein, the term "complementarity determining region" or "CDR" refers to the sequence of amino acids within the variable region of an antibody that confer antigen specificity and binding affinity. For example, in general, three CDRs (eg, CDR-H1, CDR-H2, and CDR-H3) are present in each heavy chain variable region, and three CDRs (CDR-H3) are present in each light chain variable region -L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known protocols, including those described by Kabat et al., 1991, "Sequences of Proteins of Immunological Interest" [with immunological importance]. Sequence of the Proteins], 5th ed., National Institutes of Health, Division of Public Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., 1997, JMB273:927-948 ( "Josiah" numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136 (1999); Lefranc et al., 2003, Dev. Comp. Immunol. [Developmental Immunology and Comp.] Immunology] 27:55-77 ("IMGT" numbering scheme). For example, for the classical form, the CDR amino acid residues in the variable domain (VH) of the heavy chain are numbered 31-35 (CDR- H1), 50-65 (CDR-H2) and 95-102 (CDR-H3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50 -56 (CDR-L2) and 89-97 (CDR-L3). According to Chothia, the CDR amino acids in VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2) and 95-102 ( CDR-H3); and numbered amino acid residues in VL as 26-32 (CDR-L1), 50-52 (CDR-L2) and 91-96 (CDR-L3). By combining Kabat and Josiah two The CDRs of human VH are defined by amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2) and 95-102 (CDR-H3) in human VH and amino acid residues 24-24 in human VL 34 (CDR-L1), 50-56 (CDR-L2) and 89-97 (CDR-L3). The CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51 according to IMGT -57 (CDR-H2) and 93-102 (CDR-H3) and number the CDR amino acid residues in VL at approximately 27-32 (CDR-L1), 50-52 (CDR-L2) and 89-97 (CDR-L3) (numbering according to "Kabat"). According to IMGT, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.

Single-chain Fv or scFv: As used herein, a "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Rosenburg and Moore, (1994) Springer-Verlag, New York, pp. 269-315 Page.

dsFv: The term "dsFv" refers to disulfide-stabilized Fv fragments. In dsFv, VH and VL are linked by interdomain disulfide bonds. To generate such molecules, one amino acid each in the framework regions of VH and VL is mutated to cysteines, which in turn form stable interchain disulfide bonds. Typically, position 44 in VH and position 100 in VL are mutated to cysteine. See Brinkmann, 2010, Antibody Engineering 181-189, DOI: 10.1007/978-3-642-01147-4-14. The term dsFv encompasses dsFv (molecules in which VH and VL are linked by an interchain disulfide bond rather than a linker peptide) or scdsFv (molecules in which VH and VL are linked by a linker and an interchain disulfide bond) as known in the art both.

Diabodies: As used herein, the term "diabodies" refers to small antibody fragments having two antigen-binding sites, typically formed by the pairing of scFv chains. Each scFv comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL, where VH is located at the N-terminus or C-terminus of VL). Unlike typical scFvs, in which VH and VL are separated by a linker that allows VH and VL on the same polypeptide chain to pair and form an antigen-binding domain, diabodies typically contain linkers that are too short and insufficient to allow pairing of the VH and VL domains on the same chain, thereby forcing the VH and VL domains to pair with the complementary domains of the other chain and creating two antigen binding sites. Diabodies are more fully described in, eg, EP 404,097; WO 93/11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

VH: The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv or Fab.

VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.

VH-VL or VH-VL pair: The terms "VH-VL" and "VH-VL pair" are used for convenience when referring to VH-VL pairs, whether on the same polypeptide chain or on different polypeptide chains , and are not intended to convey any particular direction unless the context dictates otherwise. Thus, a scFv comprising a "VH-VL" or "VH-VL pair" can have the VH and VL domains in any orientation, eg, VH is N-terminal to VL or VL is N-terminal to VH.

Fusion: The term "fusion" in the context of a trifunctional fusion protein refers to a functional relationship between two or more polypeptide chains. In particular, the term "fused" means that two or more polypeptides are fused to each other, eg, non-covalently through molecular interactions or covalently fused through one or more disulfide bonds or chemical cross-links, resulting in A functional trifunctional fusion protein in which the TAA antigen 1 binding molecule, the TAA antigen 2 binding molecule and the CD3 antibody fragment can bind their respective targets. Examples of possible fusions in the trifunctional fusion proteins created by the present invention include, but are not limited to, fusions between Fc regions in the Fc domain (such as homodimers or heterodimers as described in the introduction to II). body), fusions between VH and VL domains in Fab or Fv, and fusions between CH1 and IL15Ra, and between IL15 and CL in Fab.

Host cell or recombinant host cell: The term "host cell" or "recombinant host cell" refers to a cell that has been genetically engineered, eg, by the introduction of heterologous nucleic acid. It should be understood that this term refers not only to a particular subject cell, but also to the progeny of such cells. Because certain modifications may occur in progeny due to mutation or environmental influences, such progeny may differ in fact from the parental cell, but still be included within the scope of "host cell" as used herein. The host cell may transiently carry the heterologous nucleic acid, eg, on an extrachromosomal heterologous expression vector, or stably carry the heterologous nucleic acid, eg, by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing the trifunctional fusion protein created by the present invention, the host cell can be a mammalian-derived cell line or a cell line with mammalian-like characteristics, such as monkey kidney cells (COS, eg, COS-1, COS-7) , HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, horse- Darwin's bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and/or engineered variants thereof. Engineered variants include, for example, glycan profile modified and/or site-specific integration site derivatives.

Antibody Numbering System: In this specification, unless otherwise stated, references to amino acid residues numbered in antibody domains are based on the EU numbering system (eg, in Tables 8B and 8C). This system was originally designed by Edelman et al., 1969, Proc. Nat'l Acad. Sci. USA 63:78-85 and by Kabat et al., 1991, in Sequences of Proteins of Immunological Interest [with Protein sequences of immunological importance], described in detail in the US Department of Health and Human Services, NIH, USA.

Monoclonal Antibody: As used herein, the term "monoclonal antibody" refers to polypeptides derived from the same genetic source, including antibodies, antibody fragments, molecules (including TBM), and the like.

Humanized: The term "humanized" form of a non-human (eg, murine) antibody is a chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the receptor's hypervariable region are replaced by those from a non-human species (such as mouse, rat, rabbit, or non-human primate). Residue substitutions of hypervariable regions (donor antibodies) of the desired specificity, affinity and capacity (animaloid). In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will contain substantially all of at least one, typically two variable domains, wherein all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, and all Or substantially all FRs are those of the human immunoglobulin lo sequence. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. For further details, see Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol . [Current Research in Structural Biology] 2:593-596. See also the following review articles and references cited therein: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma & Immunol. [Annals of Allergy, Asthma and Immunology] 1:105-115; Harris, 1995, Biochem. Proceedings of the Society] 23: 1035-1038; Hurle and Gross, 1994, Curr. Op. Biotech. [Current Perspectives in Biotechnology] 5: 428-433.

Human antibody: As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutated forms of human germline sequences, or antibodies containing consensus framework sequences derived from analysis of human framework sequences, For example as described in: Knappik et al., 2000, J Mol Biol 296, 57-86. The structures and positions of immunoglobulin variable domains (eg, CDRs) can be defined using well-known numbering schemes (eg, Kabat numbering scheme, Josiah numbering scheme, or a combination of Kabat and Josiah) (see eg, Lazikani et al., 1997, J. Mol. Bio. [Journal of Molecular Biology] 273:927 948; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242 U.S. Department of Health and Human Resources Services; Chothia et al, 1987, J. Mol. Biol. 196:901-917; Chothia et al, 1989, Nature 342:877-883 ).

Human antibodies may include amino acid residues that are not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo, or conservative substitutions to promote stability or production. ). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted into human framework sequences.

Chimeric Antibody: The term "chimeric antibody" (or antigen-binding fragment thereof) is an antibody molecule (or antigen-binding fragment thereof) in which (a) the constant region or portion thereof is altered, substituted or replaced such that the The antigen binding site (variable region) is linked to a different or altered type, effector function and/or species of constant region, or to a completely different molecule (e.g. enzymes, toxins, hormones, growth factors, drugs, etc.); or (b) the variable region or portion thereof is altered, replaced or replaced with a variable region having a different or altered antigen specificity. For example, mouse antibodies can be modified by replacing their constant regions with constant regions from human immunoglobulins. Due to the replacement with human constant regions, the chimeric antibody can retain its specificity for recognizing an antigen while having reduced antigenicity in humans compared to the original mouse antibody.

As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" means a cell-mediated response in which non-specific cytotoxic cells expressing FcyRs recognize bound antibodies on target cells and subsequently elicit the target cells lysis. ADCC is associated with binding to FcyRIIIa; increased binding to FcyRIIIa results in an increase in ADCC activity. As discussed herein, many embodiments of the present invention completely eliminate ADCC activity.

As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis means a cell-mediated response in which non-specific cytotoxic cells expressing FcyRs recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells effect.

Effector function: The term "effector function" refers to the activity of an antibody molecule, which is mediated by binding through a domain of an antibody rather than an antigen-binding domain, usually by binding of an effector molecule. Effector functions include complement-mediated effector functions mediated, for example, by the binding of the C1 component of the complement to the antibody. Activation of complement is important in the opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity responses. Effector functions also include Fc receptor (FcR) mediated effector functions, which can be triggered by binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of antibodies to Fc receptors on the cell surface triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, and lysis of antibody-coated target cells by killer cells (termed antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. The effector function of an antibody can be altered by altering, for example, increasing or decreasing the affinity of the antibody for effector molecules such as Fc receptors or complement components. Typically the binding affinity will be altered by modifying the effector molecule binding site, and in this case, it may be appropriate to locate the site of interest and modify at least part of the site in a suitable manner. It is also envisaged that alteration of the binding site on the antibody directed against the effector molecule need not significantly alter the overall binding affinity, but may alter the geometry of the interaction, rendering the effector mechanism ineffective, as in non-productive binding. It is further contemplated that effector function can also be altered by modifying sites that are not directly involved in effector molecule binding, but are otherwise involved in the performance of effector function.

As used herein, "IgG subclass modification" or "isotype modification" means an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different, aligned IgG isotype. For example, because IgG1 contains tyrosine at EU position 296 and IgG2 contains phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification.

As used herein, "non-naturally occurring modification" means an amino acid modification that is not an isoform. For example, since none of the IgGs contain a serine at position 434, the substitution 434S in IgGl, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification.

As used herein, "IgG Fc ligand" means a molecule, preferably a polypeptide, from any organism that binds the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors homologous to FcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, incorporated by reference in its entirety). Fc ligands can include undiscovered molecules that bind Fc. Specific IgG Fc ligands are FcRn and Fcγ receptors. As used herein, "Fc ligand" means a molecule, preferably a polypeptide, from any organism, which binds the Fc region of an antibody to form an Fc/Fc ligand complex.

As used herein, "Fcy receptor", "FcyR" or "FcgammaR" means any member of the protein family that binds the Fc region of an IgG antibody and is encoded by the FcyR gene. In humans, this family includes, but is not limited to, FcyRI (CD64), including isotypes FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), including isotypes FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb- 1 and FcyRIIb-2) and FcyRIIc; and FcyRIII (CD16), including isotypes FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIb-NA1 and FcyRIIb-NA2) (Jefferis et al., 2002 , Immunol Lett 82:57-65, incorporated by reference in its entirety), and any undiscovered human FcyR or FcyR isotype or allotype. FcyRs can be from any organism, including but not limited to human, mouse, rat, rabbit and monkey. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2), as well as any undiscovered mouse FcyR or FcyR isotype or allotype.

As used herein, "FcRn" or "neonatal Fc receptor" means a protein that binds the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can be from any organism, including but not limited to human, mouse, rat, rabbit and monkey. As known in the art, functional FcRn proteins comprise two polypeptides, commonly referred to as heavy and light chains. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2-microglobulin. Various FcRn variants can be used to increase binding to the FcRn receptor and, in some cases, increase serum half-life. In general, unless otherwise stated, the Fc monomers of the invention retain binding to the FcRn receptor (and, as noted below, amino acid variants may be included to increase binding to the FcRn receptor).

As used herein, "parent polypeptide" means the starting polypeptide that is subsequently modified to generate a variant. The parent polypeptide can be a naturally occurring polypeptide, or a variant or engineered form of a naturally occurring polypeptide. Parent polypeptide can refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence encoding it. Accordingly, as used herein, "parent immunoglobulin" means an unmodified immunoglobulin polypeptide, which is modified to generate a variant, and as used herein, "parent antibody" means an unmodified antibody, which is Modifications are made to generate variant antibodies. It should be noted that "parent antibody" includes known commercial, recombinantly produced antibodies as outlined below.

As used herein, "Fc" or "Fc region" or "Fc domain" means an antibody constant region comprising part of an immunoglobulin domain (eg, CH1 ) excluding the first constant region, and in some cases, the hinge of polypeptides. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG (eg, CH2 and CH3), the last three constant region immunoglobulin domains of IgE and IgM, and for these domains the N-terminal of flexible hinges. For IgA and IgM, the Fc can include the J chain. For IgG, the Fc domain comprises the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region can vary, a human IgG heavy chain Fc region is generally defined to include residues C226 or P230 for its carboxy terminus, where numbering is according to the EU index as in Kabat. In some embodiments, as described more fully below, the Fc region is subjected to amino acid modifications, eg, to alter binding to one or more FcγR receptors or FcRn receptors.

"Heavy constant region" means herein the CH1-hinge-CH2-CH3 portion of an antibody.

"Fc fusion protein" or "immunoadhesin" herein means a protein comprising an Fc region, typically linked to a different protein such as IL-15 and/or IL-15R (optionally via a linker moiety as described herein) ), as described herein. In some cases, two Fc fusion proteins can form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, with the latter being preferred. In some cases, one monomer of a heterodimeric Fc fusion protein comprises a separate Fc domain (eg, an empty Fc domain), while the other monomer is an Fc fusion comprising a variant Fc structure Domains and protein domains such as receptors, ligands or other binding partners.

As used herein, "position" means a position in a protein sequence. Positions may be numbered sequentially, or according to a defined format such as the EU index used for antibody numbering.

As used herein, "target cell" means a cell that expresses a target antigen.

"Wild type or WT" as used herein means the amino acid sequence or nucleotide sequence found in nature, including allelic variation. The WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

The trifunctional fusion proteins of the present invention are generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified and separated and/or recovered from the cell or cell culture from which it is expressed. Typically, isolated polypeptides are prepared by at least one purification step. "Isolated protein" means a protein that is substantially free of other proteins with different binding specificities. "Recombinant" means a protein produced in a foreign host cell using recombinant nucleic acid techniques.

"Percent (%) amino acid sequence identity" in terms of protein sequences is defined as, when sequences are aligned and gaps are introduced where necessary to achieve maximum percent sequence identity, and without considering any conservative substitutions as part of sequence identity, candidates The percentage of amino acid residues in a sequence that are identical to amino acid residues in a particular (parental) sequence. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in various ways within the skill in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences to be compared. One particular procedure is the ALIGN-2 procedure outlined at paragraphs [0279] to [0280] of US Publication No. 20160244525, which is hereby incorporated by reference.

The degree of identity between an amino acid sequence of the invention ("sequence of the invention") and the parent amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences divided by the length of the "sequence of the invention", or the length of the parent sequence , whichever is the shortest. Results are expressed as percent identity.

In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

"Specifically binds" or "specifically binds" or "specifically for" a particular antigen or epitope means binding that is measurably different from a non-specific interaction. For example, specific binding can be measured by measuring molecular binding compared to binding of a control molecule, which is typically a similarly structured molecule that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target.

Recognition: As used herein, the term "recognize" refers to TAA antigen-binding molecules and anti-CD3 antibody fragments that find and interact (eg, bind) to their epitopes.

Epitope: An epitope or antigenic determinant is a portion of an antigen that can be recognized by an antibody or other antigen-binding portion as described herein. Epitopes can be linear or conformational.

Nucleic acid: The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring and non-naturally occurring, having binding properties similar to the reference nucleic acid, and Metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA) ).

Conservatively modified variants thereof (eg, degenerate codon substitutions) and complements thereof are also implicitly encompassed by a particular nucleic acid sequence, as well as explicitly indicated sequences, unless otherwise indicated. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is mixed with bases and/or Deoxyinosine residue substitution (Batzer et al., (1991) Nucleic Acid Res. [Nucleic Acids Research] 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. [Journal of Biochemistry] 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

Vector: The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve the same function.

Subject: The term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, bone marrow, lymphoma, leukemia, thyroid, endometrium, uterus, bladder, nerve Endocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma , glioblastoma, glioma, sarcoma, mesothelioma, myelodysplastic syndrome, etc., such as any TAA-positive cancer of any of the foregoing types.

Tumor: The term "tumor" is used interchangeably with the term "cancer" herein, eg, both terms encompass both solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes premalignant as well as malignant cancers and tumors.

Tumor-Associated Antigen: The term "tumor-associated antigen" or "TAA" refers to molecules (typically proteins, carbohydrates, lipids or their) expressed either in whole or as fragments (eg, MHC/peptides) on the surface of cancer cells some combinations), and it can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, the TAA is a marker expressed by both normal cells and cancer cells, eg, a lineage marker, eg, CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, eg, 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more compared to normal cells. In some embodiments, TAAs are cell surface molecules that are inappropriately synthesized in cancer cells, eg, molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, TAAs will only be expressed fully or as fragments (eg, MHC/peptides) on the cell surface of cancer cells, and not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific for cancer cells, sometimes referred to in the art as tumor specific antigens ("TSA").

Treat, Treat, and Treat: As used herein, the terms "treat, treatment, and treating" refer to the progression, severity of proliferative disorders resulting from administration of one or more TBMs of the present disclosure and/or reduction or improvement in duration, or improvement in one or more symptoms (preferably, one or more identifiable symptoms) of a proliferative disorder. In particular embodiments, the terms "treat, treatment, and treating" refer to amelioration of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible by a patient. In other embodiments, the terms "treat, treatment, and treating" refer to the inhibition of a proliferative disorder physically, eg, by stabilizing discernible symptoms, or physiologically, eg, by stabilizing physical parameters, or both. progress. In other embodiments, the terms "treat, treatment, and treating" refer to reducing or stabilizing tumor size or cancer cell count.

The present invention provides a new structural form of TAA/CD3/IL15 trifunctional fusion protein, which simultaneously realizes the three functions of tumor targeting, anti-CD3 antibody and T cell activation: the cytokine IL15 and IL15Ra have an ultra-high affinity (KD About 30-100pM), use IL15 and IL15Ra to replace the CL and CH1 domains in one of the antibody structures, respectively, to solve the mismatch of the light chain of the bispecific antibody, and to solve the mismatch of the heavy chain through the Fc heterodimer form. Biologically, the TAA/CD3/IL15 trifunctional fusion protein of the present invention is targeted to the tumor microenvironment through the TAA antibody, IL15 and IL15Ra activate the immune system, and at the same time, the CD3 antibody targets CD3 to bridge T cells and exert T cell killing. effect. The ratio of TAA antibody to CD3 antibody in the TAA/CD3/IL15 trifunctional fusion protein can be 2:1 or 1:1.

Generally speaking, the TAA/CD3/IL15 trifunctional fusion protein of the present invention has four functional components: anti-TAA antigen component, IL15 or IL-15/IL-15Rα complex, anti-CD3 component and Fc component, Each of these can take different forms, and each of them can be combined with the other components in any configuration.

A. TAA antigen binding molecules

The term "TAA" or "tumor-associated antigen" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of cancer cells, either in whole or as fragments (eg, MHC/peptides) , and it can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, the TAA is a marker expressed by both normal cells and cancer cells, eg, a lineage marker, eg, CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, eg, 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more compared to normal cells. In some embodiments, TAAs are cell surface molecules that are inappropriately synthesized in cancer cells, eg, molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, TAAs will only be expressed fully or as fragments (eg, MHC/peptides) on the cell surface of cancer cells, and not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific for cancer cells, sometimes referred to in the art as tumor specific antigens ("TSA").

"[antiTAA]", an anti-TAA antigen-binding molecule, may comprise, for example, an anti-TAA antibody, antibody derivative or polypeptide fragment. The anti-TAA antibody, antibody derivative or polypeptide fragment may comprise, for example, the CDR sequences of the antibodies listed in Table A. In some embodiments, the anti-TAA antibody or antigen binding domain thereof has the heavy and light chain variable region sequences of the antibodies listed in Table A.

Table A Exemplary Anti-Tumor-Associated Antigen Antibodies

In certain embodiments, the TAA is selected from CLDN18.2. In other embodiments, the TAA is selected from EGFR and folate receptor. Exemplary different forms of CLDN18.2/CD3/IL15 trifunctional fusion protein and EGFR/folate receptor/CD3/IL15 multifunctional fusion protein sequences are listed in Example 1 below.

B. IL-15/IL-15Rα(sushi) domain

IL-15 is produced on monocytes and dendritic cells and is predominantly presented as a membrane-bound heterodimeric complex with IL-15Rα presented on the same cells. Its effects are achieved by trans-presenting the IL-15/IL-15Rα complex to NK cells and CD8+ T cells expressing IL-2Rβ and a consensus γ chain. IL-15 has a very fast clearance rate and its half-life is measured in minutes. Additionally, IL-15 itself is less stable due to its preference for IL-15Rα-related complexes. Recombinantly produced IL-15/IL-15Rα heterodimers have also been shown to efficiently activate T cells.

As shown in the figures, the IL-15 complex can take several forms. As noted above, the IL-15 protein itself is less stable than when complexed with the IL-15Rα protein. As known in the art, the IL-15Rα protein contains a "sushi domain," which is the shortest region of the receptor that retains IL-15 binding activity. Thus, although heterodimeric fusion proteins comprising the entire IL-15Rα protein can be prepared, preferred embodiments herein include complexes using only the sushi domain.

Accordingly, IL-15 complexes typically comprise the IL-15 protein and the sushi domain of IL IL-15Rα (the full-length sequences are used unless otherwise specified, "IL-15Rα", "IL-15Rα(sushi)" and "sushi" is used interchangeably throughout). The complex can be used in three different forms. For example, IL-15 protein and IL-15Rα (sushi) are not covalently attached, but self-assemble through conventional ligand-ligand interactions. As described more fully herein, it can be an IL-15 domain or a sushi domain covalently linked to an Fc domain (generally using an optional domain linker). Finally, each of the IL-15 and sushi domains can be engineered to contain cysteine amino acids that form disulfide bonds to form complexes, again, where either the IL-15 domain or the sushi domain is covalently attached (using optional domain linker) to the Fc domain.

In some embodiments, the linker is a "domain linker" used to join any two domains together as outlined herein. While any suitable linker can be used, many embodiments utilize glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least one (and Typically 1 to 2 to 3 to 4 to 5), and any peptide sequence that allows for the recombinant attachment of the two domains is of sufficient length and flexibility to allow each domain to retain its biological function.

In some embodiments, the IL15/IL15Ra complex includes, but is not limited to: 1) IL15 and its mutants, truncations and various derivatives capable of binding IL15Ra; 2) IL15Ra and its mutants, truncations capable of binding IL15 and various derivatives; the mutations include but are not limited to those listed in Table B-1 (the counting method starts from the first amino acid of the IL15 sequence shown in SEQ ID NO: 1 as the 1st position ; the first amino acid of the IL15Ra sequence shown in SEQ ID NO: 3 starts to be counted as the 1st position).

Table B-1 Mutations Contained by Exemplary IL15/IL15Ra Complexes

In some embodiments, the IL15 includes, but is not limited to, mutations as listed in Table B-2 (counting is based on the first amino acid of the IL15 sequence shown in SEQ ID NO: 1, starting at position 1) .

Table B-2 Exemplary IL15-Contained Mutations

C. Anti-CD3 Antibody or Antibody Fragment

The term "CD3" refers to a cluster of differentiation 3 co-receptor (or co-receptor complex, or polypeptide chain of a co-receptor complex) of the T cell receptor. The amino acid sequence of the polypeptide chain of human CD3 is provided in NCBI accessions P04234, P07766 and P09693. CD3 proteins can also include variants. CD3 proteins can also include fragments. CD3 proteins also include post-translational modifications of the CD3 amino acid sequence. Post-translational modifications include, but are not limited to, N-linked and O-linked glycosylation.

As indicated herein, anti-CD3 antibodies include, but are not limited to, OKT3, SP34, UCTH1 and derivatives thereof or other specific antibodies that bind CD3, antibody fragments, single domain antibodies, and humanized forms of each, wherein single domain antibodies include Nanobodies Antibody. In some embodiments, the anti-CD3 antibody has a pair of disulfide bonds between VL and VH including, but not limited to, the mutations shown in Figure C (counted according to kabat EU).

D. Fc domain

The trifunctional fusion proteins of the present invention may include Fc domains derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

The Fc domain can be derived from any suitable class of antibodies, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgGl, IgG2, IgG3 or IgG4. In one embodiment, the Fc domain is derived from IgGl. In one embodiment, the Fc domain is derived from IgG4.

The Fc domain comprises two polypeptide chains, each referred to as the heavy chain Fc region. The two heavy chain Fc regions dimerize to generate Fc domains. The two Fc regions in the Fc domain may be the same or different from each other. In native antibodies, the Fc regions are typically identical, but for the purpose of generating multispecific binding molecules, eg, the trifunctional fusion proteins of the invention, the Fc regions may advantageously differ to allow heterologous two Polymerization, as described below.

Typically, each heavy chain Fc region comprises or consists of two or three heavy chain constant domains.

In native antibodies, the heavy chain Fc region of IgA, IgD and IgG consists of two heavy chain constant domains (CH2 and CH3) and the Fc region of IgE and IgM consists of three heavy chain constant domains (CH2, CH3 and CH4) )constitute. These antibodies dimerize to generate the Fc domain.

In the present invention, the heavy chain Fc region may comprise heavy chain constant domains from one or more different types (eg one, two or three different types) of antibodies.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgGl.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG2.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG3.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG4.

It will be appreciated that the heavy chain constant domains used to generate the heavy chain Fc regions for the trifunctional fusion proteins of the invention may include variants of the naturally occurring constant domains as described above. Such variants may contain one or more amino acid variations compared to wild-type constant domains. In one example, the heavy chain Fc region of the invention comprises at least one constant domain that differs in sequence from a wild-type constant domain. It will be appreciated that the variant constant domains may be longer or shorter than wild-type constant domains. For example, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the constant domains are at least 70% identical or similar. In another example, the constant domains are at least 75% identical or similar. In another example, the constant domains are at least 80% identical or similar. In another example, the constant domains are at least 85% identical or similar. In another example, the constant domains are at least 90% identical or similar. In another example, the constant domains are at least 95% identical or similar. In another example, the constant domains are at least 99% identical or similar.

The Fc domains incorporated into the trifunctional fusion proteins of the invention may comprise one or more modifications that alter one or more functional properties of the protein, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen dependent cytotoxicity. In addition, the trifunctional fusion proteins of the invention can be chemically modified (eg, one or more chemical moieties can be attached to the trifunctional fusion protein) or modified to alter their glycosylation, thereby again altering one of the trifunctional fusion proteins one or more functional characteristics. The structural forms of the trifunctional fusion proteins of the present invention are shown in Figures 1 to 18 .

Effector functions of antibody molecules include complement-mediated effector functions, which are mediated, for example, by the binding of the C1 component of the complement to the antibody. Activation of complement is important in opsonization and direct lysis of pathogens. Additionally, it stimulates inflammatory responses by recruiting and activating phagocytes to sites of complement activation. Effector functions include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of an antibody's constant domain to an Fc receptor (FcR). Antigen-antibody complex-mediated cross-linking of Fc receptors on the surface of effector cells triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated particles by killer cells Controlled by target cells (termed antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transport, and immunoglobulin production.

The Fc region can be altered by substituting at least one amino acid residue for a different amino acid residue to alter effector function. For example, one or more amino acids can be replaced with different amino acid residues such that the Fc region has an altered affinity for the effector ligand. The affinity-altering effector ligand can be, for example, an Fc receptor or the C1 component of complement. For example, Winter et al. describe this approach in both US Patent Nos. 5,624,821 and 5,648,260. Modified Fc regions can also alter Clq binding and/or reduce or eliminate complement-dependent cytotoxicity (CDC). For example, Idusogie et al. describe this method in US Patent No. 6,194,551. Modified Fc regions can also alter the ability of the Fc region to fix complement. For example, Bodmer et al. describe this method in PCT Publication WO 94/29351. Allotypic amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgGl, IgG2, and IgG3 subclasses and the constant regions of the light chains of the kappa isotype, as in Jefferis et al., 2009, MAbs, 1:332- 338.

The Fc region can also be modified to "silence" the effector function, eg, to reduce or eliminate the ability of the trifunctional fusion protein to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP). This can be achieved, for example, by introducing mutations in the Fc region. Such mutations have been described in the art: LALA and N297A (Strohl, 2009, Curr. Opin. Biotechnol. [Current Biotechnology Perspective] 20(6):685-691); and D265A (Baudino et al, 2008, J . Immunol. [J. Immunol.] 181:6664-69; Strohl, supra). Examples of silent Fc IgG1 antibodies include the so-called LALA mutants comprising L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgGl antibody includes the D265A mutation. Another silent IgGl antibody contains a so-called DAPA mutant comprising the D265A and P329A mutations in the IgGl Fc amino acid sequence. Another silent IgG1 antibody contained the N297A mutation, which resulted in an aglycosylated/aglycosylated antibody.

The Fc region can be modified to increase the ability of a trifunctional fusion protein containing the Fc region to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP), for example, by modifying one or more amino acids residues to increase the affinity of the trifunctional fusion protein for activating Fcγ receptors, or decrease the affinity of the trifunctional fusion protein for inhibitory Fcγ receptors. Human activating Fcy receptors include FcyRIa, FcyRIIa, FcyRIIIa, and FcyRIIIb, and human inhibitory Fcy receptors include FcyRIIb. This method is described, for example, by Presta in PCT Publication WO 00/42072. In addition, the binding sites on human IgG1 for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al., J. Biol. Chem. [J. Biol. Chem.] 276 : 6591-6604, 2001). Optimization of Fc-mediated effector functions of monoclonal antibodies such as increased ADCC/ADCP function has been described (see Strohl, 2009, Current Opinion in Biotechnology 20:685-691). Mutations that can enhance ADCC/ADCP function include one or more mutations selected from the group consisting of: G236A, S239D, F243L, P247I, D280H, K290S, R292P, S298A, S298D, S298V, Y300L, V305I, A330L, I332E, E333A, K334A , A339D, A339Q, A339T, and P396L (all positions by EU numbering).

The Fc region can also be modified to increase the ability of the trifunctional fusion protein to mediate ADCC and/or ADCP, for example, by modifying one or more amino acids to increase the affinity of the trifunctional fusion protein for activating receptors, typically The parental trifunctional fusion protein, such as FcaRI, is not recognized. This method is described, for example, in Borrok et al., 2015, mAbs. 7(4):743-751.

Thus, in certain aspects, the trifunctional fusion proteins of the invention may include disulfide modified with altered effector functions (eg, but not limited to binding to Fc receptors, such as FcRn or leukocyte receptors, binding to complement, modified Bond structure or altered glycosylation pattern of the Fc domain.Table D-1 provides various modification strategies for eliminating immune effects in the examples of the present invention.

Fc domains can also be altered to include manufacturable modifications that improve asymmetric trifunctional fusion proteins, for example by allowing heterodimerization (the preferential pairing of different Fc regions with respect to the same Fc region) . Heterodimerization allows the production of trifunctional fusion proteins in which different TAA antigen binding molecules are linked to each other by Fc domains containing Fc regions with different sequences.

Many multispecific molecular formats require dimerization between two Fc regions that, unlike native immunoglobulins, are operably linked to non-identical antigen-binding domains (or portions thereof, e.g., Fab's VH or VH-CH1). Insufficient heterodimerization of the two Fc regions forming the Fc domain has been an obstacle to improving the production of desired multispecific molecules and represents a purification challenge. Various methods available in the art can be used to enhance dimerization of the Fc region that may be present in the trifunctional fusion proteins of the invention, for example as disclosed in: EP 1870459A1; US Patent No. 5,582,996; US Patent No. 5,731,168; US Patent No. 5,910,573; US Patent No. 5,932,448; US Patent No. 6,833,441; US Patent No. 7,183,076; US Patent Application Publication No. 2006204493A1;

The present invention provides trifunctional fusion proteins comprising Fc heterodimers, ie Fc domains comprising heterologous, non-identical Fc regions. A heterodimerization strategy was used to enhance dimerization of Fc regions operably linked to different forms of [antiTAA], [antiCD3], [IL15] and decrease the dimerization of Fc regions operably linked to the same form of [antiTAA], [ antiCD3], dimerization of the Fc region of [IL15]. Typically, each Fc region in an Fc heterodimer comprises the CH3 domain of an antibody. The CH3 domains are derived from the constant regions of antibodies of any isotype, class or subclass, and in some cases of the IgG (IgGl, IgG2, IgG3, and IgG4) class, as previously described.

Typically, in addition to the CH3 domain, the trifunctional fusion protein also comprises other antibody fragments, such as the CH1 domain, CH2 domain, hinge domain, one or more VH domains, one or more of the herein described VL domains, one or more CDRs, and/or antigen-binding fragments. In some embodiments, the two heteropolypeptides are two heavy chains that form a bispecific or multispecific molecule. Heterodimerization of two different heavy chains at the CH3 domain produces the desired antibody or antibody-like molecule, whereas homodimerization of the same heavy chain will reduce production of the desired antibody or molecule. In an exemplary embodiment, the two or more heteropolypeptide chains comprise two chains comprising a CH3 domain and forming a molecule of any of the multispecific molecular formats of the invention described above. In an embodiment, the two heteropolypeptide chains comprising CH3 domains comprise modifications (relative to the unmodified chains) that facilitate heterodimeric fusion of the polypeptides. Table D-2 provides modification strategies for various heterodimers in the examples of the present invention.

2. Specific Examples

The specific embodiments of the present invention will be described below with reference to the accompanying drawings and embodiments. However, these examples do not limit the scope of the present invention.

In the following embodiments, the experimental methods that do not specify specific conditions are usually in accordance with conventional conditions, or in accordance with conditions suggested by raw material or commodity manufacturers. Such as "Molecular Cloning", "Laboratory Manual", "Cold Spring Harbor Laboratory", "Contemporary Molecular Biology Methods", "Cell Biology" and so on. Reagents with no specific source indicated are conventional reagents purchased in the market.

In the present invention, the technical scheme of the present invention is illustrated by the molecular design and molecular function detection of the trifunctional fusion protein with TAA as the specific anti-CLDN18.2 antibody and TAA as the specific anti-EGFR and folate receptor antibody.

Example 1: Molecular cloning and protein expression and purification of trifunctional fusion protein

Taking the protein QP32083209 as an example, the process of molecular cloning and expression and purification of the trifunctional fusion protein is described in detail. The same method is also used for the cloning, construction and expression and purification of other protein molecules in the present invention.

Molecular cloning, expression and purification of trifunctional fusion protein QP32083209

QD3208, QD3209 vector molecular cloning construction:

Construction of QD3208 vector encoding Hsp34VL-(G4S) _3IL15 (L52C), see SEQ ID NO: 72 and Hsp34VH-(G4S) _3IL15a Sushi(S40C)-G4S-Fc(Knob), Fc contains Knob mutation, namely T366W mutation, see SEQ ID NO: 73, the plasmid contains DHFR as a screening marker, which can be used for stable strain screening; construct the light chain sequence of the QD3209 vector encoding anti-tumor specific antigen and antibody, see SEQ ID NO: 75, and the anti-tumor specific antigen antibody light chain sequence. The heavy chain sequence, wherein Fc contains Hole mutations, namely T366S, L368A, Y407V, and H435R mutations, see SEQ ID NO: 74, and the plasmid contains GS as a selection marker, which can be used for stable strain selection. The plasmid structures are shown in Figures 19 and 20.

Transient expression of proteins in ExpiCHO-S cells:

ExpiCHO-S cells were seeded into FortiCHO medium (Gibco, A1148301) supplemented with 8 mM GlutaMax, and incubated at 37° C., 120 rpm, 8% CO ₂ . One day before transfection, the density of ExpiCHO-S cells was adjusted to 3*10E6/mL, placed in a shaker, 37°C, 120rpm, 8% _CO2 for culture; on the day of transfection, samples were taken, counted, and the cell density was diluted to 6 *10E6/ml, 40mL, placed in a 125ml shake flask; plasmids QD3208 and QD3209 were added in a 1:1 ratio with a total of 40ug mixed with 4.8mL Opti MEM, and 120ul Polyplus-FectoPRO transfection reagent was added. After the reagents were evenly mixed, they were placed at room temperature for 10 minutes, and the mixture was slowly placed in the cells. During the culture, each bottle was supplemented with 2 mL of Feed PFF05 (OPM, F81279-001) and 1 m of 30% glucose solution on the 1st, 4th, 6th, and 8th days, respectively. On the first day of transfection, reduce the temperature to 32 °C and the _CO concentration to 5%. After 13 days, samples were collected, centrifuged at 8000 rpm for 20 min, and the supernatant was taken for purification.

Purification of fusion proteins

Protein A affinity chromatography purification:

Pass the column with the equilibrium solution, at least 3CV, the actual volume is 20ml, to ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, and the flow rate is 1ml/min; the supernatant of the culture medium after centrifugation is passed through the column, and the sample is loaded with 40ml, and the flow rate is 0.33ml. /min; pass through the column with the balance solution, at least 3CV, the actual volume is 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the balance solution, and the flow rate is 0.33ml/min; use the eluent to pass through the column, and the UV280 will start when it rises to 15mAU The elution peak (PAC-EP) was collected, and the collection was stopped when the UV280 decreased to 15 mAU, and the flow rate was 1 ml/min. After sample collection, PAC-EP was adjusted to neutrality with pH adjustment solution.

CH1-XL affinity chromatography:

Centrifuge the samples treated with Protein A at 8000rpm × 15min, and take the supernatant; pass the column with the equilibrium solution, at least 3CV, the actual volume is 20ml, to ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, and the flow rate is 1ml/min; After the supernatant is passed through the column through the sample loop, the flow rate is 0.33ml/min; the balance liquid is passed through the column, at least 3CV, the actual volume is 20ml, to ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the balance liquid, and the flow rate is 0.33ml/min; Pass the column with the eluent, start collecting the elution peak (PAC-EP) when the UV280 rises to 10mAU, stop collecting when the UV280 drops to 10mAU, and the flow rate is 1ml/min. After sample collection is complete, CH1-EP is adjusted to neutrality with pH adjustment solution.

PNGase F digestion:

Take 39.5μl of sample, add 1.98μl of 10% SDS and 1.58μl of 1M DTT, mix well and boil at 100°C for 10 minutes; after the sample is cooled, add 4.8μl of 10% NP-40 and 1.0μl of PNGase F, after mixing, water bath at 4°C Overnight; the samples were heated at 75°C for 10 minutes to inactivate, and the samples were subjected to SDS-PAGE electrophoresis and SEC to detect the protein purity.

Reducing SDS-PAGE electrophoresis analysis:

Take 5 μg of samples before and after digestion, and dilute the volume to 48 μl with 1×PBS, then add 12 μl of 5×Protein Loading Dye, and heat at 95°C for 10 minutes. Pour Tris-HCl buffer (containing 0.1% SDS) at pH 8.3 into the electrophoresis tank, add samples to the sample tank with a pipette, and add 60 μl of sample to each sample tank. Set the voltage to 140V and the time to 100 minutes to start electrophoresis. After electrophoresis, the gel was taken out, soaked in staining solution for 30 minutes, and then destained with destaining solution until the protein band was clear. The results are shown in Figure 21. Among them, Lane 1 in Figure 21 represents the molecular weight standard (20.1, 29.0, 44.3, 66.4, 97.2, 116kDa); Lane2 represents the reduced SDS-PAGE analysis of the QP32083209 protein band after PNaseF digestion; Lane3 represents the reduced SDS-PAGE analysis of the QP32083209 protein No PNaseF digestion band was performed;

Band 1 represents hSP34VH-IL15Ra Sushi-Fc (Knob) and full-length heavy chain of anti-tumor-associated antigen (TAA) antibody (anti CLDN18.2VH-CH1-Fc); band 2 represents hSP34VL-IL15 protein (because it contains IL15 and its glycosylation site, the band is sporadic and larger than the full length of the light chain of the anti-TAA antibody (band 3); the band 3 represents the full length of the light chain of the anti-TAA antibody (anti CLDN18.2VL-CL ); Band 4 indicates that hSP34VL-IL15 protein was removed from the N sugar of IL15 by PNGaseF, resulting in the disappearance of band 2, and the band formed by overlapping with the light chain of anti-TAA antibody.

Non-reducing SDS-PAGE electrophoresis analysis:

Take 5 μg of sample, dilute the volume to 48 μl with 1×PBS, add 12 μl of 5×Protein Loading Dye (without reducing agent), and heat at 95°C for 10 minutes. Pour Tris-HCl buffer (containing 0.1% SDS) at pH 8.3 into the electrophoresis tank, add samples to the sample tank with a pipette, and add 60 μl of sample to each sample tank. Set the voltage to 140V and the time to 100 minutes to start electrophoresis. After electrophoresis, the gel was taken out, soaked in staining solution for 30 minutes, and then destained with destaining solution until the protein band was clear. The results are shown in Figure 22. Among them, Lane1 in Figure 22 represents the molecular weight standard (20.1, 29.0, 44.3, 66.4, 97.2, 116kDa); Lane2 represents the non-reducing SDS-PAGE analysis of QP32083209 protein; Band 1 is the non-reducing QP32083209 protein band with a molecular weight of about 150KDa.

The results showed that the trifunctional fusion protein QP32083209 was successfully obtained after the above molecular cloning construction and protein expression and purification.

Molecular Design of Control Proteins

The anti-CLDN18.2 mAb protein number is QP14611463. QP14611463 is obtained by co-transfection of plasmid QD1461 and plasmid QD1463. The expressed protein sequence is as follows:

QD1461(下划线为信号肽序列): MDMRVPAQLLGLLLLWFPGSRC DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDHSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC*

QD1463(下划线为信号肽序列): MEFGLSWLFLVAILKGVQC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIMHWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLGFTTRNAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

IL15 was designed as a positive control IL15/IL15Ra-Fc, see the article (Scientific ReporTs|(2018) 8:7675|DOI: 10.1038/s41598-018-25987-4 in protein P22339), the molecular clone was constructed, and the expression and purified protein number was QP33123313 It was obtained by co-transfection of plasmid QD3312 and plasmid QD3313. The expressed protein sequence is shown below:

QD3312 (signal peptide sequence underlined): MEFGLSWLFLVAILKGVQC NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*

QD3313(下划线为信号肽序列): MEFGLSWLFLVAILKGVQC ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

Molecular cloning design of different forms of other trifunctional fusion proteins

All other protein molecular cloning and protein expression purification methods are the same as the QP32083209 protein shown above. For example, the design of molecular clone in the form of CLDN18.2/CD3/IL15 trifunctional fusion protein is shown in Table 1.

Table 1: Cloning design of different forms of CLDN18.2/CD3/IL15 trifunctional fusion protein

The molecular clone design of EGFR/folate receptor/CD3/IL15 multifunctional fusion protein is shown in Table 2 below:

Table 2: Cloning design of different forms of EGFR/folate receptor/CD3/IL15 multifunctional fusion protein

Example 2: FACS detection of the ability of TAA antibodies in trifunctional fusion protein format to bind to cells stably or natively expressing TAA protein

Experimental sample:

CLDN18.2/CD3/IL15 trifunctional fusion protein samples: QP32083209, QP331633193320, QP34683469, QP34683475, QP34683478, QP36503651, QP36633631, QP34683472, QP36543651, QP36573651,

EGFR/folate receptor/CD3/IL15 multifunctional fusion protein samples: QP34843487, QP35293530, QP35293644, QP35293645

Positive control and negative control: QP14611463, human IgG

Experimental steps:

Prepare to express TAA protein (such as CHOS cells stably expressing CLDN18.2 to detect the ability of CLDN18.2/CD3/IL15 trifunctional fusion protein samples to bind to human CLDN18.2 protein; human epidermal cancer cell line A431 naturally expressing EGFR, natural The human ovarian cancer cell line SK-OV-3 expressing folate receptor was used to detect the ability of EGFR/folate receptor/CD3/IL15 multifunctional fusion protein samples to bind to human EGFR and folate receptor respectively), count, centrifuge at 300g for 5min, remove the upper Clean, wash once with 5ml 1XPBS, remove PBS, inoculate cells at 1E5/well to a 96-well plate, calculate the volume of blocking solution, and seal in a centrifuge tube. Blocking: blocking solution 3% FBS/PBS, 1h on ice, after blocking and plating, 50ul/well. Incubate protein: configure different concentrations according to protein molecular weight, dilute each protein in a gradient, add 50ul/well to the cell suspension, mix well by pipetting, incubate on ice for 1 h, centrifuge at 300 g, 4 °C, 5 min, wash 2 times with PBS, wash with Trigger to aspirate PBS. Incubation secondary antibody: PE goat-anti human IgG Fc (1:200) diluted, 30ul/well, incubated on ice for 45min, washed 3 times with PBS, add 200ul of 1XPBS for each wash and centrifuge at 300g at 4°C for 5min. Reading: Resuspend cells with 120ul/well of 3% FBS/PBS, and read the mean fluorescence value by FACS. The results were analyzed using graphpad prism software.

The FACS detection results are shown in Figures 23a-c and Figures 27-28.图23a，图23b，图23c的FACS实验结果表明CLDN18.2/CD3/IL15三功能融合蛋白QP32083209,QP331633193320,QP34683469,QP34683475,QP34683478,QP36503651,QP36633651,QP34683472,QP36543651,QP36573651,QP36603651均结合稳定表达CLDN18 .2 CHOS cells that bind human CLDN18.2 protein. At the same time, the positive control CLDN18.2 mAb QP14611463 binds to CHOS-CLDN18.2, while the negative control human IgG does not bind to CHOS-CLDN18.2. The results of FACS experiments in Figure 27 and Figure 28 show that the EGFR/folate receptor/CD3/IL15 multifunctional fusion proteins QP34843487, QP35293530, QP35293644, and QP35293645 all bind to A431 cells that naturally express EGFR and SK-OV-3 cells that naturally express folate receptor protein , that is, both bound to EGFR and folate receptor proteins.

Example 3: FACS detection of CD3 antibody binding to Jurkat cells naturally expressing CD3 protein in trifunctional fusion protein

CLDN18.2/CD3/IL15三功能融合蛋白样品：QP331633173318,QP331633193320,QP331633213322,QP331633233324,QP32083209,QP34683469,QP34683475,QP36503651,QP36633651,QP34683472,QP36543651,QP36573651,QP36603651

Positive control and negative control: QP14611463, human IgG

Experimental steps:

Prepare Jurkats cell resuspension, count, centrifuge at 300g for 5min, remove supernatant, wash with 5ml 1XPBS, remove PBS, inoculate cells into 96-well plate according to 1E5/well, calculate the volume of blocking solution, and seal in centrifuge tube. Blocking: blocking solution 3% FBS/PBS, 1h on ice, after blocking and plating, 50ul/well. Incubation antibody: configure the highest molar concentration of 667nM according to the molecular weight of the antibody, dilute 8 concentration gradients, 3, 3, 5, 5, 5, 10, 100 times dilution. Add 50ul/well to the cell suspension, mix well by pipetting, incubate on ice for 1h, centrifuge at 300g, 4°C, 5min for 5min, wash twice with PBS, and aspirate the PBS with a plate washer. Incubation secondary antibody: PE goat-anti human IgG Fc (1:200) diluted, 30ul/well, incubated on ice for 45min, washed 3 times with PBS, add 200ul of 1XPBS for each wash and centrifuge at 300g at 4°C for 5min. Reading: Resuspend the cells with 120ul/well of 3% FBS/PBS, read the average fluorescence value by FACS, and analyze the results using graphpad prism software.

The results of FACS detection are shown in Figure 24a-c and Figure 29.图24a,图24b，图24c的FACS实验结果表明CLDN18.2/CD3/IL15三功能融合蛋白QP331633173318,QP331633193320,QP331633213322,QP331633233324,QP32083209,QP34683469,QP34683475,QP36503651,QP36633651,QP34683472,QP36543651,QP36573651,QP36603651均Binds to Jurkat cells that naturally express CD3, that is, to human CD3 protein, while CLDN18.2 monoclonal antibody QP14611463, IL15/IL15Ra fusion protein QP33123313 and human IgG do not bind to Jurkat cells. The results of the FACS experiment in FIG. 29 show that the EGFR/folate receptor/CD3/IL15 multifunctional fusion proteins QP34843487, QP35293530, QP35293644, and QP35293645 all bind to Jurkat cells that naturally express CD3, that is, bind to human CD3 protein.

Example 4: Mo7e cell proliferation experiment

Experimental sample:

CLDN18.2/CD3/IL15 trifunctional fusion protein samples: QP34683469, QP36503651, QP36633651, QP32083209, QP33123313, QP36603651, QP36573651, QP36543651

EGFR/folate receptor/CD3/IL15 multifunctional fusion protein samples: QP35293533, QP35293644, QP35293645

Positive control and negative control: QP33123313, human IgG

Experimental reagents:

Mo7e cells, the human giant cell leukemia cell line, were purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. Available from perprotech, Cat. No. 300-03; Human IgG, from Sigma, Cat. No. I4506; other antibodies were prepared in-house.

experimental method:

Mo7e cells were cultured in RPMI1640 containing 10% FBS, 2mM L-glutamine and 8ng/ml GM-CSF in a 37°C 5% CO2 incubator; cells were resuspended in RPMI1640 medium without GM-CSF and counted, The cells were seeded in 2×104 cells, 80 μl per well in a 96-well plate; after each antibody to be tested was diluted 4-fold with the medium, 20 μl per well was mixed with the cell suspension evenly, in a 37°C 5% CO2 incubator cultured for 3 days; add 10 μl of CCK-8 reagent per well to the 96-well plate to be tested, incubate for 4 hours in a 37°C 5% CO2 incubator; absorbance value. The results were analyzed using graphpad prism software.

The results of Mo7e cell proliferation experiments are shown in Figure 25a-b and Figure 30 . Figure 25a and Figure 25b show the results of the Mo7e cell proliferation experiment that the CLDN18.2/CD3/IL15 trifunctional fusion proteins QP34683469, QP36503651, QP36633651, QP32083209, QP33123313, QP36603651, QP36573651, and QP36543651 can all promote the proliferation of Mo7e cells, indicating that the trifunctional fusion proteins can promote the proliferation of Mo7e cells. IL15/IL15Ra in the protein has biological activity, and all of them are significantly weaker than IL15/IL15Ra fusion protein QP33123313, which is consistent with our designed target to reduce the toxicity of IL15/IL15Ra. The results of the Mo7e cell proliferation assay in Figure 30 show that the EGFR/folate receptor/CD3/IL15 multifunctional fusion proteins QP35293533, QP35293644, and QP35293645 can all promote the proliferation of Mo7e cells, indicating that their IL15/IL15Ra have biological activity, and they are significantly weaker than IL15/ IL15Ra fusion protein QP33123313, which is consistent with our designed target to reduce IL15/IL15Ra toxicity.

Example 5: Cytotoxicity assays

Purpose:

It is determined that our molecule to be tested can achieve the function of bridging T cell TCR receptors by targeting CD3 at one end, and bridging the target cell at the other end by targeting the target cell surface antigen to activate T cells to kill target cells.

experimental method:

Prepare target cells (such as human gastric cancer cell NUGC4-hCLDN18.2 (stably expressing human CLDN18.2), human epidermal cancer cell A431 (naturally expressing human EGFR), human epidermal cancer cell SK-OV-3 (naturally expressing human folate receptor) ), digested with 0.25% trypsin, and centrifuged at 300 g for 5 min. Change fresh medium (containing 10% FBS) to plate 96-well plate, 15000 cells/well, and incubate overnight. On the same day, before adding antibodies, cells were washed once with pre-warmed 1×PBS, and then 40 μl/well of pre-warmed medium (RPMI1640 containing 5% low-IgG FBS) was added for incubation. Prepare the antibody: Dilute the protein with different concentration gradients in the medium. Add 40 μl/well of the above-diluted antibodies of each concentration. Prepare PBMCs: revive PBMCs, incubate overnight, wash, resuspend in medium, and count. According to PBMC:Target cell=10:1, add 40 μl/well to the above well plate, and incubate at 37°C for 48h. Add 13.3 μl lysis buffer (1% Triton-X100) to the largest lysis well, and incubate at 37°C for 45 min. Detection: Centrifuge the 96-well plate at 250 × g for 4 min, and transfer 50 μl/well of the supernatant to a new transparent 96-well plate. Take out CytoTox

Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000assays), prepare Reaction Solution, add 50μl/well, and let stand at room temperature for 30min. The reaction was stopped by adding 50 μl of stop solution. 490 nm reading, data were analyzed according to the formula %Cytotoxicity=[(Experimental-Effector Spontaneous-Target Spontaneous)/(Target Maximum-Target Spontaneous)]×100. Data entry and analysis were performed using Graphpad Prism software analysis.

The results of the PBMC killing experiments are shown in Figures 26a-b and 31-32. As shown in Figure 26a and Figure 26b, the PBMC killing assay detected the biological function activity of antiCLDN18.2/antiCD3 in the CLDN18.2/CD3/IL15 trifunctional fusion protein.

Figure 26a, Figure 26b PBMC killing experiment results show that CLDN18.2/CD3/IL15 trifunctional fusion proteins QP34683478, QP36503651, QP36633651, QP32083209, QP34683469, QP34683475 can cause T cells in PBMC to kill human gastric cancer stably expressing CLDN18.2 Cells NUGC4-CLDN18.2 cells, and CD3 monoclonal antibody OKT3 and human IgG did not kill.

Figure 31 and Figure 32 show that EGFR/folate receptor/CD3/IL15 multifunctional fusion protein QP35293530 can cause T cells in PBMC to kill the human epidermal cancer cell line A431 that naturally expresses EGFR and cause T cells in PBMC to kill the natural expression of EGFR The human epidermal cancer cell line SK-OV-3 of the folate receptor, and the CD3 monoclonal antibody OKT3 and human IgG did not kill.

Claims

A trifunctional fusion protein, characterized in that the trifunctional fusion protein comprises: an anti-tumor surface antigen (TAA) antibody part, an anti-CD3 antibody part, an IL15/IL15Rα complex part, and a heterodimeric Fc part.

The trifunctional fusion protein of claim 1, wherein the IL15 comprises the following mutations, and the counting method starts from the first amino acid of IL15 shown in SEQ ID No.1 and counts as the first position:

combination IL15 mutation 1 N1D 2 N4D 3 D8N 4 D30N 5 D61N 6 E64Q 7 N65D 8 Q108E 9 N1D/D61N 10 N1D/E64Q 11 N4D/D61N 12 N4D/E64Q 13 D8N/D61N 14 D8N/E64Q 15 D61N/E64Q 16 E64Q/Q108E 17 N1D/N4D/D8N 18 D61N/E64Q/N65D 19 N1D/D61N/E64Q/Q108E 20 N4D/D61N/E64Q/Q108E

Or the IL15 and IL15Rα contain the combination of mutations shown below, and the counting method starts from the first amino acid of IL15 shown in SEQ ID No.1 and counts as the first position; the first amino acid of IL15Rα shown in SEQ ID No.3 starts to count as 1st place.

The trifunctional fusion protein according to claim 1, wherein the anti-TAA antibody and the anti-CD3 antibody comprise variable regions VL and VH, respectively, and the anti-TAA antibody and/or the anti-CD3 antibody has a difference between VL and VH. There is a pair of disulfide bonds between them, including the following combinations of mutations, according to EU counts.
The trifunctional fusion protein of claim 1, wherein the heterodimeric Fc comprises different mutated A and B chains; the A and B chains have the following combinations of mutations, counted according to EU:
The trifunctional fusion protein of claim 1, wherein the Fc segment comprises Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc and mutants thereof; A chain and B of the Fc segment chain, one of which is capable of binding protein A and the other is a mutant that is not capable of binding protein A, the mutation comprising H435R or H435R/Y436F, according to EU counts.
The trifunctional fusion protein of claim 1, wherein the Fc segment is in a form to eliminate immune effects, comprising the following combination of mutations:
The trifunctional fusion protein of claim 1, wherein the anti-CD3 antibody is OKT3, SP34, UCTH1 and derivatives thereof, or other antibodies or antibody fragments that bind to CD3.
The trifunctional fusion protein of claim 1, wherein the anti-tumor surface antigen antibody portion is an antibody that binds at least one TAA antigen; the anti-TAA antibody is a monovalent or multivalent molecule, and the anti-TAA antibody The bound antigen or antigen-specific mutations include CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD217, CD239, CD274, CD276, PD-1, CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, IL-6R, IL-2R, CCR4, VEGFR-2, CD6, integrin α4, PDGFRα, NeuGcGM3, IL-4Rα, IL-6Rα.
The trifunctional fusion protein of claim 1, wherein the anti-TAA antibody or the anti-CD3 antibody is a chimeric, humanized or fully human antibody.
The trifunctional fusion protein of claim 1, wherein the anti-TAA antibody, anti-CD3 antibody, IL15/IL15Rα, and heterodimeric Fc are connected by a linker sequence, and the linker sequence is low in immunogenicity amino acid sequence.
The trifunctional fusion protein according to any one of claims 1-10, wherein it has the structure shown in formula I:

Chain 1: [VL1A/VH1A], [antiCD3 VH/VL], [IL15/IL15Ra] fusion;

Chain 2: [antiCD3 VL/VH], [VH1A/VL1A], [IL15Ra/IL15] fusion, C-terminal fusion [Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[CL]

(Formula I)

The chain 1: TAA-1 binding antibody variable region VL1A or VH1A and CD3 binding antibody variable region VL or VH and IL15 or IL15Ra fusion; the chain 2: CD3 binding antibody variable region VH or VL and The antibody variable region VH1A or VL1A that binds to TAA-1 is fused with IL15Ra or IL15, and the C-terminal is fused to the Fc of the heterodimer; the chain 3: from the N-terminal to the C-terminal, the antibody that binds to TAA-2 can be Variable region VH1B, CH1 fragment, Fc that can form heterodimers; said chain 4: C-terminal fusion CL fragment of antibody variable region VL1B that binds TAA-2.
The trifunctional fusion protein according to any one of claims 1-10, characterized in that it has the structure shown in formula II:

Strand 1: [VL1A/VH1A], [IL15/IL15Ra] fusion

Chain 2: [VH1A/VL1A], [IL15Ra/IL15] fusion, C-terminal fusion [Fc (heterodimeric A chain or B chain)]

Chain 3: [VL1B/VH1B], [antiCD3 VH/VL] fusion, C-terminal fusion [CH1]-[Fc (heterodimeric A chain or B chain)]

Strand 4: [antiCD3 VL/VH], [VH1B/VL1B] fusion, C-terminal fusion [CL]

(Formula II)

The chain 1: TAA-1 binding antibody variable region VL1A or VH1A fused to IL15 or IL15Ra; the chain 2: TAA-1 binding antibody variable region VH1A or VL1A fused to IL15Ra or IL15; at its C-terminus Fusion can form a heterodimer Fc; the chain 3: TAA-2 binding antibody variable region VH1B or VL1B and CD3 binding antibody variable region VL or VH fusion, fused at its C-terminus [CH1]- [Fc (heterodimeric A chain or B chain)]; chain 4: fusion of TAA-2-binding antibody variable region VL1B or VL1B and CD3-binding antibody variable region VH or VL, C-terminal fusion of CL fragment.
The trifunctional fusion protein according to any one of claims 1-10, characterized in that it has the structure shown in formula III:

Strand 1: [antiCD3 VH/VL], [IL15/IL15Ra] fusion

Chain 2: [antiCD3 VH/VL], [IL15Ra/IL15] fusion, C-terminal fusion [Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1]-[CL]

(Formula III)

Said chain 1: fusion of CD3-binding antibody VL or VH with IL15 or IL15Ra; said chain 2: fusion of CD3-binding antibody VH or VL with IL15Ra or IL15, and fusion of heterodimeric Fc at its C-terminus; The chain 3: from the N-terminus to the C-terminus, the TAA-binding antibody variable region VH1 is fused to the CH1 fragment, and the C-terminus is fused with an Fc that can form a heterodimer to form an antibody heavy chain; the chain 4: binding The C-terminal of the antibody variable region VL1 of TAA is fused to the CL fragment to form the antibody light chain.
The trifunctional fusion protein according to any one of claims 1-10, characterized in that, it has the structure shown in formula IV:

Chain 1: [VL1/VH1], [antiCD3 VH/VL] fusion, C-terminal link [IL15/IL15Ra];

Chain 2: [antiCD3 VL/VH], [VH1/VL1] fusion, C-terminal link [IL15Ra/IL15]-[Fc (heterodimer A chain or B chain)]

Chain 3: [Fc (heterodimeric A chain or B chain)]

(Formula IV)

The chain 1: TAA-binding antibody variable region VL1 or VH1 and CD3-binding antibody variable region VL or VH fusion, C-terminal is connected to IL15 or IL15Ra; the chain 2: CD3-binding antibody variable region VH or VL It is fused to the variable region VH1 or VL1 of the antibody that binds to TAA, and the C-terminus is connected to [IL15Ra/IL15]-[Fc (heterodimer A chain or B chain)]; the chain 3: the Fc of the heterodimer chain A or chain B.
The trifunctional fusion protein of any one of claims 11-14, wherein the chain 1 and the chain 2 comprise the combination forms described in the following table:
The trifunctional fusion protein of claim 1, wherein the TAA is CLDN18.2; or the TAA is EGFR and folate receptor alpha.
The trifunctional fusion protein of claim 11, wherein the TAA-1 and TAA-2 are both CLDN18.2; the Fc is IgG4 subtype; IL15 and IL15Ra of the chain 1 and chain 2 Complex fusion proteins have disulfide bond formation.
The trifunctional fusion protein of claim 17, wherein the 4 chains of the trifunctional fusion protein respectively comprise the sequences SEQ ID NO:36, SEQ ID NO:35, SEQ ID NO:34, SEQ ID NO: :33; or SEQ ID NO:38, SEQ ID NO:37, SEQ ID NO:34, SEQ ID NO:33; or SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:34, SEQ ID NO: :33; or SEQ ID NO:42, SEQ ID NO:41, SEQ ID NO:34, SEQ ID NO:33.
The trifunctional fusion protein of claim 11, wherein the TAA-1 and TAA-2 are respectively one of EGFR and folate receptor α; Fc is an IgG4 heterodimer; chain 1 and chain 2 The IL15 and IL15Ra complex fusion proteins have disulfide bond formation.
The trifunctional fusion protein of claim 19, wherein the 4 chains of the trifunctional fusion protein respectively comprise the sequences SEQ ID NO:46, SEQ ID NO:45, SEQ ID NO:44, SEQ ID NO: :43; or SEQ ID NO:48, SEQ ID NO:47, SEQ ID NO:44, SEQ ID NO:43; or SEQ ID NO:50, SEQ ID NO:49, SEQ ID NO:44, SEQ ID NO:43; :43; or SEQ ID NO:52, SEQ ID NO:51, SEQ ID NO:44, SEQ ID NO:43; or SEQ ID NO:56, SEQ ID NO:55, SEQ ID NO:54, SEQ ID NO: : 53; or SEQ ID NO: 58, SEQ ID NO: 57, SEQ ID NO: 54, SEQ ID NO: 53; or SEQ ID NO: 60, SEQ ID NO: 59, SEQ ID NO: 54, SEQ ID NO: 53; : 53; or SEQ ID NO:62, SEQ ID NO:61, SEQ ID NO:54, SEQ ID NO:53.
The trifunctional fusion protein of claim 14, wherein the TAA is CLDN18.2; the Fc is IgG4 subtype; the IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bond formation.
The trifunctional fusion protein of claim 21, wherein the three chains of the trifunctional fusion protein comprise the sequence SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:63; or SEQ ID NO: :67, SEQ ID NO:66, SEQ ID NO:63; or SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:63; or SEQ ID NO:71 SEQ ID NO:70, SEQ ID NO: 63.
trifunctional fusion protein as claimed in claim 13, is characterized in that, it is arranged from N-terminal to C-terminal, and it has the structure shown in formula V:

Strand 1: [antiCD3 VL]-[IL15]

Chain 2: [antiCD3 VH]-[IL15Ra]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1]-[CL]

(Formula V) wherein said VH1 and VL1 are anti-TAA antibody variable regions.
The trifunctional fusion protein of claim 23, wherein the TAA is CLDN18.2; the Fc is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bonds Formed, the 4 chains comprise the sequences SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75.
The trifunctional fusion protein of claim 12, wherein the chain 3 and chain 4 are fused to heavy chains in the form of a diabody by a CD3-binding antibody variable region and a TAA-2-binding antibody variable region in the form of a diabody Constant region CH1-CH2-CH3 or antibody light chain constant region CL composition.
The trifunctional fusion protein of claim 25, wherein:

The TAA-1 and TAA-2 are both CLDN18.2; the Fc is IgG4 subtype; the IL15 and IL15Ra complex fusion proteins of the chain 1 and chain 2 have disulfide bond formation; the trifunctional fusion protein Contains the sequences SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:77, SEQ ID NO:76; or SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:80 ; or SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:83, SEQ ID NO:82; or SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:84 .
The trifunctional fusion protein of claim 12, wherein chain 3 and chain 4 are constant by fusion of a heavy chain in the form of a DVD-Ig by an antibody variable region that binds CD3 and an antibody variable region that binds TAA-2 The region CH1-CH2-CH3 or the antibody light chain constant region CL is composed of the structure shown in formula VI:

Strand 1: [VL1A/VH1A]-[IL15/IL15Ra];

Chain 2: [VH1A/VL1A]-[IL15Ra/IL15/]-[Fc (heterodimeric A chain or B chain)]

Chain 3: [VH1B]-[antiCD3 VH]-[CH1]-[Fc (heterodimeric A chain or B chain)]

Chain 4: [VL1B]-[antiCD3 VL]-[CL]

(Formula VI)

Wherein, the chain 1: the C-terminus of the antibody variable region VL1A or VH1A that binds to TAA-1 is fused to IL15 or IL15Ra; the chain 2: the C-terminus of the antibody variable region VH1A or VL1A that binds to TAA-1 is fused to IL15Ra or IL15, whose C-terminus can be re-fused to form a heterodimeric Fc; the chain 3: the C-terminus of the antibody variable region VH1B that binds to TAA-2 is fused to the antibody variable region VH that binds to CD3, and the C-terminus is re-assembled. The CH1 fragment and the Fc that can form a heterodimer are fused; the chain 4: the C-terminus of the antibody variable region VL1B that binds TAA-2 is fused to the variable region VL of the CD3-binding antibody, and the CL fragment is fused to its C-terminus.
The trifunctional fusion protein of claim 27, wherein:

The TAA-1 and TAA-2 are both CLDN18.2; the Fc is an IgG4 heterodimer; the IL15 and IL15Ra complex fusion proteins of the chain 1 and chain 2 have disulfide bond formation; the three The functional fusion protein comprises the sequences SEQ ID NO:79, SEQ ID NO:78, SEQ ID NO:87, SEQ ID NO:86.
The trifunctional fusion protein of any one of claims 11-14, wherein the IL15 sequence is shown in SEQ ID No.1 or SEQ ID No.2; the IL15Rα sequence is shown in SEQ ID No.3 , SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9.
The trifunctional fusion protein of any one of claims 11-14, wherein the sequence of the anti-CD3 antibody comprises SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: :13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:1 twenty one.
The trifunctional fusion protein of any one of claims 11-14, wherein the human Fc fragment sequence comprises SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: :25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.
Use of the trifunctional fusion protein according to any one of claims 1 to 31 in the preparation of medicines for cancer, infection or immunomodulatory diseases.
Use of the trifunctional fusion protein according to any one of claims 1-31 in the preparation of a medicament for inhibiting tumor growth.
The application according to claim 32 or 33, wherein the cancer or tumor comprises: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, intrauterine cancer Membrane cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, bone marrow cancer, lymphoma, leukemia, small cell lung cancer, non-small cell lung cancer, melanoma, basal Cellular skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.