WO2023284733A1

WO2023284733A1 - GITR/TGF-β DUAL-TARGETED FUSION PROTEIN AND USE THEREOF

Info

Publication number: WO2023284733A1
Application number: PCT/CN2022/105175
Authority: WO
Inventors: 周雅琼; 高章照; 吴振华; 聂磊; 胡锋; 齐健; 王海彬
Original assignee: 海正生物制药有限公司; 浙江博锐生物制药有限公司
Priority date: 2021-07-14
Filing date: 2022-07-12
Publication date: 2023-01-19
Also published as: CN113429488B; CN113429488A

Abstract

The present invention relates to the field of biomedicine, and provides a GITR/TGF-β dual-targeted fusion protein, a conjugate and pharmaceutical composition comprising same, and a use of the fusion protein or conjugate in the preparation of a drug.

Description

GITR/TGF-β dual targeting fusion protein and its application

technical field

The invention relates to the field of biomedicine. Specifically, the present invention discloses a GITR/TGF-β dual-targeting fusion protein, a conjugate and a pharmaceutical composition comprising the same, and the use of the fusion protein or the conjugate in preparing medicines.

Background technique

Immunotherapy treatment options are broad and include a variety of approaches. One of the most successful treatment options to date has been treatment with immune checkpoint inhibitors (such as anti-PD-L1 antibodies and anti-PD-1 antibodies), which have demonstrated highly durable responses and significantly prolonged survival in a variety of advanced tumors. overall lifetime. Despite the progress of checkpoint inhibitors in anticancer therapy, the vast majority of patients remain unresponsive to checkpoint inhibitors.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine belonging to the transforming factor superfamily. The TGF-β signaling pathway is mediated by the TGF-β receptor complex composed of TGF-β type I receptor (TGF-βRⅠ) and TGF-β type II receptor (TGF-βRⅡ), and regulates cell growth, proliferation, differentiation, apoptosis Important cellular processes such as death and migration. TGF-β signaling pathway is closely related to tumor occurrence and development. In one aspect, TGF-β contributes to epithelial-mesenchymal transition (EMT) of tumor cells, promoting tumor cell invasion and metastasis. On the other hand, TGF-β inhibits the proliferation of T cells and B cells and inhibits the production of immune factors by B lymphocytes. It can also weaken the activity of T cells by inhibiting the function of antigen-presenting cells (such as dendritic cells). This mechanism suppresses the antitumor response of the immune system. In addition, TGF-β can promote the growth and metastasis of tumor cells by stimulating angiogenesis.

Some TGF-β inhibitors have been developed in the prior art, such as TGF-β Trap (dimeric extracellular domain of TGF-βRII, see for example WO2015118175A2 and WO2018205985A1) and anti-TGF-β monoclonal antibody fresolimumab (see for example US7723486B2 ). Clinically, the combination therapy of TGF-β inhibitors and anti-PD-L1 antibodies or anti-PD-1 antibodies can relieve the suppression of the immune system, thereby enhancing the effect of anti-tumor therapy. For example, WO2018205985A1 and WO2015118175A2 disclose bifunctional molecules of anti-PD-L1 antibody and TGF-βTrap, which can simultaneously block TGF-β signaling pathway and inhibit immune checkpoint for cancer treatment.

Glucocorticoid-induced tumor necrosis factor receptor (GITR or AITR) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). GITR is highly expressed on the surface of regulatory T cells (Treg), and has lower expression on the surface of naive T cells and memory T cells. When effector T cells (Teff) are activated, the expression level of GITR will increase rapidly in a short time. GITR signaling plays an important role in immune regulation. On the one hand, the activation of GITR on the surface of Teff can promote the proliferation and survival of Teff cells, thereby enhancing immune function. On the other hand, the activation of GITR on the surface of Treg promotes the failure of Treg cells, thereby indirectly activating the function of Teff.

Contents of the invention

In one aspect, the present invention provides a dual targeting fusion protein targeting GITR and TGF-β, which comprises from N-terminus to C-terminus:

The first polypeptide-the second polypeptide-the third polypeptide Formula (1);

or

The third polypeptide-the second polypeptide-the first polypeptide formula (2);

in

The first polypeptide comprises an extracellular domain of TGF-βRII (TGFBR2-ECD);

The second polypeptide has the following structure from N-terminus to C-terminus:

Lk ₁ –Dd–Lk ₂ ,

in

Dd is a dimerization domain;

Lk ₁ and Lk ₂ are each independently a linker or absent; and

The third polypeptide has the following structure from the N-terminus to the C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ,

in

Sd ₁ , Sd ₂ and Sd ₃ are each independently an extracellular domain of GITRL;

Ln ₁ and Ln ₂ are each independently a linker or absent.

In one embodiment, the TGFBR2-ECD comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

In one embodiment, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In a specific embodiment, said third polypeptide comprises the amino acid sequence of SEQ ID NO:3.

In one embodiment, the dimerization domain comprises from N-terminus to C-terminus the CH2 and CH3 domains of an immunoglobulin, preferably the CH2 and CH3 domains of human IgG1.

In one embodiment, the second polypeptide has the following structure from the N-terminus to the C-terminus:

Lk ₁ -Dd -Lk ₂ ;

in

Dd is a dimerization domain comprising from N-terminus to C-terminus the hinge region or part thereof, CH2 and CH3 domains of an immunoglobulin, preferably the Fc fragment of human IgG1; preferably, the hinge region or part thereof comprises The amino acid sequence of SEQ ID NO:9, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8;

Lk ₁ and Lk ₂ are each independently a linker.

In a specific embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

In another aspect, the invention provides a polynucleotide encoding a fusion protein of the invention. The present invention also provides an expression vector or host cell comprising a polynucleotide encoding the fusion protein of the present invention.

The invention also relates to a conjugate comprising a fusion protein of the invention conjugated to at least one therapeutic agent. Preferably, the therapeutic agent is selected from detectable markers, chemotherapeutic agents, cytotoxins, radionuclides, immune checkpoint inhibitors, cytokines and enzymes.

In yet another aspect, the present invention provides a pharmaceutical composition comprising:

(i) fusion protein of the present invention, and

(ii) A pharmaceutically acceptable carrier.

In another aspect, the present invention also relates to the use of said fusion protein, polynucleotide, conjugate or pharmaceutical composition.

Description of drawings

Figure 1: Analysis of fusion proteins C15 and C2 by reducing protein gel electrophoresis (SDS-PAGE). Molecular weights (kDa) indicated using protein standards are shown on the left. Lane 1: C15; Lane 2: C2.

Figure 2: Binding activity of B22, C15, C12 and C2 to TGF-β analyzed by ELISA.

Figure 3: In vitro binding affinity and kinetics of C12, 336B11 and 36E5 to hGITR analyzed by Fortebio.

Figure 4: In vitro binding activity of C12 and C15 to CHOS-hGITR cells analyzed by FACS.

Figure 5: In vitro activation of GITR by C12, C15 and 36E5 analyzed by GITR Blockade Bioassay. IgG Isotype was used as negative control.

Figure 6: In vitro blocking of TGF-β activity by C12 and anti-TGF-β antibody BMK-R2 analyzed by TGF-β receptor reporter assay. Human IgG1 was used as a negative control.

Figure 7: The activities of C12, 336B11 and 36E5 to activate T cells in vitro analyzed by ELISA. Protein samples: 336B11, C12, IgG Isotype (IgG isotype, negative control) and 36E5. For each group of samples, the protein concentration is from left to right: 10, 1, 0.1 μg/ml. medium (culture medium) was used as a blank control.

Figure 8: The activities of C12, C15 and 36E5 to activate T cells in vitro. Protein samples: C12, C15, 36E5 and IgG Isotype (IgG isotype, negative control). For each group of samples, protein concentration from left to right: 50, 10, 2, 0.4, 0.08 μg/ml. Medium (culture medium) was used as a blank control.

Figure 9: Growth curves of tumor volume in mice treated with control and fusion protein C12 in the murine colon cancer MC38-OVA model. NOTE: Tumor volumes are expressed as "mean ± standard error (SEM)".

detailed description

definition

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "at least one (species)" or "one (species) or more (species)" may mean 1, 2, 3, 4, 5, 6, 7, 8 (species) or more ( kind).

As used herein, the terms "about" and "approximately" when used in conjunction with a numerical variable generally mean that the value of the variable and all values of the variable are within experimental error (e.g., within a 95% confidence interval for the mean) or within Within ±10% of the specified value, or within a wider range.

As used herein, the expression "comprises" or its synonymous similar expressions "comprising", "containing" and "having", etc. are open-ended and do not exclude additional unrecited elements, steps or components. The expression "consisting of" excludes any element, step or ingredient not specified. The expression "consisting essentially of" means limiting the scope to the specified elements, steps or ingredients, plus the optional presence of elements, steps or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that description includes that said event or circumstance occurs and that it does not. For example, a fusion protein optionally comprising a linker encompasses the inclusion or absence of a linker.

As used herein, the term "polypeptide" refers to a polymer comprising at least two amino acids or derivatives thereof linked by peptide bonds. The terms "polypeptide" and "protein" are often used interchangeably herein. In some embodiments, proteins can be formed from one or more polypeptides covalently or non-covalently, eg, dimeric proteins.

Herein, when specifying the position of an amino acid in a polypeptide, the n-th amino acid refers to the n-th amino acid counted from the 1st amino acid at the amino terminal (N-terminal) of the polypeptide.

Herein, the amino terminus (N terminus) and the carboxyl terminus (C terminus) may indicate the relative positions of two or more amino acid sequences in a polypeptide, and do not mean that the two or more amino acid sequences are in close proximity, for example, in the present invention In the fusion protein, the first polypeptide can be located at the N-terminus of the second polypeptide, the second polypeptide can be located at the N-terminus of the third polypeptide, and between the first polypeptide and the second polypeptide and/or the second Additional components (eg, amino acid sequences, eg, linkers) may be included between the polypeptide and the third polypeptide. Alternatively, the amino terminus (N terminus) and the carboxyl terminus (C terminus) can also indicate the position of the specified amino acid or amino acid sequence in the polypeptide in which it is located, for example, the first polypeptide of the present invention can be located at the N terminus of the fusion protein of the present invention It can also be located at the C-terminus of the fusion protein of the present invention.

As used herein, the term "fusion protein" or "fusion polypeptide" refers to a protein comprising at least two polypeptides covalently linked that are not normally covalently linked in nature. Fusion proteins can be formed by covalently linking at least two polypeptides by chemical, enzymatic or recombinant DNA techniques.

As used herein, the term "mutation" refers to a substitution, deletion or addition comprising one or more amino acids in a polypeptide. The substitutions may be conservative or non-conservative. In polypeptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art, and conservative amino acid substitutions can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter the desired biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.co., p.224). For example, the Fc fragment may contain mutations such as deletion of the C-terminal lysine in the CH3 domain.

As used herein, a "wild-type" polypeptide or protein refers to a naturally occurring polypeptide or protein that has not been artificially modified. A "mutant" or "mutant" of a polypeptide or protein carries a mutation relative to a "wild-type" polypeptide or protein. For example, the wild-type TGF-βRII extracellular domain comprises the sequence of SEQ ID NO:4. TGF-βRII ectodomain mutants carry mutations relative to SEQ ID NO:4.

As used herein, the term "polynucleotide" or "nucleic acid" refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxygenated nucleotides, usually linked together by phosphodiester bonds. Ribonucleic acid (DNA) and ribonucleic acid (RNA).

A fusion protein or polynucleotide of the invention may be "isolated". The expression "isolated" means that the polypeptide or polynucleotide may, for example, be artificially engineered, and/or separated from other substances in its naturally occurring environment. An "isolated" polynucleotide, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.

As used herein, a "vector" is a vehicle for introducing exogenous nucleic acid into a host cell, and when the vector is transformed into an appropriate host cell, the exogenous nucleic acid is amplified or expressed. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those comprising nucleic acid encoding a polypeptide. Vectors usually remain episomal, but can be designed to allow integration of a gene, or part thereof, into the chromosome of the genome. Also contemplated are vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (eg, yeast artificial chromosomes and mammalian artificial chromosomes), and the like. As used herein, a vector can be expressed or replicated in a host cell.

As used herein, "expression" refers to the process by which a polypeptide is produced by the transcription and translation of a polynucleotide. An "expression vector" includes a vector capable of expressing a polypeptide comprising a polynucleotide sequence encoding a polypeptide of interest. The polynucleotide sequence encoding the polypeptide of interest is operably linked to regulatory sequences capable of affecting its expression. Such regulatory sequences may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector may refer to a recombinant DNA or RNA construct, such as a plasmid, phage vector, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA or RNA. Appropriate expression vectors are well known to those skilled in the art and include expression vectors replicable in eukaryotic cells and/or prokaryotic cells as well as expression vectors that remain episomal or integrate into the host cell genome.

As used herein, a "host cell" is a cell used to receive, maintain, replicate or amplify a vector. Host cells can also be used to express nucleic acids or polypeptides encoded by vectors. Host cells can be eukaryotic or prokaryotic. Suitable host cells include, but are not limited to, CHO cells, COS cells, HeLa cells, and HEK cells (eg, HEK293 cells).

As used herein, "affinity" or "binding affinity" is used to measure the strength of the binding between a molecule and its ligand through non-covalent forces, such as the binding strength between the fusion protein of the present invention and its target, such as GITR and Binding strength between extracellular domains of GITRL or between TGFBR2-ECD and TGF-β. The magnitude of affinity is usually reported as the equilibrium dissociation constant, K _D , which is often determined by measuring the association rate constant (k _on ) and the dissociation rate constant (k _dis ) and calculating the quotient of k _dis divided by k _on (K _D = k _dis / k _on ). _KD can be easily determined using conventional techniques, for example, methods that can be used include but are not limited to: equilibrium dialysis; biofilm layer interferometry, for example by Octet RED96 detection system; enzyme-linked immunosorbent assay (ELISA); surface plasmon Resonance (SPR) method; or by other methods known to the skilled person.

"Specific binding" means that two molecules bind to each other with a relatively high affinity. Usually, the K _D value between two molecules that specifically binds can be 10 ^-6 , 10 ^-7 , 10 ^-8 to 10 ^-9 M or lower. For example, the K D value between GITR and the extracellular domain of GITRL can be 10 ^-6 to 10 ^-9 M or lower, and the K _D value between TGFBR2-ECD and TGF-β can be 10 ^-6 to 10 ^-9 _A KD value of M or lower binds specifically.

The term "treating" means preventing, curing, ameliorating, alleviating, arresting or partially arresting the symptoms of a disease or disorder. Thus, subjects in need of treatment include, but are not limited to: those with a disease or condition; those at risk of having a disease; and those in whom the condition is to be prevented. In the case of cancer or tumors, a subject is successfully "treated" according to the methods of the invention if the subject exhibits one or more of the following: increased immune response, increased anti-tumor response, lysis of immune cells Increased cellular activity, increased killing of tumor cells by immune cells, decreased number or absence of cancer cells; reduced tumor size; suppressed or absent cancer cell infiltration into surrounding organs (including spread of cancer cells into soft tissue and bone); tumor or inhibition of cancer cell metastasis or absence; inhibition or absence of cancer growth; relief of one or more symptoms associated with a particular cancer; reduction in morbidity and mortality; improved quality of life; reduction in tumorigenicity; A decrease in number or frequency; or some combination of effects.

As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material or composition which is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder. For example, for the treatment of tumors, a therapeutically effective amount of a fusion protein or pharmaceutical composition of the invention preferably inhibits cell growth or tumor growth by at least about 10%, at least about 20%, at least About 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, preferably at least about 80%. The effect of inhibiting tumor growth can be evaluated in animal model systems predictive of therapeutic effect on human tumors; alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of the fusion protein or pharmaceutical composition of the present invention can reduce tumor size, or alleviate symptoms of a subject in other ways such as preventing and/or treating metastasis or recurrence. Those skilled in the art understand that a therapeutically effective amount will be influenced by factors such as the subject's weight, severity of symptoms and the particular composition or route of administration chosen. A therapeutically effective amount can be administered in one or more administrations.

fusion protein

In one aspect, the invention provides a kind of fusion protein, it comprises from N-terminus to C-terminus:

The first polypeptide-the second polypeptide-the third polypeptide Formula (1);

or

The third polypeptide-the second polypeptide-the first polypeptide formula (2);

Wherein the first polypeptide comprises a TGF-βRII extracellular domain, the second polypeptide comprises a dimerization domain, and the third polypeptide comprises three GITRL extracellular domains.

The fusion protein of the present invention is a GITR/TGF-β double-targeting fusion protein, which specifically binds TGF-β and GITR at the same time. The fusion protein of the present invention inhibits the TGF-β signaling pathway by binding to TGF-β, and acts as a TGF-β inhibitor; meanwhile, the fusion protein of the present invention activates the GITR signaling pathway by binding to GITR, and acts as a GITR agonist. The fusion protein of the present invention can be used as an immunotherapeutic agent to induce, promote, enhance, activate or prolong immune response, especially induce, promote, enhance, activate or prolong immune response against tumor cells.

first polypeptide

In some embodiments, the first polypeptide comprises a TGF-βRII extracellular domain (TGFBR2-ECD).

As used herein, the term "TGF-βRII extracellular domain" or "TGFBR2-ECD" refers to the extracellular region of the TGF-β type II (TGF-βRII) receptor, which has TGF-β binding activity. Dimerized TGFBR2-ECD can act as a TGF-βTrap to bind and capture TGF-β dimers, thereby inhibiting TGF-β signaling pathway and eliminating TGF-β signaling pathway-related immunosuppression. Inhibition of the TGF-β signaling pathway can be determined using various detection methods known in the art, such as detecting the expression of downstream genes controlled by the TGF-β signaling pathway.

TGFBR2-ECD contained in the first polypeptide may be wild type or a mutant thereof. For the description of wild-type TGFBR2-ECD, see, for example, WO2015118175A2. The amino acid sequence of an exemplary wild-type TGFBR2-ECD is shown in SEQ ID NO:4.

The inventors found that the wild-type TGFBR2-ECD is prone to breakage in amino acids 1-24 of the N-terminus, which is not conducive to the subsequent development of druggability. To solve this problem, the inventors developed TGFBR2-ECD mutants, which are improved polypeptides relative to wild-type TGFBR2-ECD. Compared with the fusion protein comprising the wild-type TGFBR2-ECD, the fusion protein comprising the TGFBR2-ECD mutant is less likely to be degraded during the production process and has better druggability.

In one embodiment, the TGFBR2-ECD mutant comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

In some embodiments, the first polypeptide comprises wild-type TGFBR2-ECD. In a specific embodiment, the wild-type TGFBR2-ECD comprises the amino acid sequence of SEQ ID NO:4.

In some embodiments, the first polypeptide comprises a TGFBR2-ECD mutant. In a specific embodiment, the TGFBR2-ECD mutant comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

In a specific embodiment, the first polypeptide comprises TGFBR2-ECD comprising the amino acid sequence of SEQ ID NO:4. In yet another specific embodiment, the first polypeptide comprises TGFBR2-ECD comprising the amino acid sequence of SEQ ID NO:5. In yet another specific embodiment, the first polypeptide comprises TGFBR2-ECD comprising the amino acid sequence of SEQ ID NO:6.

second polypeptide

As used herein, "dimerization domain" refers to a domain capable of facilitating the combination of two or more polypeptides into dimers or multimers (e.g., three, four, five, six, seven) through covalent or non-covalent interactions. , octa or nine-mer) polypeptides. It is believed that the dimerized TGF-βRII extracellular domain can act as a TGF-β Trap (see eg WO2015118175A2 and WO2018205985A1 ) to bind TGF-β dimers and inhibit TGF-β signaling pathway. The dimerization domain promotes the fusion protein of the present invention to form dimers or multimers, so that the fusion protein of the present invention can function as TGF-βTrap.

Non-limiting examples of dimerization domains may include the heavy chain constant region 3 (CH3) of immunoglobulins IgG, IgA, IgD, and the heavy chain constant region 4 (CH4) of IgM and IgE. The dimerization domain may further comprise other domains of a particular type of immunoglobulin, such as the hinge region or part thereof and/or the heavy chain constant region 2 (CH2) of IgG1, IgG2, IgG3 or IgG4, to obtain a protein having the desired Specific fusion proteins, such as enhancing antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular endocytosis (ADCP), complement-dependent cytotoxicity (CDC), and binding activity to FcRn to prolong the half-life of fusion proteins Wait.

In one embodiment, the dimerization domain comprises the CH2 and CH3 domains of an immunoglobulin, preferably the CH2 and CH3 domains of a human IgGl.

In preferred embodiments, the dimerization domain further comprises an immunoglobulin hinge region or portion thereof. Preferably, the hinge region is the hinge region of human IgG1. In a specific embodiment, the dimerization domain comprises the Fc fragment of an immunoglobulin, preferably the Fc fragment of human IgGl.

As used herein, "immunoglobulin Fc fragment" or "Fc fragment" refers to the hinge region or part thereof and two or more domains in the heavy chain constant region CH2, CH3, CH4 of an immunoglobulin. For example, the Fc fragment of IgG may comprise the hinge region or part thereof and the CH2 and CH3 domains of an immunoglobulin, while the Fc fragments of IgM and IgE may comprise the hinge region or part thereof, CH2, CH3 and CH4 domains of an immunoglobulin . The Fc fragment can be derived from any immunoglobulin, e.g., any class or subclass of IgG, IgM, IgD, IgE, IgA, and IgY, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgG2a, and IgG2b. Portions in the Fc fragment (eg, hinge region, CH2 and CH3) may be derived from the same or different immunoglobulins. Preferably, each portion in the Fc fragment is derived from the same immunoglobulin, preferably human IgG, eg human IgGl, IgG2, IgG3 or IgG4. Fc fragments can be antibody derivatives produced by enzymatic treatment of full-length antibodies (such as non-antigen-binding parts after papain cleavage), and derivatives produced by chemical synthesis or genetic engineering techniques (such as DNA recombinant technology).

Those skilled in the art can judge the position and amino acid sequence of CH2, CH3, CH4 and hinge regions in the immune protein according to known algorithms and software, and the description of applicable algorithms and software can be found in, for example, William R. Strohl, Lila M. Strohl, (2012), Antibody structure–function relationships, In Woodhead Publishing Series in Biomedicine, Therapeutic Antibody Engineering, Woodhead Publishing, pp.37-56. The amino acid sequences of various types of immunoglobulin Fc fragments can be obtained by methods well known to those skilled in the art, for example, using public databases (eg Uniprot or NCBI) to obtain the amino acid sequences of various parts of the immunoglobulin heavy chain constant region. Moreover, those skilled in the art know that the Fc fragment can be modified as needed. For example, one or more amino acids may be added, substituted or deleted in the hinge region, CH2 and/or CH3 domains, for example modifying CH2 to alter the glycosylation modification of the Fc fragment, or additions or deletions in the hinge region for formation of molecules One or more cysteines between disulfide bonds.

In other embodiments, the second polypeptide further comprises a linker fused to the N-terminus and/or C-terminus of said dimerization domain. The linker is as described herein.

In some embodiments, the second polypeptide has the following structure from N-terminus to C-terminus:

Lk ₁ –Dd–Lk ₂ ,

in

Dd is a dimerization domain;

Lk ₁ and Lk ₂ are each independently a linker or absent.

In some embodiments, the dimerization domain comprises a CH2, CH3, CH4 domain, or a combination thereof. In some embodiments, the dimerization domain comprises at least one CH3 domain. In some embodiments, the dimerization domain comprises CH2 and CH3 domains from N-terminus to C-terminus, preferably the CH2 and CH3 domains of human IgGl. In some embodiments, the CH2 and CH3 domains are derived from human IgGl. In some embodiments, the dimerization domain comprises, from N-terminus to C-terminus, CH2 and CH3 domains derived from human IgGl. In one embodiment, the C-terminal lysine of the CH3 domain is deleted. In one embodiment, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. In one embodiment, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:8.

In some embodiments, the dimerization domain comprises the hinge region of an immunoglobulin or a portion thereof and a CH2, CH3, CH4 domain or a combination thereof. In some embodiments, the dimerization domain comprises from N-terminus to C-terminus the hinge region or part thereof, CH2 and CH3 domains of an immunoglobulin, preferably the Fc fragment of human IgGl. The CH2 and CH3 domains are described above. In some embodiments, the hinge region is derived from human IgGl. In some embodiments, the hinge region does not comprise one or more cysteines. In one embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO:9.

In a specific embodiment, the dimerization domain comprises, from N-terminus to C-terminus, the CH2 and CH3 domains of an immunoglobulin, wherein said CH2 and CH3 domains comprise SEQ ID NO: 7 or SEQ ID NO: 8 amino acid sequence.

In a specific embodiment, the dimerization domain comprises from N-terminus to C-terminus the hinge region or part thereof, CH2 and CH3 domains of an immunoglobulin, wherein said hinge region or part thereof comprises the sequence of SEQ ID NO:9 Amino acid sequence, the CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. In a preferred embodiment, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:8.

In some embodiments, the linker comprises 5-25 amino acids each independently selected from glycine, serine, and alanine. In a specific embodiment, the linker comprises (GGGGS) _n , wherein n is an integer selected from 1-10. In a specific embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In a specific embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, neither Lk ₁ nor Lk ₂ is present, and the second polypeptide has the following structure from N-terminus to C-terminus:

Dd;

in

Dd is a dimerization domain, said dimerization domain being as previously defined.

In some embodiments, Lk ₁ and Lk ₂ are each independently a linker, and the second polypeptide has the following structure from the N-terminus to the C-terminus:

Lk ₁ -Dd -Lk ₂ ;

in

Dd is a dimerization domain, and the dimerization domain is as defined above;

Lk ₁ and Lk ₂ are each independently a linker, the linker being as defined above.

In some embodiments, Lk ₁ is a linker, Lk ₂ is absent, and the second polypeptide has the following structure from N-terminus to C-terminus:

Lk ₁ -Dd;

or

Lk ₁ does not exist, Lk ₂ is a linker, and the second polypeptide has the following structure from N-terminus to C-terminus:

Dd–Lk ₂ ;

in

Dd is a dimerization domain, and the dimerization domain is as defined above;

Each of Lk ₁ and Lk ₂ is a linker, the linker being as defined above.

In a specific embodiment, the second polypeptide has the following structure from N-terminus to C-terminus:

Lk ₁ -Dd -Lk ₂ ;

in

Dd is a dimerization domain, which comprises a hinge region, CH2 and CH3 domains from the N-terminal to the C-terminus, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:9, and the CH2 and CH3 domains comprise SEQ ID The amino acid sequence of NO:7 or SEQ ID NO:8;

Lk ₁ -Dd -Lk ₂ ;

in

Lk ₁ and Lk ₂ are each independently a linker comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

Lk ₁ -Dd -Lk ₂ ;

in

Dd is a dimerization domain, which comprises a hinge region, CH2 and CH3 domains from the N-terminal to the C-terminus, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:9, and the CH2 and CH3 domains comprise SEQ ID NO: the amino acid sequence of 8;

Lk ₁ and Lk ₂ are each independently a linker, wherein Lk ₁ comprises the amino acid sequence of SEQ ID NO:10, and Lk ₂ comprises the amino acid sequence of SEQ ID NO:11.

third polypeptide

In an embodiment of the invention, the third polypeptide comprises 3 GITRL extracellular domains. Without being bound by any theory, a single-chain polypeptide comprising 3 GITRL ectodomains (single-chain GITRL ectodomain trimer) compared to a single GITRL ectodomain (monomeric GITRL ectodomain) Trimers similar to the full-length GITRL protein are formed on the cell surface, and such trimers have higher GITR activation activity as GITR agonists.

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). Without being bound by any theory, it is believed that activation (or agonism) of GITR signaling can activate effector T cells and suppress the immunosuppressive activity of regulatory T cells. As used herein, "GITR agonist" refers to a substance capable of inducing, promoting, enhancing, activating or prolonging GITR signaling. Activation of GITR signaling can be determined using various assays known in the art, such as by the assays described herein. GITR agonists include, but are not limited to, GITRL, GITRL extracellular domain, GITR agonist antibodies. The fusion protein or conjugate of the present invention can act as a GITR agonist to induce, promote, enhance, activate or prolong an immune response, such as inducing, promoting, enhancing, activating or prolonging an immune response against a tumor or tumor cells.

As used herein, "GITR ligand (Glucocorticoid-induced TNF-related ligand, GITRL)" or "glucocorticoid-induced TNF receptor ligand" refers to glucocorticoid-induced tumor necrosis factor receptor (Glucocorticoid- induced tumor necrosis factor receptor (GITR) protein binding ligand. In some embodiments, GITRL refers to human GITRL protein. The human GITRL (hGITRL) protein is encoded by the TNFSF18 gene, and its amino acid sequence is shown in GenBank accession number NP_005083.2 (or Uniprot ID: Q9Y5U5). As used herein, "GITRL extracellular domain" refers to the extracellular region of the human GITRL protein, which specifically binds and agonizes GITR. Those skilled in the art will understand that the exact amino acid sequence that the extracellular domain of GITRL can comprise can vary without affecting its ability to agonize GITR. Typically, the GITRL extracellular domain may comprise about amino acids 71-199 of NP_005083.2, such as amino acids 72-199 (SEQ ID NO: 1) or amino acids 75-199 (SEQ ID NO ) of NP_005083.2 :2). In some embodiments, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The third polypeptide may further comprise a linker between said GITRL extracellular domains. The linker is as described herein.

In some embodiments, the third polypeptide has the following structure from N-terminus to C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ;

in

Ln ₁ and Ln ₂ are each independently a linker or absent.

In some embodiments, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO:2.

In some embodiments, the linker comprises 5-25 amino acids each independently selected from glycine, serine, and alanine. In a specific embodiment, the linker comprises (GGGGS) _n , wherein n is an integer selected from 1-10. In a specific embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In a specific embodiment, said linker comprises the amino acid sequence of SEQ ID NO:12.

In some embodiments, neither Ln ₁ nor Ln ₂ is present, and the third polypeptide has the following structure from N-terminus to C-terminus:

Sd ₁ -Sd ₂ -Sd ₃ ;

Wherein Sd ₁ , Sd ₂ and Sd ₃ are each independently an extracellular domain of GITRL, and the extracellular domain of GITRL is as defined above.

In other embodiments, Ln ₁ and Ln ₂ are each independently a linker, and the third polypeptide has the following structure from the N-terminus to the C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ;

in

Sd ₁ , Sd ₂ and Sd ₃ are each independently a GITRL extracellular domain, and the GITRL extracellular domain is as defined above;

Ln ₁ and Ln ₂ are each independently a linker, the linker being as defined above.

In some embodiments, Ln ₁ is a linker, Ln ₂ is absent, and the third polypeptide has the following structure from N-terminus to C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Sd ₃ ;

or

Ln ₁ does not exist, Ln ₂ is a linker, and the third polypeptide has the following structure from N-terminus to C-terminus:

Sd ₁ -Sd ₂ -Ln ₂ -Sd ₃ ;

in

Each of Ln ₁ and Ln ₂ is a linker, the linker being as defined above.

In a specific embodiment, the third polypeptide has the following structure from the N-terminus to the C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ;

in

Sd ₁ , Sd ₂ and Sd ₃ are each independently an extracellular domain of GITRL, which comprises the amino acid sequence of SEQ ID NO:2;

In yet another specific embodiment, the third polypeptide has the following structure from the N-terminus to the C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ;

in

Ln ₁ and Ln ₂ are each independently a linker comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

In another specific embodiment, the third polypeptide has the following structure from the N-terminus to the C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ;

in

Ln ₁ and Ln ₂ are each independently a linker comprising the amino acid sequence of SEQ ID NO:12.

In a specific embodiment, the third polypeptide comprises the amino acid sequence of SEQ ID NO:3.

connector

Between the components of the fusion protein (for example between the first and second polypeptide, between the second and the third polypeptide, between the first, second and third GITRL extracellular domains in the third polypeptide Between) are optionally connected by a linker. Preferably, the linker may comprise 1-50 amino acids, preferably 1-25, 3-25, 5-25, 4-10 amino acids, for example 4, 5, 6, 7, 8, 9 or 10 amino acids. Amino acids are generally unhindered amino acids such as glycine (G), alanine (A) and serine (S). Suitable linkers are well known to those skilled in the art, for example linkers may comprise glycine, alanine, serine or combinations thereof. Exemplary linkers may include, but are not limited to: polyglycine, polyalanine, GGA, GS, GGSG, GGGS, GGGSGGG, and GGGGSSGS.

In one embodiment, the linker comprises (GGGGS) _n , wherein n is an integer selected from 1-10. In a preferred embodiment, n is an integer selected from 2-5. In one embodiment, n is 4 (SEQ ID NO: 10). In another embodiment, n is 2 (SEQ ID NO: 11).

In yet another specific embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

signal peptide

Fusion proteins of the invention optionally include a signal peptide. Signal peptides (also known as signal sequences, transit peptides, leader sequences, or leader peptides) are short peptides (typically comprising 5-30 amino acids) that direct the localization, transfer, and/or secretion of nascent proteins. In the present invention, the preferred signal peptide improves the expression level of the fusion protein and ensures the correct folding of the fusion protein, and helps to secrete the fusion protein out of cells. Secretory signal peptides are usually cleaved during protein secretion, allowing the protein to be released outside the cell as a mature protein. The fusion protein of the present invention comprising a secretion signal peptide can also be called a fusion protein precursor, which is also included in the scope of the fusion protein of the present invention.

The signal peptide can be located anywhere in the fusion protein. Preferably, the signal peptide is located at the N-terminus of the fusion protein.

The signal peptide may be any signal peptide known in the art, preferably a secretory signal peptide commonly used in expression in mammalian cells (eg, CHO cells). In a specific embodiment, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

Specific embodiments of fusion proteins

In some embodiments, the fusion protein of the present invention comprises from N-terminus to C-terminus:

The first polypeptide-the second polypeptide-the third polypeptide Formula (1);

or

The third polypeptide-the second polypeptide-the first polypeptide formula (2);

in

Lk ₁ –Dd–Lk ₂ ,

in

Dd is a dimerization domain;

Lk ₁ and Lk ₂ are each independently a linker or absent; and

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ,

in

Sd ₁ , Sd ₂ and Sd ₃ are each independently a GITRL extracellular domain; and

Ln ₁ and Ln ₂ are each independently a linker or absent.

In some embodiments, the TGFBR2-ECD comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. In a preferred embodiment, the TGFBR2-ECD comprises the amino acid sequence of SEQ ID NO:5.

In some embodiments, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In a preferred embodiment, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO:2.

In some embodiments, the dimerization domain comprises CH2 and CH3 domains. In a preferred embodiment, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the dimerization domain comprises a hinge region, CH2 and CH3 domains. In one embodiment, the hinge region comprises the amino acid sequence of SEQ ID NO:9. In one embodiment, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. In a preferred embodiment, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:8. In some embodiments, the dimerization domain comprises a hinge region, CH2 and CH3 domains, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 9, and the CH2 and CH3 domains comprise SEQ ID NO: 8 amino acid sequence.

In some embodiments, the linker comprises 5-25 amino acids each independently selected from glycine, serine, and alanine. In a preferred embodiment, the linker comprises (GGGGS) _n , wherein n is an integer selected from 1-10. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

In one embodiment, the third polypeptide has the following structure from the N-terminus to the C-terminus:

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ;

in

Sd ₁ , Sd ₂ and Sd ₃ are each independently a GITRL extracellular domain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and

Ln ₁ and Ln ₂ are each independently a linker.

In some embodiments, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO:2, and each of Ln ₁ and Ln ₂ is independently a linker comprising the amino acid sequence of SEQ ID NO:12. In a specific embodiment, said third polypeptide comprises the amino acid sequence of SEQ ID NO:3.

Lk ₁ -Dd -Lk ₂ ;

in

Dd is a dimerization domain, which comprises a hinge region, CH2 and CH3 domains from the N-terminus to the C-terminus, the hinge region comprises the amino acid sequence of SEQ ID NO: 9, and the CH2 and CH3 domains comprise SEQ ID NO :7 or the amino acid sequence of SEQ ID NO:8;

Lk ₁ and Lk ₂ are each independently a linker.

In some embodiments, the dimerization domain comprises a hinge region, CH2 and CH3 domains from the N-terminus to the C-terminus, the hinge region comprises the amino acid sequence of SEQ ID NO: 9, the CH2 and CH3 domains Comprising the amino acid sequence of SEQ ID NO: 8, Lk ₁ and Lk ₂ are each independently a linker, wherein Lk ₁ contains the amino acid sequence of SEQ ID NO: 10, and Lk ₂ contains the amino acid sequence of SEQ ID NO: 11.

The first polypeptide-the second polypeptide-the third polypeptide Formula (1);

or

The third polypeptide-the second polypeptide-the first polypeptide formula (2);

in

The first polypeptide comprises TGFBR2-ECD comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;

Lk ₁ –Dd–Lk ₂ ,

in

Lk ₁ and Lk ₂ are each independently a linker comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12; and

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ,

in

Sd ₁ , Sd ₂ and Sd ₃ are each independently an extracellular domain of GITRL, which comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

In a specific embodiment, the fusion protein of the present invention comprises from the N-terminus to the C-terminus:

The first polypeptide-the second polypeptide-the third polypeptide Formula (1);

in

Lk ₁ –Dd–Lk ₂ ,

in

Dd is a dimerization domain, which comprises a hinge region, CH2 and CH3 domains from the N-terminus to the C-terminus, the hinge region comprises the amino acid sequence of SEQ ID NO: 9, and the CH2 and CH3 domains comprise SEQ ID NO : the amino acid sequence of 8;

Lk ₁ and Lk ₂ are each independently a linker, wherein Lk ₁ comprises the amino acid sequence of SEQ ID NO: 10, and Lk ₂ comprises the amino acid sequence of SEQ ID NO: 11; and

Sd ₁ -Ln ₁ -Sd ₂ -Ln ₂ -Sd ₃ ,

in

In other embodiments, any fusion protein as described above further comprises a signal peptide. Preferably, the signal peptide is located at the N-terminus of the fusion protein. In a specific embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

In one embodiment, any fusion protein as described above comprises a signal peptide at the N-terminus, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

In some embodiments, the fusion protein forms dimers or multimers.

Nucleic acids, vectors and host cells

In another aspect, the invention provides isolated polynucleotides encoding fusion proteins of the invention. In some preferred embodiments, the polynucleotide encodes a precursor of a fusion protein of the invention. The polynucleotides can be obtained by methods known in the art, for example, recombinant DNA techniques, chemical synthesis, and the like. Polynucleotides can generally be codon-optimized for the host cell used for expression.

The present invention also provides an expression vector comprising the polynucleotide of the present invention. Methods for constructing suitable expression vectors using the polynucleotides of the present invention are well known to those skilled in the art, including but not limited to in vitro recombinant DNA, chemical synthesis, and in vivo recombination (genetic recombination).

The present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention. Suitable host cells include, but are not limited to, prokaryotic cells (eg, E. coli) and eukaryotic cells (eg, yeast cells, insect cells, and mammalian cells). In preferred embodiments, the host cells are simian COS cells, Chinese Hamster Ovary (CHO) cells, HEK293F cells or immune effector cells (eg effector T cells). In one embodiment, the host cell is a yeast cell, an insect cell or a mammalian cell. In one embodiment, the host cell is an effector T cell.

Production method of fusion protein

Another aspect of the present invention relates to a method for producing the fusion protein of the present invention, the method comprising:

(i) culturing the host cell of the invention under conditions suitable for expression of the polynucleotide or expression vector, and

(ii) recovering the fusion protein of the present invention from said host cell or its culture.

Various methods known in the art can be used to introduce the polynucleotide or expression vector of the present invention (especially the polynucleotide or expression vector encoding the expression precursor of the fusion protein of the present invention) into suitable host cells. Such methods include, but are not limited to, lipofection, electroporation, stable transfection, calcium phosphate transfection, and the like. In some embodiments, to further purify the fusion protein of the present invention, conventional affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration or a combination thereof can be used.

Conjugate

The invention further provides a conjugate comprising a fusion protein of the invention conjugated to at least one therapeutic agent.

As used herein, "conjugate" refers to the linking of two or more moieties to each other by covalent or non-covalent interactions. Preferably, fusion proteins of the invention and at least one therapeutic agent are covalently conjugated.

In some embodiments, the therapeutic agent is selected from detectable markers, chemotherapeutic agents, cytotoxins, radionuclides, immune checkpoint inhibitors, cytokines, and enzymes.

In some embodiments, the therapeutic agent is a detectable label, such as a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent.

In some embodiments, the therapeutic agent is a cytotoxin. Examples of cytotoxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thienenoside, Vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxendione, mitoxantrone, mitoxantrone, actinomycin D, 1-dehydrotestosterone, sugar Corticosteroids, procaine, tetracaine, lidocaine, carcinomacin, calicheamicin, maytansin, alistatin, propranolol and puromycin and their analogs or homologues.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, carbamide), alkylating agents (e.g., nitrogen mustard, chlorambucil, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, Mitomycin C and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthraninomycins (eg, daunorubicin and doxorubicin), antibiotics (eg, actinomycin D , bleomycin, mithromycin, and antramycin (AMC)) and antimitotic agents (eg, vincristine and vinblastine). In some embodiments, the therapeutic agent is a biologically active protein, such as a toxin with enzymatic activity or an active fragment thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin.

In some embodiments, the therapeutic agent is a cytokine. Examples of cytokines include, but are not limited to, tumor necrosis factors (such as TNFα and TNFβ), interferons (such as INFα, INFβ, and INFγ), interleukins (such as IL-1, IL-2, IL-4, IL-5, IL- 6. IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL21 and IL-23), colony-stimulating factors (such as macrophage colony stimulatory factors, granulocyte colony stimulating factor, granulocyte and macrophage colony stimulating factor, multiple colony stimulating factor, stem cell factor), growth factors (such as VEGF, HGF, FGF, FGF-2, PDGF, IGF, TGF, NGF, EPO and EGF) and chemokines.

In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. Examples of immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof against immune checkpoint molecules such as PD-L1, PD-1, CTLA4, LAG-3, BTLA, TIM-2, LAIR1, HVEM, and TIM-4 . The antibody or antigen-binding fragment thereof may be selected from synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single domain antibodies , Fab fragment, Fab' fragment, F(ab') ₂ fragment, Fv fragment, dsFv fragment, Fd fragment, Fd' fragment, scFv, scFab, diabody.

In some embodiments, the therapeutic agent is a radionuclide. Examples of radionuclides include, but are not limited to, iodine-125, iodine-131, indium-111, indium-131, yttrium-90, rhodium-105, bismuth-212, and lutetium-177.

The therapeutic agent can also be an enzyme capable of producing a detectable product (e.g., horseradish peroxidase), gold nanoparticles/nanorods, virosomes, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-cardiac diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) and nanoparticles in any form, etc.

Methods of preparing conjugates are known to those skilled in the art. For example, the therapeutic agents described above can be conjugated to fusion proteins of the invention using linker technology used in the art. Examples of linker types that have been used for cytotoxin conjugation include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide linkers. Alternatively, polynucleotides encoding protein therapeutics (such as cytokines, antibodies or antigen-binding fragments thereof, biologically active proteins or enzymes as described above) and polynucleotides encoding fusion proteins of the present invention can be combined using recombinant DNA technology. Operably linked and expressed in a suitable host cell.

pharmaceutical composition

The present invention further provides a pharmaceutical composition, which comprises: (i) the fusion protein or conjugate of the present invention; and (ii) a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all substances that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include: solvents (such as water or oils), dispersants, suspending agents, coatings, diluents, adjuvants, antioxidants (such as ascorbic acid, ascorbyl palmitate, sodium sulfite, , butylated hydroxyanisole (BHA), propyl gallate, sodium metabisulfite, α-tocopherol, etc.), excipients, preservatives (such as antibacterial and antifungal agents), surfactants (such as polysorbates , poloxamer, triton, sodium octyl glycoside, sodium lauryl sulfate, sodium lauryl sulfate, lauryl-sulfobetaine, polyethylene glycol, polypropylene glycol, etc.), wetting agent, emulsifier, filler , carbohydrates (such as fructose, sucrose, trehalose, mannose, dextran, cyclodextrin, etc.), amino acids (such as alanine, arginine, asparagine, aspartic acid, cysteine, Glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine , valine), buffers (such as acetic acid, hydrochloric acid, sulfuric acid, histidine, citrate, phosphate, succinate), stabilizers, chelating agents (such as EDTA), isotonic and absorption delaying agents ( Such as monostearate and gelatin), etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).

The fusion protein, conjugate or pharmaceutical composition of the present invention can be administered through one or more administration routes using one or more methods known in the art. Those skilled in the art will appreciate that the route and/or manner of administration will vary depending on the desired result. Preferred routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration such as injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, intraorbital, Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the fusion protein, conjugate or pharmaceutical composition of the present invention may also be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually or partial.

The pharmaceutical composition can be prepared in various dosage forms suitable for administration, such as solutions, suspensions, microemulsions, emulsions, liposomes, tablets, pills, capsules, powders, sustained-release formulations and the like. In some embodiments, the pharmaceutical composition is prepared as injection, powder or liposome.

treat

The fusion protein, polynucleotide, conjugate or pharmaceutical composition provided by the present invention is used for treating and/or preventing cancer. In one embodiment, the fusion protein, polynucleotide, conjugate or pharmaceutical composition is used to activate GITR signaling and inhibit TGF-β signaling. In one embodiment, the fusion protein, polynucleotide, conjugate or pharmaceutical composition is used to induce, promote, enhance, activate or prolong an immune response in a subject in need thereof. In one embodiment, the fusion protein, polynucleotide, conjugate or pharmaceutical composition is used to induce, promote, enhance, activate or prolong an immune response against a tumor or tumor cells in a subject in need thereof. In one embodiment, the fusion protein, polynucleotide, conjugate or pharmaceutical composition is used to activate effector T cells, such as CD8 ⁺ effector T cells.

The present invention also relates to the use of the fusion protein, polynucleotide, conjugate or pharmaceutical composition of the present invention in the preparation of medicines. In one embodiment, the medicament is for the treatment and/or prevention of cancer. In one embodiment, the medicament is for activating GITR signaling and inhibiting TGF-beta signaling. In one embodiment, the medicament is for inducing, promoting, enhancing, activating or prolonging an immune response in a subject in need thereof. In one embodiment, the medicament is for inducing, promoting, enhancing, activating or prolonging an immune response against a tumor or tumor cells in a subject in need thereof. In one embodiment, the drug is used to activate effector T cells. In one embodiment, the T cells are CD8 ⁺ effector T cells.

The present invention further provides a method for treating and/or preventing cancer, which comprises administering a therapeutically effective amount of the fusion protein, polynucleotide, conjugate or pharmaceutical composition of the present invention to a subject in need. In one embodiment, the method induces, promotes, enhances, activates or prolongs an immune response against a tumor or tumor cells in a subject in need thereof.

The present invention also provides a method for inducing, promoting, enhancing, activating or prolonging an immune response, which comprises administering the fusion protein, polynucleotide, conjugate or pharmaceutical composition of the present invention.

The present invention also provides a method for activating GITR signal transduction and inhibiting TGF-β signal transduction, and/or a method for activating effector T cells, which comprises administering the fusion protein, polynucleotide, conjugate or pharmaceutical composition of the present invention .

As used herein, the term "cancer" or "tumor" refers to or describes the physiological condition in mammals that is often characterized by unregulated cell growth. Non-limiting examples of cancer include, but are not limited to, adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More specific examples of cancer include: squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin and non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, Cervical cancer, ovarian cancer, liver cancer such as hepatocellular carcinoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland cancer, kidney cancer such as renal cell carcinoma and Kleman's tumor, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer and various types of head and neck cancer.

In one embodiment, the cancer is selected from adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-small cell lung cancer. Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, bone marrow cancer, multiple myeloma, salivary gland cancer, kidney cancer, renal cell carcinoma, Wellman's tumor, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, and head and neck cancer.

As used herein, the term "subject" refers to mammals, including, but not limited to, goats, sheep, pigs, rats, mice, rabbits, guinea pigs, cows, horses, dogs, cats, non-human Long animals (such as gorillas, baboons and chimpanzees) and humans. In some embodiments, the subject is a human.

combination therapy

The fusion proteins, conjugates or pharmaceutical compositions of the invention can be used in combination with other therapeutic agents. Other therapeutic agents include, for example, chemotherapeutic agents, immune checkpoint inhibitors, or antibodies targeting tumor-associated antigens. In one embodiment, the other therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, or a combination thereof.

The fusion protein, conjugate or pharmaceutical composition of the invention and the other therapeutic agent can be administered all at once or separately. When administered separately (in the case of mutually different administration regimens), they may be administered continuously without interruption or at predetermined intervals. The fusion protein of the present invention or the pharmaceutical composition of the present invention may also be used in combination with radiotherapy, for example comprising administering ionizing radiation to the patient earlier than, during and/or after the fusion protein of the present invention or pharmaceutical composition Application process.

Reagent test kit

The present invention also relates to a kit, which includes the fusion protein of the present invention, its encoding polynucleotide or expression vector or a combination thereof, and instructions for use. Kits generally include a label indicating the intended use and/or method of use of the kit contents. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

Beneficial effect

The fusion protein of the present invention realizes the following beneficial effects:

1) Bind TGF-β and GITR with higher affinity activity;

2) Excellent activity of activating GITR signal transduction and inhibiting TGF-β signal transduction;

3) excellent T cell activation activity; and

4) Excellent tumor suppressive activity.

Further, the fusion protein of the present invention may comprise a TGFBR2-ECD mutant. Compared with a fusion protein comprising a wild-type TGFBR2-ECD, a fusion protein comprising a TGFBR2-ECD mutant is less likely to be broken and degraded during production, showing Greater stability.

In addition, the fusion protein of the present invention targets GITR and TGF-β dual targets, and can suppress TGF-β while activating GITR. Compared with the GITRL extracellular domain trimer protein 336B11 and the anti-GITR agonist antibody 36E5 that single-target GITR, the fusion protein of the present invention has a higher activity of activating effector T cells. The present invention proposes this dual-target fusion protein product for the first time, which has great application potential in clinic.

Example

A further understanding of the invention may be obtained by reference to certain specific examples which are given herein for illustration only and are not intended to limit the scope of the invention in any way. Apparently, various modifications and changes can be made to the present invention without departing from the essence of the present invention. Therefore, these modifications and changes are also within the protection scope of the present application.

Reagents and equipment used in the Examples section are commercially available unless otherwise indicated.

Example 1 Construction and production of GITR/TGF-β dual targeting fusion protein

The wild-type TGFBR2-ECD (SEQ ID NO: 4) and the single-chain GITRL extracellular domain trimer (SEQ ID NO: 3, which contains 3 GITRL extracellular domains connected by a linker) are connected through the Fc fragment, Construct and produce GITR/TGF-β dual targeting fusion protein C2 (SEQ ID NO: 17) (see Table 2).

The fusion protein C2 (as shown in Example 2) with wild-type TGFBR2-ECD is prone to degradation, resulting in a large fragment comprising a single-chain GITRL extracellular domain trimer and an Fc fragment and a small fragment containing TGFBR2-ECD ( see picture 1). It was found by mass spectrometry that the degradation site of the fusion protein was mainly located in three regions of the N-terminus of wild-type TGFBR2-ECD, namely the first region Q-K-S and the second region N-NDM-I (where "-" indicates the break point), And the third area "DNNG" (see Table 1). In addition, due to the complete structure of the Fc part of the main degradation products produced by the fragmentation, conventional Protein A media cannot effectively remove the degradation products, which poses a huge challenge to the development of downstream purification processes and seriously affects the development of druggability of molecules. Therefore, when using TGFBR2-ECD as a part of the fusion protein, regardless of the order in the fusion protein, it is necessary to simultaneously carry out targeted and effective mutations for the three regions or the three regions and their upstream and downstream sequences, so as to obtain high-quality fusion protein.

Based on the sequence of wild-type TGFBR2-ECD (SEQ ID NO: 4), the above three regions and their upstream and downstream regions were mutated and transformed to obtain TGFBR2-ECD mutants for the preparation of fusion proteins. Specifically, hydrophilic glycine (G) and/or hydrophilic serine (S) that can undergo potential O-glycosylation modification are introduced into the three regions of wild-type TGFBR2-ECD, and the obtained TGFBR2- The amino acid sequence of the ECD mutant is shown in SEQ ID NO:5; a wide range of hydrophilic amino acids are introduced into the above three regions of wild-type TGFBR2-ECD and their upstream and downstream regions, especially serine, which can undergo potential O-sugar modification (S) and threonine (T), the amino acid sequence of the obtained TGFBR2-ECD mutant is shown in SEQ ID NO: 6 (see Table 1).

Table 1

TGFBR2-ECDTGFBR2-ECD	序列结构sequence structure	SEQ ID NO:SEQ ID NO:
野生型Wild type	IPPHV QKSV NNDMIVT DNNGAVKF……NIIFSEEYNTSNPD IPPHV QKS V NNDMI VT DNNG AVKF…NIIFSEEYNTSNPD	44
突变体mutant	IPPHV GGSV GGSGIVT GGSGAVKF……NIIFSEEYNTSNPD IPPHV GGS V GGSG IVT GGSG AVKF…NIIFSEEYNTSNPD	55
突变体mutant	TPPHT QTST NNSMITT GTSGATKY……NIIFSEEYNTSNPD TPPHT QTS T NNSMI TT GTSG ATKY…NIIFSEEYNTSNPD	66

Note: Three regions are underlined; amino acid mutations contained in TGFBR2-ECD mutants are shown in bold.

Finally construct GITR/TGF-β dual-targeting fusion protein C12 (the TGFBR2-ECD mutant is located at the N-terminus of the fusion protein, formula (1), SEQ ID NO: 15), C15 (the TGFBR2-ECD mutant is located at the C-terminal of the fusion protein). terminal, formula (2), SEQ ID NO:16) and C2 (wild-type TGFBR2-ECD is located at the C-terminal of the fusion protein, formula (2), SEQ ID NO:17) (see Table 2).

Simultaneously construct and produce single-targeting protein 336B11 (see 336B11 in CN107406878A for amino acid sequence) and B22 as contrasting proteins, wherein 336B11 is GITR single-targeting protein (single-chain GITRL extracellular domain trimer fused to the N-terminal of Fc fragment ), B22 is a TGF-β single-targeting protein (wild-type TGFBR2-ECD fused to the N-terminus of the Fc fragment) (see Table 2).

Utilize gene total synthesis and molecular cloning technology to encode GITR/TGF-β dual-targeting fusion protein or contrast protein (both contain secretion signal peptide at N-terminus, the exemplary sequence of signal peptide is shown in SEQ ID NO: 13 and SEQ ID NO The polynucleotide sequence of :14) was cloned into the expression vector pCDNA3.4 (purchased from Thermo Fisher), and the obtained eukaryotic expression vector was transiently transformed into ExpiCHOS for routine expression. After about 10 days of serum-free cell culture, the collected cultured Purified by Protein A medium column chromatography, the structures of the fusion protein of the present invention and the comparison protein obtained are shown in Table 2.

Table 2

Example 2 SDS-PAGE detection of the purity of GITR/TGF-β dual targeting fusion protein

After the fusion proteins C15 and C2 as described in Example 1 were purified in one step by Protein A medium, the protein purity was detected by reducing protein gel electrophoresis (SDS-PAGE).

Reducing protein gel electrophoresis: take a sample and add an appropriate amount of sample buffer, add DTT to one of the samples for reduction, heat the reduced sample in a 70°C water bath for 10 minutes, load 2 μg of the sample, and separate by 10% protein gel electrophoresis. The experimental results are shown in FIG. 1 , lane 1 is C15 (containing TGFBR2-ECD mutant), and lane 2 is C2 (containing wild-type TGFBR2-ECD).

As can be seen from Figure 1, a major protein band is shown in swimming lane 1, which is consistent with the expected molecular weight of the target protein C15 (SEQ ID NO: 15). Lane 2 shows three main protein bands, the band with the largest molecular weight is consistent with the expected molecular weight of the target protein C2 (SEQ ID NO: 16), and the other two protein bands are C2 in the N-terminus of TGFBR2-ECD. The degradation products produced by the fragmentation correspond to the large fragment (degradation band 1) containing single-chain GITRL ectodomain trimer and Fc fragment (degradation band 1) and the small fragment (degradation band 2) containing TGFBR2-ECD, respectively. It can be seen that C2 is significantly degraded compared to C15. Degradation of the GITR/TGF-β dual targeting fusion protein was effectively controlled by inclusion of the TGFBR2-ECD mutant.

Example 3 The activity of GITR/TGF-β dual-targeting fusion protein binding to TGF-β in vitro

Test purpose: To detect the binding activity of single-target and dual-target fusion proteins to TGF-β by ELISA, to compare the pros and cons of dual-target fusion protein samples with different structural designs for TGF-β binding in vitro, and B22 is used as a positive control.

Test samples: B22, C15, C12 and C2 prepared as in Example 1.

Test steps:

a. Human TGF-β (purchased from Acro) at a concentration of 0.5 μg/ml was directly coated onto a 96-well plate, 100 μl/well, overnight at 4°C.

b. Wash 3 times with 250 μl/well 1×PBS, add 250 μl/well 2% BSA-PBS, and block at 37°C for 2 hours.

c. Wash 3 times with 250 μl/well 1×PBS, add serially diluted samples B22, C15, C12 and C2, 100 μl/well, and incubate at 37°C for 1 hour.

d. Wash 3 times with 250 μl/well 1×PBST, add 100 μl/well anti-human Fc antibody-HRP (1:5000), and incubate at 37°C for 1 hour.

e. Wash 3 times with 250 μl/well 1×PBST, add 100 μl/well TMB, incubate at 37°C for 10 minutes, and stop the reaction with 2M hydrochloric acid.

f. Detect the absorbance at 450nm on a microplate reader, and analyze the data with Graphpad Prism5.

Results: The ELISA results of the fusion protein binding to TGF-β in vitro are shown in Figure 2 and Table 3, and the results showed that C2, C12 and C15 all had TGF-β binding activity. In terms of _EC50 (nM), the TGF-β binding activities of C12 and C15 were comparable and comparable to B22, indicating that the binding activity of dual targeting proteins C12 and C15 to TGF-β was comparable to that of wild-type TGFBR2-ECD. In addition, the binding activity of C12 and C15 to TGF-β was better than that of C2, and the combination sequence of C15 was consistent with that of C2, but both C12 and C15 used the mutant TGFBR2-ECD, while C2 used the wild-type TGFBR2-ECD. As shown in Example 2, the dual-targeting fusion protein C2 containing wild-type TGFBR2-ECD is prone to degradation, which may be one of the reasons for its low TGF-β binding activity.

table 3

Example 4 Affinity and kinetics of GITR/TGF-β dual-targeting fusion protein binding to hGITR in vitro

Detection purpose: pass

Octet RED96 measures the affinity of GITR/TGF-β dual targeting fusion protein to hGITR.

Test samples: C12 and 336B11 prepared as in Example 1 and anti-GITR agonist antibody 36E5 (see CN103951753A for the amino acid sequence).

Detection process:

a. Coupled ligand: This method uses HIS1K (Anti-Penta-HIS biosensor, ForteBio) chip, soak the chip with SD buffer for 10-15 minutes in advance, and load the ligand (hGITR) at a concentration of 50 μg/ml for a preset time For 300 seconds, the ligand loading preset value is 1nm, and the chip is infiltrated with running buffer (SD buffer) until the baseline is stable to remove free ligand.

b. Kinetic experiment: C12, 336B11 and 36E5 were each diluted to 50 nM with SD buffer. Using the sample as the analyte and hGITR as the ligand, the affinity detection experiment was carried out. The ligand and analyte binding time (Association) is 200 seconds, and the analyte dissociation time (Dissociation) is 300 seconds.

c. Analyze with ForteBio Data Analysis 9.0 using static fitting model.

Detection results: the kinetic analysis process is shown in Figure 3, and the affinity results are shown in Table 4. It can be seen from Table 4 that the affinity of C12 to hGITR is significantly better than that of 36E5 and comparable to that of 336B11.

Table 4

样品sample	K _D(M) K _D (M)	k _on(1/Ms) k _on (1/Ms)	k _dis(1/s) k _dis (1/s)
C12C12	5.17E-105.17E-10	6.34E+056.34E+05	3.28E-043.28E-04
336B11336B11	5.24E-105.24E-10	5.39E+055.39E+05	2.82E-042.82E-04
36E536E5	3.56E-093.56E-09	2.07E+052.07E+05	7.38E-047.38E-04

Example 5 The activity of GITR/TGF-β dual-targeting fusion protein binding to CHOS-hGITR cells in vitro

Test samples: C12 and C15 prepared as in Example 1.

Detection process:

a. CHOS cells were transfected with the recombinant vector of the full sequence of membrane protein hGITR (Uniprot ID: Q9Y5U5), pressurized selection after 24 hours of transfection, and selected medium (CD FortiCHO ^TM medium + 15 μmol/L MSX + 10 μg/ml purine Mycin), until the cell viability increased. The monoclonal cell line was screened by the limiting dilution method, and the monoclonal cell line was incubated with 336B11, and then the supernatant was removed, and anti-human IgG-488 (Invitrogen, 1:2000) was added as a detection antibody, and was screened by flow cytometry. Cell lines with higher mean fluorescence intensity (MFI) and better peak shape are used as stable transfection CHOS-hGITR cell lines.

b. Incubate samples C12 and C15 with CHOS-hGITR monoclonal cell line according to the concentration gradient for 1 hour at room temperature; rinse twice with PBS, add anti-human IgG-488 (Invitrogen, 1:2000) and mix with cells at room temperature Incubate for 45 minutes, and then wash twice with PBS; then detect the fluorescent signal by flow cytometry and calculate the EC ₅₀ .

Test results: the results are shown in Figure 4 and Table 5. From the results of EC ₅₀ (nM), it can be seen that the binding activity of C12 to CHOS-hGITR cells is slightly better than that of C15.

table 5

Example 6 GITR/TGF-β dual targeting fusion protein activates GITR activity in vitro

GITR Blockade Bioassay is a cell-dependent bioluminescent detection method. Its detection principle is: when GITR agonists are added to the genetically engineered cell line GS-H3/GITR cells expressing GITR (provided by GenScript), GITR activation and Expression of the luciferase gene under the control of NFAT, which can subsequently be detected by adding a luciferase detection reagent (Bio-Glo, Promega; containing the luciferase substrate luciferin and reaction buffer) The luminescence signal generated by the catalytic substrate can be used to evaluate the activity of GITR agonists to activate GITR.

Test samples: C12 and C15 prepared as in Example 1, anti-GITR agonist antibody 36E5 (positive control) and IgG isotype (negative control).

The experimental steps for detecting the functional activity of GITR agonists are as follows:

a. Collect the target cells by centrifugation, resuspend the cells in EMEM Nutrient Mixture medium containing 10% FBS and adjust the cell density, and inoculate them in 384-well experimental plates.

b. Transfer the experimental plate to a cell culture incubator (37° C./5% CO ₂ ) and incubate for 16 hours.

c. Use the assay buffer to configure 2×concentrations of the reference substance and the test substance.

d. Collect the effector cells by centrifugation, resuspend the cells in assay buffer and adjust the cell density.

e. Take out the incubated 384-well experimental plate from the cell culture incubator, remove the medium, and transfer the control substance, the test product working solution and the effector cell suspension to the 384-well experimental plate in turn.

f. Transfer the experimental plate to a cell culture incubator (37° C./5% CO ₂ ) and incubate for about 6 hours.

g. Prepare luciferase substrate working solution.

h. Take out the incubated 384-well experimental plate from the cell culture incubator, and transfer the luciferase substrate working solution to the 384-well experimental plate.

i. Read the chemiluminescence value on PHERA Star FSX and record the data.

j. Data analysis: According to the corresponding relationship between the relative chemiluminescence signal value (Relative Luminescence Unit) and the final detection concentration, the corresponding dose-effect curve is established.

The test results are shown in Figure 5 and Table 6. The anti-GITR agonist antibody 36E5 can effectively bind to GITR and activate downstream pathways to generate signals. The negative control IgG isotype cannot bind to GITR and cannot activate downstream pathways to generate signals. Both C12 and C15 can activate downstream signaling pathways with similar activities, and the dose-response curve EC ₅₀ and dose-response maximum of the samples are significantly better than 36E5.

Table 6

Example 7 In vitro inhibition of TGF-β activity by GITR/TGF-β dual targeting fusion protein

TGF-β receptor reporter gene assay (TGF-β receptor Reporter Gene Bioassay) is a bioluminescent detection method that relies on genetically engineered cell lines. When the TGF-β protein binds to the TGF-β receptor on the cell surface, the receptor-mediated signal transduction pathway is activated to induce the expression of luciferase, and then by adding the luciferase detection reagent (Bio-Glo , Promega) to read the luminescent signal generated by the luciferase-catalyzed substrate. The addition of anti-TGF-β antibodies or inhibitors blocks the binding of TGF-β to its receptor, resulting in a decrease in the luminescent signal. In this experiment, A549/SBE cells (provided by GenScript) were used as a functional cell line, and the antibody TGF-β antibody BMK-R2 (GenScript) was used as a positive control to evaluate the GITR/TGF-β dual-targeting fusion protein Block the activity of TGF-β in vitro.

Detection samples: C12 prepared as in Example 1 and anti-TGF-β antibody BMK-R2.

The experimental procedure is described as follows:

a. Collect the target cells by centrifugation, resuspend the cells in F12K Nutrient Mixture medium containing 10% FBS and adjust the cell density, and inoculate them in 96-well experimental plates.

c. Use the assay buffer to configure the EC100 reference substance and the test substance at a concentration of 7×.

d. Take out the incubated 96-well experimental plate from the cell culture incubator, and transfer TGF-β1 (EC100), the control substance and the working solution of the test product to the 96-well experimental plate in turn.

e. Transfer the experimental plate to a cell culture incubator (37° C./5% CO ₂ ) and incubate for about 24 hours.

f. Prepare luciferase substrate working solution.

g. Take out the incubated 96-well experimental plate from the cell culture incubator, and transfer the luciferase substrate working solution to the 96-well experimental plate.

h. Read the chemiluminescence value on the PHERA Star FSX and record the data.

i. Data analysis, based on the corresponding relationship between the relative chemiluminescence signal value (Relative Luminescence Unit) and the final detection concentration to establish a corresponding dose-effect curve.

The experimental results are shown in Figure 6 and Table 7. From Figure 6 and Table 7, it can be seen that the positive control (BMK-R2) can effectively bind to TGF-β, and inhibit the downstream pathway to generate signals, and the _IC50 of the dose-effect curve is 0.226nM, and the negative control (Human IgG1) cannot bind to TGF-β. -β binding, unable to inhibit signaling from downstream pathways. Sample C12 can effectively bind to TGF-β, and inhibit downstream pathways to generate signals. The IC ₅₀ of the dose-effect curve is 0.29nM, which is comparable to the activity of BMK-R2.

Table 7

Example 8 GITR/TGF-β dual-targeting fusion protein activates the activity of effector T cells in vitro

Test samples: C12, C15, 336B11 and anti-GITR agonist antibody 36E5 prepared as in Example 1.

In the experiment, the Raji cell cross-linking method was used to detect the activity of the fusion protein to activate T cells. The detection index was IFN-γ, and the steps were as follows:

1. Coat 0.2μg/ml OKT3 onto a 96-well plate;

2. Add isolated T cells (purchased from Wisdom Chemicals) and culture for 4 days;

3. Take out T cells, stop pre-stimulation, and leave for 2 days;

4. Add Raji cells at 1×10 ⁵ cells/well to 96 wells pre-bound with 2 μg/ml OKT3;

5. Add sample proteins C12, C15, 36E5 and 336B11, the highest concentration is 50 μg/ml or 10 μg/ml, 5-fold gradient dilution, 4 concentration gradients for each sample, and 3 replicate wells for each concentration;

6. Mix T cells, add to 96-well plate at a concentration of 1E5/well, and culture for 4 days;

7. Collect the supernatant and detect the IFN-γ content by ELISA.

The results are shown in Figure 7 and Figure 8: According to the content of the cytokine IFN-γ released after T cells are activated, it can be known that the activity of C12 activated effector T cells is much higher than that of the positive control antibody 36E5 and the single targeting protein 336B11, and higher than C15. It can be seen that the dual-targeting fusion protein C12 of the present invention has better activity in activating effector T cells than the GITRL extracellular domain trimer protein 336B11 of single-targeting GITR and anti-GITR agonist antibody 36E5. In addition, the effect of C12 is better than that of C15. It is speculated that the structure of C12 is the most favorable for the dual-targeting fusion protein, and C15 may have steric hindrance in its function, which is consistent with the results of Examples 5 and 6.

Example 9 In vivo tumor suppressive activity of GITR/TGF-β dual targeting fusion protein

The anti-tumor effect of the fusion protein of the present invention was tested in the mouse model of colon cancer MC38-OVA cell line subcutaneously implanted with C57BL/6-hGITR female transgenic mice.

Collect MC38-OVA cells in the exponential growth phase (Crown Biotechnology Co., Ltd.), and subcutaneously inoculate C57BL/6-hGITR on the right back on Day 0 at a dose of 1×10 ⁶ cells/mouse For mice, on Day 3 after tumor cell inoculation (Day 3), the body weight of all animals was weighed. The mice were grouped according to their body weight to ensure that the body weights of different groups were similar, with 9 mice in each group, 2 groups in total. The intraperitoneal injection was administered on the 3rd, 6th, 9th, and 12th day after the inoculation of tumor cells, and the administration was administered four times in total. The dosage of the test group was C12 30mg/kg, and the control group was an equal volume of vehicle (PBS solution). From the 6th day (Day 6), the body weight and tumor size of the mice were measured twice a week.

The results showed that during the experiment, the animals were in good health and the mice tolerated each treatment antibody well. The tumor growth of the treatment group and the control group is shown in FIG. 9 . The average tumor volume of mice in the vehicle control group was 897.16 mm ³ on the 8th day (Day 20) after administration. The average tumor volume of the fusion protein C12 (30mg/kg) treatment group on Day 20 was 363.98mm ³ , and the relative tumor inhibition rate TGI (%) was 59%, which was statistically significantly different from that of the control group (p=0.0498) . The average tumor volume of mice in the vehicle control group was 2025.13 mm ³ on day 12 (Day 24) after administration. The average tumor volume of the fusion protein C12 (30 mg/kg) treatment group on Day 24 was 1039.94 mm ³ , and the relative tumor inhibition rate TGI (%) was 49%. It shows that the dual targeting fusion protein C12 of the present invention has significant tumor suppressing activity.

The calculation formula of relative tumor inhibition rate TGI (%) is as follows:

TGI (%)=(1-T/C)×100%, where T and C are the tumor volume (TV) of the treatment group and the control group at a specific time point, respectively.

sequence listing

Claims

A fusion protein comprising from N-terminus to C-terminus:

The first polypeptide-the second polypeptide-the third polypeptide Formula (1);

or

The third polypeptide-the second polypeptide-the first polypeptide Formula (2);

in

The first polypeptide comprises an extracellular domain of TGF-βRII (TGFBR2-ECD);

The second polypeptide has the following structure from N-terminus to C-terminus:

Lk 1 –Dd–Lk 2 ,

in

Dd is a dimerization domain;

Lk 1 and Lk 2 are each independently a linker or absent; and

The third polypeptide has the following structure from the N-terminus to the C-terminus:

Sd 1 -Ln 1 -Sd 2 -Ln 2 -Sd 3 ,

in

Sd 1 , Sd 2 and Sd 3 are each independently an extracellular domain of GITRL;

Ln 1 and Ln 2 are each independently a linker or absent.
The fusion protein of claim 1, wherein the TGFBR2-ECD comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; preferably, the TGFBR2-ECD comprises the amino acid sequence of SEQ ID NO:5 amino acid sequence.
The fusion protein of claim 1 or 2, wherein the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2; preferably, the GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO:2 amino acid sequence.
The fusion protein according to any one of claims 1-3, wherein the third polypeptide comprises the amino acid sequence of SEQ ID NO:3.
The fusion protein according to any one of claims 1-4, wherein the dimerization domain comprises the CH2 and CH3 domains of an immunoglobulin from the N-terminus to the C-terminus, preferably the CH2 and CH3 domains of human IgG1; more preferably Preferably, the CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.
The fusion protein according to any one of claims 1-5, wherein the second polypeptide has the following structure from the N-terminus to the C-terminus:

Lk 1 -Dd -Lk 2 ;

in

Dd is a dimerization domain comprising from N-terminus to C-terminus the hinge region or part thereof, CH2 and CH3 domains of an immunoglobulin, preferably the Fc fragment of human IgG1; preferably, the hinge region or part thereof comprises The amino acid sequence of SEQ ID NO:9, said CH2 and CH3 domains comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8;

Lk 1 and Lk 2 are each independently a linker.
The fusion protein of any one of claims 1-6, wherein

The TGFBR2-ECD comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6;

The GITRL extracellular domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and

The dimerization domain comprises a hinge region or part thereof, CH2 and CH3 domains of an immunoglobulin from the N-terminus to the C-terminus, the hinge region or part thereof comprises the amino acid sequence of SEQ ID NO: 9, the CH2 and the CH3 domain comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.
The fusion protein according to any one of claims 1-7, wherein the linker comprises 5-25 amino acids, each of which is independently selected from glycine, serine and alanine; preferably, the linker comprises (GGGGS) n , wherein n is an integer selected from 1-10; more preferably, the linker comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.
The fusion protein of claim 1, which comprises the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.
A polynucleotide encoding the fusion protein of any one of claims 1-9.
An expression vector comprising the polynucleotide of claim 10.
A host cell comprising the polynucleotide of claim 10 or the expression vector of claim 11;

Preferably, the host cell is a eukaryotic cell; more preferably, the host cell is a yeast cell, a mammalian cell or an immune effector cell; further preferably, the host cell is an effector T cell.
A conjugate comprising the fusion protein of any one of claims 1-9 conjugated to at least one therapeutic agent; preferably, the therapeutic agent is selected from the group consisting of detectable markers, chemotherapeutic agents, cytotoxins, Radionuclides, immune checkpoint inhibitors, cytokines and enzymes.
A pharmaceutical composition comprising:

(i) the fusion protein of any one of claims 1-9, and

(ii) a pharmaceutically acceptable carrier;

Preferably, the pharmaceutical composition is prepared as an injection.
Use of the fusion protein according to any one of claims 1-9, the polynucleotide according to claim 10, the conjugate according to claim 13 or the pharmaceutical composition according to claim 14 in the preparation of medicines;

Preferably, the drug is used to treat and/or prevent cancer;

or

The drug is used to activate GITR signaling and inhibit TGF-beta signaling; or

The medicament is used to induce, promote, enhance, activate or prolong an immune response in a subject in need thereof; or

The medicament is for inducing, promoting, enhancing, activating or prolonging an immune response against a tumor or tumor cells in a subject in need thereof;

Preferably, the cancer is selected from adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's Chickkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, myeloma , multiple myeloma, salivary gland cancer, kidney cancer, renal cell carcinoma, Wellman's tumor, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, and head and neck cancer.