TWI815184B - TGFBR2-ECD mutants and fusion proteins containing them and their applications - Google Patents

Abstract

The invention relates to the field of biomedicine. Specifically, the present invention discloses TGFBR2-ECD mutants, as well as fusion polypeptides, targeting molecules or fusion proteins containing them, and the use of the mutants, fusion polypeptides, targeting molecules or fusion proteins in the preparation of drugs, In particular the use in the preparation of medicaments for the treatment and/or prevention of neoplastic diseases.

Description

TGFBR2-ECD mutants and fusion proteins containing them and their applications

The invention relates to the field of biomedicine. Specifically, the present invention provides a polypeptide that is a TGF-β RII extracellular domain (TGFBR2-ECD) mutant, a fusion protein containing the TGFBR2-ECD mutant, and its use in the preparation of medicines.

Transforming growth factor-β (TGF-β) has three highly homologous subtypes: TGF-β1, TGF-β2 and TGF-β3, which together belong to the TGF-β superfamily. The high-affinity binding of TGF-β to membrane receptors can initiate the downstream SMAD protein phosphorylation pathway to participate in the development of the disease.

In the process of tumor development, TGF-β regulates the abnormal expression of genes related to tumor cell proliferation, differentiation, and migration, and participates in enhancing cancer processes such as tumor tissue angiogenesis, cell metastasis, and mesenchymal transformation. It also participates in the suppression of the human immune monitoring system and ultimately exert a cancer-promoting effect. Abnormal expression of TGF-β changes the tumor tissue microenvironment, and its high expression level in vivo is associated with poor prognosis of various tumors. In colorectal cancer, SMAD activation of the TGF-β downstream pathway can initiate the expression of biomarkers related to poor prognosis. The high expression of TGF-β1 and TGF-β3 in patients is significantly related to their poor prognosis. Studies have shown that TGF-β can promote the generation of Treg cells, inhibit the initiation and differentiation of CD8+ T cells and NK cells, and is also involved in inhibiting the proliferation of first-generation CAR-T and CD137 costimulatory signals. Furthermore, TGF-β is closely associated with a variety of inflammatory and autoimmune diseases: in models of inflammatory bowel disease, TGF-β By inducing a large number of Th17 cell differentiation, it leads to the exacerbation of Th17 cell-related autoimmune diseases; in asthma models, IL-13 participates in the pulmonary fibrosis process of patients by inducing the production of TGF-β.

TGF-β signaling is mediated by the TGF-β receptor complex. The TGF-β receptor complex is composed of a TGF-β type I receptor (TGF-βRI) homodimer and a TGF-β type II receptor (TGF-βRII) homodimer. Studies have found that two extracellular domains of TGF-βRII (TGFBR2-ECD) directly bind to TGF-β dimers to form a complex. Therefore, TGFBR2-ECD can be used to develop TGF-β Trap to capture TGF-β, thus blocking the TGF-β signaling pathway.

In tumor treatment, TGF-β inhibition of the tumor microenvironment has been recognized as an effective therapeutic target, but TGF-β inhibitors alone have shown poor efficacy in clinical studies. Some bifunctional molecules containing TGF-β Trap have been developed in the prior art, such as bifunctional molecules containing anti-PD-L1 antibodies and TGF-β Trap (see, for example, WO2018/205985 and WO2015/118175), which can simultaneously block TGF -β signaling pathway and inhibit immune checkpoints for cancer treatment. Among them, the solution of WO2015/118175 uses wild-type TGFBR2-ECD as the TGF-β Trap. The bifunctional molecule formed by fusion with the PD-L1 antibody is prone to serious degradation and breakage during the production process, which greatly affects the drug development. , which also poses challenges to the effectiveness and safety of drugs. The scheme of WO2018/205985 obtains a more stable bifunctional molecule by truncating the breakable N-terminal region of wild-type TGFBR2-ECD. However, the truncated TGFBR2-ECD may fail in the in vivo environment due to the lack of the original N-terminal region. The sequence results in a structure that is significantly different from the wild-type protein, and there are some unknown risks.

This application claims priority to Chinese Patent Application No. 202011011927.7, titled "TGFBR2-ECD Mutants and Fusion Proteins Containing the Same and Applications", submitted on September 23, 2020. The entire content of this application is incorporated herein by reference.

The present invention provides a mutant of the extracellular region of TGF-βRII, which is capable of binding TGF-β and carries one or more amino acid mutations at the N-terminus, so that compared with the extracellular region of wild-type TGF-βRII, the mutant The mutants comprise amino acid mutations in the cleavage-prone region and/or improvements in hydrophilicity.

In one embodiment, the mutant of the present invention includes the amino acid sequence of SEQ ID NO: 1 and carries one or more amino acid mutations in the amino acids at positions 1-24 at its N-terminus.

In some preferred embodiments, the mutants comprise amino acid mutations in each of three regions, and at least one S or T in each of the three regions, wherein the three Each region includes: the first region QKS (amino acids at positions 6-8), the second region NNDMI (amino acids at positions 10-14), and the third region DNNG (amino acids at positions 17-20).

In some embodiments, the amino acid mutations include amino acid substitutions in each of the three regions with a hydrophilic amino acid, an N-X-S/T motif, or a combination thereof, where X is proline Any amino acid other than acid (P). Preferably, there is at least one substitution in each of the three regions. More preferably, there are at least two substitutions in each of the three regions. In some embodiments, the hydrophilic amino acid is selected from G, T, S, N, E, Y, or combinations thereof. In some embodiments, the N-X-S/T motif is N-A-S.

In some embodiments, the amino acid mutations include mutating the first region QKS to GGS, TGS or NAS, mutating the second region NNDMI to GGSGI, TGSGI or NASGI, and mutating the third region DNNG to GGSG, TGSG Or GNAS. In a specific embodiment, the mutant of the invention has an amino acid sequence selected from SEQ ID NO: 2-6.

In other preferred embodiments, the mutants comprise amino acid mutations in each of the three regions, and at least one S or T in each of the three regions, and Amino acid mutations in the upstream and downstream regions of the three regions are also included. In these embodiments, the amino acid mutations include substituting at least one amino acid in each of the three regions with G, S, E, T, or combinations thereof, and substituting T, Y, or combinations thereof in At least one amino acid is substituted in the upstream and downstream regions of each region. In some embodiments, the amino acid mutation includes mutating the first amino acid I to T, mutating the 5-8 amino acid (VQKS) to TQES or TQTS, and mutating the 9-15 amine The amino acid (VNNDMIV) was mutated to TSESMIT or TNNSMIT, and the amino acid at positions 17-24 (DNNGAVKF) was mutated to GESGATKY or GTSGATKY. In a specific embodiment, the mutant has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

In another aspect, the invention provides a targeting molecule comprising a mutant of the invention and a targeting moiety, wherein the targeting moiety is covalently linked to the N-terminus of the mutant, and the targeting moiety targets The targeted molecules are selected from: growth factors and their receptors, cytokines and their receptors, cell surface antigens, immune checkpoint molecules, hormones and other biologically active components in the body. In one embodiment, the targeting moiety is an antibody or an antigen-binding fragment thereof, a mimetic antibody, a ligand, a ligand-binding domain or a functionally active domain of an in vivo active protein. In one embodiment, the targeting moiety is an Fc fragment, preferably an Fc fragment of human IgGl. In one embodiment, the Fc fragment of human IgG1 is fused to the N-terminus of the TGFBR2-ECD mutant of the invention to provide an Fc-TGFBR2-ECD fusion protein. In a specific embodiment, the Fc-TGFBR2-ECD fusion protein comprises the amino acid sequence of SEQ ID NO: 52.

In a specific embodiment, the targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof.

In another aspect, the invention provides a fusion polypeptide comprising a mutant of the invention and a first polypeptide, and the C-terminus of the first polypeptide is covalently linked to the N-terminus of the mutant. exist In one embodiment, the first polypeptide comprises at least one heavy chain variable region of an antibody or at least one light chain variable region of an antibody. In a specific embodiment, the antibody is an anti-PD-L1 antibody.

In yet another aspect, the invention provides a fusion protein comprising a fusion polypeptide of the invention and a second polypeptide, wherein the second polypeptide, when combined with the first polypeptide, forms a targeting moiety. In one embodiment, the first polypeptide comprises at least one heavy chain variable region of an antibody and the second polypeptide comprises at least one light chain variable region of an antibody. In yet another embodiment, the first polypeptide comprises at least one light chain variable region of an antibody and the second polypeptide comprises at least one heavy chain variable region of an antibody. In a specific embodiment, the antibody is an anti-PD-L1 antibody. In a specific embodiment, the fusion proteins of the invention form dimers.

On the other hand, the present invention also provides a pharmaceutical composition, which contains the targeting molecule, fusion polypeptide or fusion protein of the present invention, and a pharmaceutically acceptable carrier. The present invention also provides the use of the targeting molecule, fusion polypeptide, fusion protein or pharmaceutical composition in the preparation of drugs for the treatment and/or prevention of tumor diseases.

In yet another aspect, the present invention also provides nucleic acid molecules encoding the mutants, targeting molecules, fusion polypeptides or fusion proteins, expression vectors comprising the nucleic acid molecules, and host cells comprising the nucleic acid molecules or expression vectors.

Figure 1A: Schematic diagram of the structure of anti-PD-L1/TGF-β Trap and schematic diagram of the degradation of wild-type anti-PD-L1/TGF-β Trap.

Figure 1B: Reduced protein electrophoresis of wild-type anti-PD-L1/TGF-β Trap and mutant anti-PD-L1/TGF-β Trap, using silver staining to develop protein bands: Lane 1 is anti-PD-L1 antibody , Lane 2 is wild-type anti-PD-L1/TGF-β Trap, Lane 3 is Mu5; HC, heavy chain; LC, light chain; fusion protein-HC, Fusion polypeptide of anti-PD-L1 antibody heavy chain and TGFBR2-ECD; fusion protein-LC, antibody light chain in anti-PD-L1/TGF-β Trap, consistent with anti-PD-L1 antibody light chain; degradation product, fusion Protein-HC is a missing TGFBR2-ECD product resulting from cleavage of the N-terminus of TGFBR2-ECD.

Figure 2: SEC-HPLC spectrum shows the proportion of each component in wild-type anti-PD-L1/TGF-β Trap (WT) (2A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (2B) ; Mono, monomer; LMW, low molecular weight degrader.

Figure 3: Non-reducing CE-SDS capillary electrophoresis pattern shows wild-type anti-PD-L1/TGF-β Trap (WT) (3A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (3B) The proportion of each component: Fragment, incomplete structural protein; Intact, complete protein.

Figure 4: Reduced CE-SDS capillary electrophoresis pattern shows each of wild-type anti-PD-L1/TGF-β Trap (WT) (4A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (4B) Ratio of components: HC, fusion polypeptide of antibody heavy chain in anti-PD-L1/TGF-β Trap and TGFBR2-ECD; LC, antibody light chain in anti-PD-L1/TGF-β Trap; Fragment, incomplete Structural protein fragment; Intact, complete protein.

Figure 5: Binding of wild-type anti-PD-L1/TGF-β Trap (WT) and mutant anti-PD-L1/TGF-β Trap (Mu5-11) to hPD-L1 (5A) and hTGF-β1 (5B) Activity ELISA test results.

Figure 6: Molecular dynamics detection results of the interaction between wild-type anti-PD-L1/TGF-β Trap (WT) (6A) and mutant anti-PD-L1/TGF-β Trap (Mu5) (6B) and hTGF-β1 .

Figure 7: Results of detecting the inhibition of expression of EMT pathway genes by wild-type anti-PD-L1/TGF-β Trap and mutant anti-PD-L1/TGF-β Trap in A549 cell line: in the presence of hTGF-β1, respectively Detect the expression inhibition of EMT pathway genes by wild-type anti-PD-L1/TGF-β Trap and mutant PD-L1/TGF-β Trap. The genes detected from left to right are: CDH2, MMP2, MMP3, MMP9, TWIST1, VIMENTIN; cell samples are: (1) TGF-b1 2ng: hTGF-β1 with a final concentration of 2ng/ml was added to the culture medium and cultured for 48 hours as a negative control; (2) TGF-b1+WT: TGF-b1 2ng/ml, add 5μg/ml wild-type PD-L1/TGF-β Trap at the same time, culture for 48h; (3) TGF-b1+Mu5: TGF-b1 2ng/ml, add 5μg/ml Mu5 at the same time, culture for 48h.

Figure 8: Detection of in vivo tumor inhibitory activity of mutant anti-PD-L1/TGF-β Trap (Mu5) in MC38/hPD-L1 mouse colon cancer cell subcutaneous xenograft tumor model; the curve shows the in vivo tumor inhibitory activity of each group of mice over time relative tumor volume.

definition

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

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

As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetic (eg, recombinant) produced, that specifically bind to an epitope of an antigen through at least one antigen-binding site. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies encompass antigen-binding fragments. As used herein, the term antibody therefore includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single domain Antibodies and antigen-binding fragments, such as but not limited to Fab fragments, Fab' fragments, F(ab') _fragments , Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv ), single-chain Fab (scFab), diabody, disulfide-linked Fab (dscFab), or antigen-binding fragment of any of the above antibodies. Antibodies provided herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b) members.

As used herein, an "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of said antibody that binds an antigen (e.g., one or more CDRs and/or one or more antigen-binding site), and therefore retains at least part of the full-length antibody's ability to specifically bind the antigen. Antigen-binding fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as derivatives produced by chemical synthesis or genetic engineering techniques (eg, recombinant DNA techniques). Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see , for example Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may include multiple chains linked together, for example by disulfide bonds and/or by peptide linkers. Antigen-binding fragments include any antibody fragment which, when inserted into an antibody framework (eg, by substitution of corresponding regions), results in an antibody that immunospecifically binds an antigen.

"Traditional" or "full-length" antibodies typically contain four polypeptides: two heavy chains and two light chains. Each chain may contain variable and constant regions. Each light chain contains a light chain variable region (V _L ) and a light chain constant region ( _CL ) from N-terminus to C-terminus. Each heavy chain includes a heavy chain variable region ( _VH ) and one or more heavy chain constant regions ( _CH ) from the N-terminus to the C-terminus. The heavy chain constant region from the N-terminus to the C-terminus may include _CH1 , _CH 2 and _CH 3, or _CH 1, _CH 2, _CH 3 and _CH 4. Each _VL and each _VH can contain three highly variable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)". In this article, the CDRs of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3. Those skilled in the art can identify CDRs using methods well known in the art, such as using Kabat or Chothia encoding (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). It is generally considered that an Fv fragment containing six CDRs (i.e., three heavy chain CDRs and three light chain CDRs) is the smallest unit of an antigen-binding site, but in some cases, it contains less than six CDRs (e.g., three, Four or five) other fragments (such as single domain antibodies, also known as nanobodies) also have the ability to bind antigens.

As used herein, "Fv fragment" refers to an antigen-binding fragment comprising a pair of VH and VL. "Single-chain Fv (scFv)" can be obtained by connecting VH and VL through a peptide linker. By introducing disulfide bonds into Fv or scFv, "disulfide bond-stabilized Fv (dsFv)" or "single-chain disulfide bond-stabilized Fv (scdsFv or dsscFv)" can be obtained respectively.

As used herein, "Fab" includes an intact antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also known as Fd). Single-chain "Fab (scFab)" can be obtained by connecting CL and CH1 in "Fab" using a peptide linker. "F(ab') ₂ " essentially consists of two Fab fragments linked by a disulfide bond in the hinge region. "Fab'" is half of F(ab') ₂ , which can be obtained by reducing the disulfide bond in the hinge region of F(ab') ₂ .

As used herein, "Fc fragment" refers to the papain-digested crystallizable fragment of a full-length antibody. Generally speaking, the Fc fragment of IgG may contain part of the hinge region, CH2, and CH3. As used herein, the Fc fragment may comprise at least part of the hinge region (eg all or part of the hinge region), CH2 and CH3. Those skilled in the art can determine the positions of CDR, FR, VH, VL, CL, CH1, CH2, CH3 and hinge regions in antibodies based on known algorithms and software. For descriptions of algorithms and software that can be applied, see for example William R. Strohl, Lila M. Strohl, (2012), Therapeutic Antibody Engineering, Woodhead Publishing, pp.37-56.

As used herein, "diabody" refers to an antibody that contains two scFvs in which the _VH and _VL in each scFv are connected by a short peptide linker (approximately 5-10 amino acid residues). base) are connected, so that the V _H and V _L chains are paired (i.e., the V _H of the first scFv is paired with the V _L of the second scFv, and the V _L of the first scFv is paired with the V _H of the second scFv) to form an antigen binding site. .

As used herein, "chimeric antibody" refers to an antibody in which a portion (e.g., CDRs, FRs, variable regions, constant regions, or a combination thereof) is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remainder A portion is identical or homologous to a corresponding sequence in an antibody derived from another species. Chimeric antibodies can be produced through antibody engineering. Methods for antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be generated by DNA recombinant techniques (see, for example, Sambrook, J., et al. (1989). Molecular cloning: a laboratory manual , 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

As used herein, the term "humanized antibody" refers to an antibody in which a non-human antibody has been modified to increase sequence homology with a human antibody. Humanized antibodies can be obtained by antibody engineering of any non-human species antibody or an antibody containing sequences derived from a non-human species (eg, a chimeric antibody). Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art.

As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. The definition of human antibodies encompasses intact or full-length antibodies, fragments thereof and/or antibodies comprising at least one human heavy chain and/or light chain polypeptide.

As used herein, "single domain antibody (sdAb)", also known as "nanobody", refers to an antibody that contains a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Similar to the variable regions of full-length antibodies, single variable domains typically contain CDR1, CDR2, and CDR3 that form the antigen-binding site and a supporting framework region.

"Affinity" or "binding affinity" is used to measure the strength of the binding between a molecule and its ligand through non-covalent interactions, such as the binding strength between the targeting moiety of the present invention and its target, such as an antibody or antigen-binding fragment. The binding strength between the antigen or the TGFBR2-ECD mutant of the present invention 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 constant (ka) and dissociation constant (kd) and calculating the quotient of kd divided by ka (K _D =kd/ka). _KD can be readily determined using conventional techniques, such as by equilibrium dialysis; by using the Octet RED96 detection system, using the general methods listed by the manufacturer; by using an enzyme-linked immunosorbent assay (ELISA); by using a radiolabeled target antigen Radioimmunoassay; or by other methods known to the skilled person.

"Specific binding" means that two molecules bind to each other with a high affinity. Usually, the K _D value between two molecules that specifically bind can be 10 ^-6 , 10 ^-7 , 10 ^-8 to 10 ^-9 M or lower. For example, the antibody or antigen-binding fragment can specifically bind to the antibody-binding site of the antigen through the antigen-binding site with a K _D value of 10 ^-6 to 10 ^-9 M, and TGFBR2-ECD can specifically bind to TGF-β with a K D value of 10 -6 to 10 -9 M. Specific binding with a K _D value of 10 ^-6 to 10 ^-9 M.

Suitable conservative amino acid substitutions in polypeptides or proteins are known to those skilled in the art and 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 biological activity (see, e.g., Watson et al., Molecular Biology of the Gene , 4th Edition, 1987, The Benjamin/Cummings Pub.co., p. 224).

As used herein, the term "polypeptide" refers to a polymer comprising at least two amino acids or derivatives thereof linked by peptide bonds. A "protein" can be formed covalently or non-covalently from one or more polypeptides.

As used herein, the term "mutation" refers to a polypeptide or protein that contains a substitution, deletion, or addition of one or more amino acids. The substitutions may be conservative or non-conservative substitutions. In embodiments of the invention, one amino acid may be substituted for another amino acid (e.g., G is substituted for Q), or one amino acid moiety may be substituted for another amino acid moiety (e.g., N-A-S moiety is substituted). sequence instead of the Q-K-S motif). As used herein, the event of substituting an amino acid motif at one position or an amino acid at a position is described as a single substitution. As used herein, when specifying a mutation position, the n-th amino acid refers to the n-th amino acid counting from the 1st amino acid at the amino terminus (ie, N-terminus) of the polypeptide. In this article, the designation of mutation positions is based on SEQ ID NO:1.

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" form of a polypeptide or protein contains mutations relative to the "wild-type" polypeptide or protein. For example, the wild-type TGF-βRII extracellular region contains the sequence of SEQ ID NO:1. The mutant of the extracellular region of TGF-βRII carries mutations relative to the sequence of SEQ ID NO:1.

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including those linked together usually by a phosphodiester bond. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

A polypeptide, protein or nucleic acid molecule of the invention may be "isolated". The expression "isolated" means that the polypeptide, protein or nucleic acid molecule may, for example, be artificially engineered and/or separated from other materials in its naturally occurring environment. An "isolated" nucleic acid molecule, 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, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The level of expression of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from the host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue or silver staining, Lowry protein assay, and Bradford protein assay.

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 polypeptides encoded by nucleic acids or vectors. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

As used herein, a "vector" is a vehicle used to introduce exogenous nucleic acid into a host cell so that when the vector is transformed into an appropriate host cell, the exogenous nucleic acid is amplified or expressed. Vectors include those into which nucleic acid encoding a polypeptide or fragment thereof can be introduced, usually by restriction enzyme digestion and ligation. Vectors also include those containing nucleic acid encoding a polypeptide. Vectors usually remain episomal, but can be designed to integrate the gene or part thereof into the chromosome of the genome. Vectors for artificial chromosomes are also considered, e.g. 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 vector" includes a vector capable of expressing a polypeptide that contains 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 a cloned DNA or RNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of the host cell.

As used herein, the term "targeting moiety" refers to a molecule capable of specifically binding to a target. A targeting moiety can target a molecule to which it is covalently linked (eg, a TGFBR2-ECD mutant of the invention) to a specific target site. A targeting moiety can recognize one or more targets.

As used herein, the term "ligand" refers to an active substance that can regulate specific cellular physiological activities, which can bind to specific active proteins in the body and initiate physiological and biochemical reactions in cells.

As used herein, "in vivo active protein" refers to the ligand itself as well as proteins that can modulate cellular physiological activities, such as accept extracellular or intracellular ligands and mediate signal transduction, which can have ligand-binding domains and/or functions. sexual domain. Active proteins in the body can be ligands, receptors, kinases, ion subchannels or other proteins involved in signal transduction. As used herein, the term "ligand-binding domain" refers to the domain of an active protein in vivo that is capable of specifically recognizing and binding a ligand. "Functional domain" refers to the domain of active proteins in the body that can mediate signal transduction and initiate physiological and biochemical reactions in cells.

As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material or composition containing a compound that is at least sufficient to produce a therapeutic effect upon application to an article. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or condition. Likewise, as used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material or composition containing a compound that, when applied to an article, has the desired prophylactic effect, e.g., preventing or delaying disease or the occurrence or recurrence of symptoms, and reduce the likelihood of the occurrence or recurrence of the disease or symptoms. Complete prevention of an effective dose does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "individual" refers to a mammal, such as a human.

TGFBR2-ECD mutant

In one aspect, the invention provides a mutant of TGF-βRII extracellular domain (TGFBR2-ECD), which is capable of binding TGF-β and carries one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) amino acid mutations, whereby the mutant is contained in a breakable region compared to the wild-type TGF-βRII extracellular region Amino acid mutations and/or improvements in hydrophilicity.

As used herein, the terms "TGF-βRII extracellular domain" or "TGFBR2-ECD" have the same meaning and refer to the extracellular domain of TGF-β type II receptor (TGF-βRII) that has the activity to bind TGF-β . Dimeric TGFBR2-ECD can bind and trap TGF-β dimers as a TGF-β Trap (see, e.g., WO2015/118175 and WO2018/205985), thereby inhibiting the TGF-β signaling pathway and eliminating In addition to immunosuppression related to the TGF-β signaling pathway. 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.

Wild-type TGFBR2-ECD is a peptide segment of 136 amino acids (SEQ ID NO: 1), which can be combined with other polypeptides to form bifunctional molecules containing TGF-β Trap, such as anti-PD-L1/TGF-β Trap ( See for example WO2015/118175 and WO2018/205985). However, bifunctional molecules containing wild-type TGFBR2-ECD are prone to fragmentation, which is not conducive to subsequent drug development. The TGFBR2-ECD mutant of the present invention is an improved polypeptide relative to wild-type TGFBR2-ECD. Compared with bifunctional molecules containing wild-type TGFBR2-ECD, bifunctional molecules containing the TGFBR2-ECD mutant of the present invention are less prone to degradation during the production process and have better druggability.

The inventors found that wild-type TGFBR2-ECD is prone to cleavage in the N-terminal amino acids 1-24. In one embodiment, the mutant comprises the amino acid sequence of SEQ ID NO: 1, and has one or more amino acid mutations in the amino acids at positions 1-24 at its N-terminus, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 9 or more. In another embodiment, the number of amino acid mutations is 16 or less.

In some preferred embodiments, the mutants comprise amino acid mutations in at least one, preferably each, of the three regions. Preferably the mutant contains at least one S or T in each of the three regions. The three regions include: the first region QKS (amino acids at positions 6-8), the second region NNDMI (amino acids at positions 10-14), and the third region DNNG (amino acids at positions 17-20). ).

In some embodiments, the amino acid mutations include amino acid substitutions in each of the three regions with a hydrophilic amino acid, an N-X-S/T motif, or a combination thereof, where X is proline Any amino acid other than acid (P). Preferably, in each of the three regions, at least Generation once. More preferably, there are at least two substitutions in each of the three regions. In some embodiments, the hydrophilic amino acid is selected from G, T, S, N, E, Y, or combinations thereof. In some embodiments, the N-X-S/T motif is N-A-S.

In some embodiments, the amino acid mutations include mutating the first region QKS to GGS, TGS or NAS, mutating the second region NNDMI to GGSGI, TGSGI or NASGI, and mutating the third region DNNG to GGSG, TGSG Or GNAS. In a specific embodiment, the mutant of the invention has an amino acid sequence selected from SEQ ID NO: 2-6.

In other preferred embodiments, the mutants comprise amino acid mutations in each of the three regions, and at least one S or T in each of the three regions, and Amino acid mutations in the upstream and downstream regions of the three regions are also included. In these embodiments, the amino acid mutations include substituting at least one amino acid in each of the three regions with G, S, E, T, or combinations thereof, and substituting T, Y, or combinations thereof in At least one amino acid is substituted in the upstream and downstream regions of each region. In some preferred embodiments, the amino acid mutations include mutating the first amino acid I to T, mutating the 5-8 amino acids (VQKS) to TQES or TQTS, and mutating the 9-15 amino acids The amino acid at position 17-24 (DNNGAVKF) was mutated into GESGATKY or GTSGATKY. In a specific embodiment, the mutant has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

Targeting molecules

In yet another aspect, the invention provides a targeting molecule comprising a TGFBR2-ECD mutant of the invention and a targeting moiety covalently linked to the N-terminus of the mutant. In one embodiment, the targeting moiety targets a target selected from the group consisting of growth factors and their receptors, Cytokines and their receptors, cell surface antigens, immune checkpoint molecules, hormones and other bioactive components in the body. The targeting molecule of the present invention is also called a bifunctional molecule, which can play the dual role of simultaneously blocking the TGF-β signaling pathway and specifically binding to a specific target.

The targeting moiety and the TGFBR2-ECD mutant can be covalently linked in any manner to form the targeting molecule. For example, DNA recombinant technology can be used to covalently connect one or more polypeptides in the targeting moiety to the mutant in the form of a recombinant polypeptide or protein, or chemical coupling or enzymatic coupling can be used to covalently link the targeting moiety. Valence connection.

In one embodiment, the targeting moiety comprises one or more polypeptides, and wherein the C-terminus of at least one polypeptide is covalently linked to the N-terminus of the mutant to form a fusion polypeptide.

In some embodiments, the targeting molecule further comprises a linker between the targeting moiety and the mutant. Preferred linkers are peptide linkers. Preferably, the linker is (G ₄ S) _n G, where n is an integer from 2 to 6, preferably an integer from 3 to 5, and more preferably 4.

targeting moiety

A targeting moiety can specifically bind to its target. In preferred embodiments, the targeting moiety may target tumor-specific markers, tumor stem cells, tumor microenvironment, immune checkpoints, or tumorigenesis regulatory mechanisms. Targets of the targeting moiety may include, for example, growth factors or their receptors, cytokines or their receptors, cell surface antigens, immune checkpoint molecules, hormones or other biologically active components in the body. Immune checkpoint molecules may include, for example, PD1, CTLA4, LAG-3, BTLA, TIM-2, LAIR1, PD-L1, B7-DC, B7-H3, B7-H4, HVEM, and TIM-4. In a preferred embodiment, the targeting moiety targets the tumor microenvironment and may, for example, target a cytokine or its receptor or a growth factor or its receptor as described herein. Examples of targets of the targeting moiety may include, but are not limited to: CD13, CD19, CD22, CD25, CD27, CD30/TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117/KIT, CD123, CD138, CD142, CD174, CD227/MUC1, CD352, CLDN18.2, DLL3, CN33, GPNMB, ENPP3, Nectin-4, HER2/ErbB2, EGFR, FGFR, VEGFR, HGFR, PDGFR, VEGF, HGF, FGF, FGF-2, PDGF, EGF, Galectin-3, SLC44A4/AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, SLITRK6, SLAMF7/CS1, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, CA6, Integrin αV, TDGF1, Ephrin A4, PTK7 , NOTCH3, C4.4A, FLT3, PD1, CTLA4, LAG-3, BTLA, TIM-2, LAIR1, PD-L1, B7-DC, B7-H3, B7-H4, HVEM and TIM-4.

Targeting moieties that can be used in the present invention include, but are not limited to, for example, antibodies or antigen-binding fragments thereof, mimetic antibodies, ligands, ligand-binding domains or functionally active domains of in vivo active proteins, and the like. In some embodiments, the targeting moiety is a ligand. In one embodiment, the ligand is a cell surface antigen, cytokine, growth factor or hormone. Cell surface antigens may include tumor-specific antigens and tumor-associated antigens. Cytokines can be selected from: G-CSF, GM-CSF, eotaxin/CCL11, MCP-1/CCL2, MIP-1α/CCL4, RANTES/CCL5, 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, IL-23, INFα, INFβ, INFγ and TNFα and TNFβ. Growth factors may be selected from: VEGF, HGF, FGF, FGF-2, PDGF, IGF, TGF, NGF, EPO and EGF. The hormone may be a peptide hormone or a steroid hormone, preferably a peptide hormone. In some embodiments, the targeting moiety is the ligand binding domain of an in vivo active protein. In vivo active proteins can be receptors (such as T cell receptors or their co-receptors, cytokine receptors, growth factor receptors, hormone receptors, etc.), kinases, ion channels or other proteins involved in signal transduction. Preferably, the ligand binding domain or functional domain is a T cell receptor, a cytokine receptor, a growth factor receptor, Ligand binding domain or functional domain of a hormone receptor. The ligand, ligand binding domain or functionally active domain may be wild type or mutant.

In one embodiment, the targeting moiety is an antibody or an antigen-binding fragment thereof, a mimetic antibody, a ligand, a ligand-binding domain or a functionally active domain of an in vivo active protein.

In some embodiments, the targeting moiety is an Fc fragment. Preferably, the targeting moiety is the Fc fragment of human IgG1. The Fc fragment can be modified to achieve desired in vivo activity, such as extending the serum half-life of the targeting molecule, or enhancing or weakening the effector function of the targeting molecule (e.g., antibody-dependent cell-mediated toxicity (ADCC) and complement-dependent cytotoxicity) function (CDC)). In some embodiments, the Fc fragment of human IgG1 contains the N297A mutation, that is, asparagine (N) at position 297 of the human IgG1 heavy chain is mutated to alanine (A). The Fc fragment containing the N297A mutation loses the ability to bind to FcγR, thereby eliminating Fc fragment-mediated ADCC. In one embodiment, the Fc fragment of human IgGl comprises the amino acid sequence of SEQ ID NO: 53. In one embodiment, the Fc fragment of human IgGl is fused to the N-terminus of the TGFBR2-ECD mutant of the invention, optionally via a linker, to provide an Fc-TGFBR2-ECD fusion protein. In a specific embodiment, the Fc-TGFBR2-ECD fusion protein comprises the amino acid sequence of SEQ ID NO: 52.

Preferably, the targeting moiety is an antibody or an antigen-binding fragment thereof, such as an antibody or an antigen-binding fragment thereof that specifically binds to the above target. The antibody or antigen-binding fragment thereof may be selected from the group consisting of 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 a preferred embodiment, the targeting moiety is an antibody or antigen-binding fragment thereof that targets the tumor microenvironment. In a specific embodiment, the targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof. The anti-PD-L1 antibodies include, but are not limited to, MSB0010718C, MEDI4736, BMS-936559, MPDL3280A, and anti-PD-L1 antibodies described in WO 2019/233462.

In some embodiments, the targeting moiety comprises an anti-PD-L1 antibody or antigen-binding fragment thereof. Preferably, the anti-PD-L1 antibody or antigen-binding fragment thereof is as described above.

In one embodiment, the targeting moiety comprises _VH and _VL of the antibody, and the C-termini of _VH and _VL are each covalently linked to the N-terminus of both mutants. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein.

In one embodiment, the targeting moiety comprises VH _{and VL} of the first antibody and _VH and _VL of the second antibody, and the C-termini of the two _VHs are each covalently linked to the N-termini of the two mutants . The first and second antibodies may be the same or different. In one embodiment, the first and second antibodies are anti-PD-L1 antibodies as described herein.

In one embodiment, the targeting moiety comprises the _VH - _CH 1 and light chain of the antibody, and the C-termini of CH ₁ and _CL are each covalently linked to the N-termini of the two mutants. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein.

In one embodiment, the targeting moiety comprises the _VH - _CH 1 and light chain of the first antibody and the _VH - _CH 1 and light chain of the second antibody, and the C terminus of both _VH - _CH 1 Each is covalently linked to the N-terminus of both mutants. The first and second antibodies may be the same or different. In one embodiment, the first and second antibodies are anti-PD-L1 antibodies as described herein.

In one embodiment, the targeting moiety comprises the heavy and light chains of the first antibody and the heavy and light chains of the second antibody, and the C-termini of the two heavy chains are each covalently linked to the N-termini of the two mutants . The first and second antibodies may be the same or different. In one embodiment, the first and second antibodies are anti-PD-L1 antibodies as described herein.

anti-PD-L1 antibody

In some preferred embodiments, the targeting moiety is an anti-PD-L1 antibody or antigen-binding fragment thereof. Exemplary anti-PD-L1 antibodies include, but are not limited to, MSB0010718C, MEDI4736, BMS-936559, MPDL3280A, and the anti-PD-L1 antibodies described in WO 2019/233462. In some preferred embodiments, the anti-PD-L1 antibodies are the anti-PD-L1 antibodies B10, H12 and B11 described in WO 2019/233462, the relevant contents of which are incorporated herein by reference in their entirety.

In some embodiments, the anti-PD-L1 antibody is a B10 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising SEQ ID NO: 16 The amino acid sequence of HCDR2, which includes the amino acid sequence of SEQ ID NO: 17, and HCDR3, which includes the amino acid sequence of SEQ ID NO: 18; the light chain variable region includes: LCDR1, which includes The amino acid sequence of SEQ ID NO:20, LCDR2, which contains the amino acid sequence of SEQ ID NO:21, and LCDR3, which contains the amino acid sequence of SEQ ID NO:22.

In yet another embodiment, the anti-PD-L1 antibody is a B11 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising SEQ ID NO: The amino acid sequence of 24, HCDR2, comprising the amino acid sequence of SEQ ID NO: 25, and HCDR3, which includes the amino acid sequence of SEQ ID NO: 26; the light chain variable region includes: LCDR1, which includes the amino acid sequence of SEQ ID NO: 28, LCDR2, which includes the amine of SEQ ID NO: 29 amino acid sequence, and LCDR3 comprising the amino acid sequence of SEQ ID NO:30.

In another embodiment, the anti-PD-L1 antibody is an H12 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising SEQ ID NO: The amino acid sequence of 32, HCDR2, which includes the amino acid sequence of SEQ ID NO: 33, and HCDR3, which includes the amino acid sequence of SEQ ID NO: 34; the light chain variable region includes: LCDR1, which comprises the amino acid sequence of SEQ ID NO:36, LCDR2, which comprises the amino acid sequence of SEQ ID NO:37, and LCDR3, which comprises the amino acid sequence of SEQ ID NO:38.

In yet another embodiment, the B10 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19. In yet another embodiment, the B10 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:23. In one embodiment, the B11 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:27. In yet another embodiment, the B11 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:31. In one embodiment, the H12 antibody comprises a heavy chain variable region, said heavy chain variable region Contains the amino acid sequence of SEQ ID NO: 35. In yet another embodiment, the H12 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:39.

In yet another embodiment, the B10, B11 or H12 antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:40. In yet another embodiment, the B10, B11 or H12 antibody further comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:41.

In one embodiment, the B11 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, the B11 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43.

fusion peptide

In another aspect, the invention provides a fusion polypeptide comprising a TGFBR2-ECD mutant and a first polypeptide, and the C-terminus of the first polypeptide is covalently linked to the N-terminus of the mutant.

In some embodiments, the first polypeptide is a targeting moiety as described above. In some embodiments, the first polypeptide and other polypeptides form a targeting moiety as described above.

In some embodiments, the first polypeptide comprises at least one heavy chain variable region of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises the heavy chain variable region of an antibody, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 27, and 35. In yet another specific embodiment, the first polypeptide further comprises a heavy chain constant region of an antibody, said heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41.

In some embodiments, the first polypeptide comprises the heavy chain of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises the heavy chain of an antibody, the heavy chain comprising the amino acid sequence of SEQ ID NO: 43.

In some embodiments, the first polypeptide comprises at least one light chain variable region of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises the light chain variable region of an antibody, the light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 31 and 39. In yet another specific embodiment, the first polypeptide further comprises a light chain constant region of an antibody, said light chain constant region comprising the amino acid sequence of SEQ ID NO: 40.

In some embodiments, the first polypeptide comprises the light chain of an antibody. In one embodiment, the antibody is an anti-PD-L1 antibody as described herein. In a specific embodiment, the first polypeptide comprises a light chain of an antibody, the light chain comprising the amino acid sequence of SEQ ID NO: 42.

In some embodiments, the fusion polypeptide further comprises a linker between the first polypeptide and the mutant. Preferred linkers are peptide linkers. Preferably, the linker is (G ₄ S) _n G, where n is an integer from 2 to 6, preferably an integer from 3 to 5, and more preferably 4.

In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from SEQ ID NO: 9-15.

fusion protein

In yet another aspect, a fusion protein is provided, comprising a fusion polypeptide of the invention and a second polypeptide. The fusion protein of the present invention can be a targeting molecule as described above, which contains a targeting moiety and a mutant of the present invention. In one embodiment, the second polypeptide, when combined with the first polypeptide, forms a targeting moiety as described herein.

In some embodiments, the first polypeptide comprises at least one heavy chain variable region of an antibody and the second polypeptide comprises at least one light chain variable region of an antibody. In some embodiments, the first polypeptide comprises at least one light chain variable region of an antibody and the second polypeptide comprises at least one heavy chain variable region of an antibody.

In a specific embodiment, the first polypeptide comprises the heavy chain of an antibody and the second polypeptide comprises the light chain of an antibody.

In one embodiment, the antibody is an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein. Therefore, the present invention also provides a bifunctional fusion protein targeting PD-L1 and TGF-β, which can also be called a mutant anti-PD-L1/TGF-β Trap.

In one embodiment, the fusion protein comprises: (1) a fusion polypeptide, from the N-terminus to the C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region, the heavy chain variable region The region includes HCDR1, which includes the amino acid sequence of SEQ ID NO: 16, HCDR2, which includes the amino acid sequence of SEQ ID NO: 17, HCDR3, which includes the amino acid sequence of SEQ ID NO: 18; and (ii ) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising LCDR1 comprising The amino acid sequence of SEQ ID NO:20, LCDR2, which contains the amino acid sequence of SEQ ID NO:21, and LCDR3, which contains the amino acid sequence of SEQ ID NO:22.

In yet another embodiment, the fusion protein comprises: (1) a fusion polypeptide, from N-terminus to C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 25, HCDR3, It comprises the amino acid sequence of SEQ ID NO: 26; and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) a second polypeptide comprising a light chain A variable region, the light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprising SEQ ID NO :30 amino acid sequence.

In another embodiment, the fusion protein comprises: (1) a fusion polypeptide, from the N-terminus to the C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region, the heavy chain may The variable region includes HCDR1, which includes the amino acid sequence of SEQ ID NO: 32, HCDR2, which includes the amino acid sequence of SEQ ID NO: 33, HCDR3, which includes the amino acid sequence of SEQ ID NO: 34; and ( ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) a second polypeptide comprising a light chain variable region comprising LCDR1, which comprises the amino acid sequence of SEQ ID NO:36, LCDR2, which comprises the amino acid sequence of SEQ ID NO:37, and LCDR3, which comprises the amino acid sequence of SEQ ID NO:38.

In one embodiment, the fusion protein comprises: (1) a fusion polypeptide, from the N-terminus to the C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a mutant comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 19 The amino acid sequence of SEQ ID NO: 23; and (2) a second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.

In yet another embodiment, the fusion protein comprises: (1) a fusion polypeptide, from the N-terminus to the C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region, the heavy chain may The variable region comprises the amino acid sequence of SEQ ID NO: 27, and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) a second polypeptide comprising a light A chain variable region, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 31.

In another embodiment, the fusion protein comprises: (1) a fusion polypeptide, from the N-terminus to the C-terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region, the heavy chain may The variable region comprises the amino acid sequence of SEQ ID NO: 35, and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) a second polypeptide comprising a light A chain variable region, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 39.

In yet another embodiment, the fusion protein comprises: (1) a fusion polypeptide, from N-terminus to C-terminus, comprising: (i) A first polypeptide comprising a heavy chain variable region and a heavy chain constant region, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 27 and 35, the heavy chain The constant region comprises the amino acid sequence of SEQ ID NO: 41, and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) a second polypeptide comprising a light chain variable region and a light chain constant region, the light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 23, 31 and 39, the light chain constant region comprising the amine group of SEQ ID NO: 40 acid sequence.

In a specific embodiment, the fusion protein comprises: (1) a fusion polypeptide, from the N-terminus to the C-terminus, comprising: (i) a first polypeptide comprising a heavy chain comprising SEQ ID NO. : the amino acid sequence of 43, and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) a second polypeptide comprising a light chain, the light chain Contains the amino acid sequence of SEQ ID NO: 42.

In some embodiments, the fusion protein further comprises a linker between the first polypeptide and the mutant. Preferably, the linker is (G ₄ S) _n G, where n is 2-6. An integer, preferably an integer from 3 to 5, more preferably 4.

In one embodiment, the fusion protein forms a dimer.

neoplastic disease

The targeting molecules, fusion polypeptides, fusion proteins and pharmaceutical compositions of the present invention can be used to treat and/or prevent tumors, inflammation, autoimmunity and other diseases caused by TGF-β family related pathways.

The present invention also relates to the use of the targeting molecule, fusion polypeptide, fusion protein or pharmaceutical composition of the present invention in the preparation of medicaments for the treatment and/or prevention of neoplastic diseases.

The present invention also relates to a method for treating and/or preventing neoplastic diseases, which includes administering the targeting molecule, fusion polypeptide, fusion protein or pharmaceutical composition of the present application to an individual in need.

The targeting molecules, fusion polypeptides, fusion proteins (especially anti-PD-L1/TGF-β Trap) or pharmaceutical compositions of the present invention can be used to treat and/or prevent tumor diseases. Preferred neoplastic diseases (or cancers) that can be prevented and/or treated include cancers that are generally responsive to immunotherapy, particularly PD-L1 positive cancers. Non-limiting examples of treatable cancers include, but are not limited to, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, colloid tumors, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus tumors and osteosarcoma. Examples of other cancers include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal area cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva Cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethra cancer, penile cancer, chronic or acute leukemia , including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system Systemic (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environment Induced cancers, including asbestos-induced cancers, and combinations of said cancers. The fusion protein and pharmaceutical composition of the present invention can also be used to treat metastatic cancer, especially metastatic cancer expressing PD-L1. In yet another embodiment, the neoplastic disease (or cancer) is lung cancer, ovarian cancer, colorectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer Carcinoma, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus cancer or osteosarcoma, especially colorectal cancer or non-small cell lung cancer.

In some embodiments, the anti-PD-L1/TGF-β Traps of the invention can be used to treat cancer. In some embodiments, the cancer is a PD-L1 positive cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, ovarian cancer, colorectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasal cancer, Pharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus cancer, osteosarcoma, colorectal cancer and non-small cell lung cancer.

Nucleic acids, vectors and host cells

In another aspect, the invention provides an isolated nucleic acid molecule encoding a mutant, targeting molecule, fusion polypeptide or fusion protein of the invention. The nucleotide sequence of the nucleic acid molecule can be codon-optimized for the host cell used for expression.

The invention also provides an expression vector comprising at least one nucleic acid molecule of the invention.

One aspect of the invention also provides a host cell comprising at least one nucleic acid molecule or expression vector of the invention.

Methods for producing fusion polypeptides

A further aspect of the invention also relates to a method for producing the mutant, targeting molecule, fusion polypeptide or fusion protein of the invention, said method comprising: (i) in the case of a suitable nucleic acid molecule or expression vector for expression Culturing the host cells of the invention, and (ii) isolating and purifying the mutants, targeting molecules, fusion polypeptides or fusion proteins of the invention.

pharmaceutical composition

The present invention also provides a pharmaceutical composition, which contains the fusion protein of the present invention and a pharmaceutically acceptable carrier. Preferably, the medicine is used to treat and/or prevent tumors, inflammation, autoimmunity and other diseases caused by TGF-β family related pathways. More preferably, the medicament is used to treat and/or prevent neoplastic diseases as described above.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion). Depending on the route of administration, the active compound, such as a fusion protein of the invention, may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate the compound.

The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbic acid palmitate esters, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

Protection against the presence of microorganisms can be ensured through sterilization procedures or through the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc. In many cases it is preferred to include isotonic agents in the composition, for example sugars, polyols such as mannitol, sorbitol or Sodium oxide. Prolonged absorption of injectable drugs can be brought about by incorporating into the composition an agent which delays absorption, such as monostearate salts and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of these media and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional media or agent is incompatible with the active compound, conventional media or agents may be used in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions generally must be sterile and stable under the conditions of preparation and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersant containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

Sterile injectable solutions may be prepared by mixing the active compound in the required amount in an appropriate solvent and adding, as required, one or a combination of ingredients enumerated above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other required ingredients enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), from which previously sterile-filtered solutions are obtained as a powder of the active ingredient plus any additional required ingredients.

The amount of active ingredient that may be combined with the carrier materials to prepare a single dosage form will vary depending upon the object treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to prepare a single dosage form is generally that amount of the composition that produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% active ingredient, on a 100% basis, and is pharmaceutically acceptable combination of carriers.

Dosage regimens can be adjusted to provide optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as required by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the articles to be treated; each unit contains a predetermined quantity of active compound calculated for combination with the required pharmaceutical carrier. Produce the desired therapeutic effect. The specific specifications for dosage unit forms of this invention are limited by, and directly dependent on (a) the unique properties of the active compounds and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the art of formulating such formulations for use in treating individual sensitivities. Limitation of active compounds.

For administration of a fusion protein or pharmaceutical composition of the invention, the dosage range is about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of recipient body weight. For example, the dosage may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens call for weekly dosing, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or with initial dosing intervals slightly The dosing interval is lengthened in the short term. The method of administration can be intravenous drip.

Alternatively, tumor-targeted fusion proteins can also be administered as sustained-release formulations, in which case less frequent dosing is required. Dosage and frequency vary based on the half-life of the drug molecule in the patient. Dosage and frequency of administration vary depending on whether treatment is prophylactic or therapeutic. In prophylactic use, relatively lower doses are given at less frequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, the patient can be administered on a prophylactic regimen.

Actual dosage levels of the active ingredients in pharmaceutical compositions may vary to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response in a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particular combination employed. other drugs, compounds and/or materials used in combination with the drug, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical field.

"Effective amount" means a dose sufficient to reduce the severity of disease symptoms, increase the frequency and duration of symptom-free periods of disease, or prevent impairment or disability caused by distress from disease. For example, for the treatment of tumors, an "effective amount" of a fusion protein or pharmaceutical composition of the invention preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 20%, relative to an untreated subject. Preferably it is at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in animal model systems that predict efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro by assays well known to those skilled in the art. An effective amount of the fusion protein or pharmaceutical composition of the present invention can reduce tumor size, or otherwise alleviate the symptoms of the object such as preventing and/or treating metastasis or recurrence. One skilled in the art can determine this amount based on factors such as the size of the article, the severity of the symptoms of the article, and the particular composition or route of administration chosen.

The fusion protein or pharmaceutical composition of the present invention can be administered by one or more administration routes using one or more methods well known in the art. It will be understood by those skilled in the art that the route and/or mode of administration will vary depending on the desired results. 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" refers to modes of administration other than enteral and topical administration, usually injection, including but Not limited to intravenous, intramuscular, intraarterial, intrathecal, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, Epidural and intrasternal injections and infusions.

Alternatively, the fusion protein 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 topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are protected by patents or are generally known to those skilled in the art.

The fusion proteins of the invention may also be conjugated to therapeutic moieties such as cytotoxins, radioactive isotopes or bioactive proteins.

Cytotoxicants include any agent that is harmful to cells (eg, cell-killing).

Examples include, but are not limited to: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecac, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thioside, vinblastin Erythromycin, vincain, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, radimycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, Procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs.

Therapeutic agents useful for conjugation also include, for example: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, aminopyridine), alkylating agents (For example, nitrogen mustard, chlorambucil, chlorambucil, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotomycin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anisomycins (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics antibiotics (e.g., actinomycin D (formerly known as actinomycin), bleomycin, mithramycin, and antromycin (AMC)), and antimitotic agents (e.g., vincain and vincain).

Other preferred examples of therapeutic cytotoxins that can be conjugated to the fusion proteins of the present invention include beclomycin, califorcin, maytansene, aristatin, and their derivatives.

Cytotoxins can be conjugated to the fusion proteins of the invention using linker technology used in the art, for example to a targeting moiety (eg, an antibody) in the fusion protein. Examples of linker types that have been used for cytotoxic conjugation include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. One can choose, for example, a linker that is susceptible to low pH cleavage within the lysosomal compartment or to a protease that is preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsins B, C, D) .

The fusion protein of the present invention can also be conjugated with radioactive isotopes to produce cytotoxic radiopharmaceuticals, also known as radioactive conjugates. Examples of radioactive isotopes that can be conjugated to the fusion proteins of the invention include, but are not limited to, iodine-131, indium-111, yttrium-90, and gallium-177. Methods of preparing radioconjugates are well known in the art.

The fusion proteins of the invention can also be conjugated to proteins with desired biological activity. Such biologically active proteins include, for example: enzymatically active toxins or active fragments thereof, such as abrin, ricin A, pseudomonal exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon -γ; or biological response regulators, such as immune regulatory factors, such as lymphokines, interleukins (such as IL-1, IL-2, IL-6, IL-10), chemokines (such as ccl3, ccl26, cxcl7) , granulocyte macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF") or other immune regulatory factors such as IFN, etc.

combination therapy

The fusion proteins or pharmaceutical compositions of the invention can be administered in combination with chemotherapeutic agents or antibodies targeting other tumor antigens. The fusion protein of the present invention and the chemotherapeutic agent or antibody targeting other tumor antigens 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 invention or the pharmaceutical composition of the invention may also be combined with radiotherapy, for example including the administration of ionizing radiation to the patient before, during and/or after the administration of the fusion protein or pharmaceutical composition of the invention. Process.

Test kit

Also included within the scope of the invention are kits that include the mutants, fusion polypeptides, targeting molecules, fusion proteins of the invention, or nucleic acid molecules or vectors encoding the same, or combinations thereof, and instructions for use. Kits generally include labels indicating the intended use and/or method of use of the contents of the kit. The term label includes any written or recorded material on or provided with the kit or otherwise provided with the kit.

beneficial effects

Bifunctional molecules containing wild-type TGFBR2-ECD are prone to fragmentation, which is not conducive to subsequent drug development. However, bifunctional molecules containing truncated TGFBR2-ECD may form a structure that is significantly different from the wild-type protein in the in vivo environment due to the lack of the original N-terminal sequence, and there are some unknown risks.

In view of the easy-to-break characteristics of wild-type TGFBR2-ECD, the inventor adopted a mutation optimization strategy that is completely different from existing technologies (such as WO2018/205985), and retained the original sequence of TGFBR2-ECD as much as possible while solving the risk of druggability. .

Compared with wild-type TGFBR2-ECD, the TGFBR2-ECD mutant of the present invention contains amino acid mutations in the breakable regions (QKS, NNDMI and DNNG) and/or improvements in hydrophilicity, but does not affect the ability to TGF-β1 Affinity activity. Furthermore, the TGFBR2-ECD mutant of the present invention (especially SEQ ID NO: 2-8), compared with wild-type TGFBR2-ECD, has three regions (see Example 1) and/or three regions. The upstream and downstream regions contain hydrophilic amino acids (eg, G, T, S, N, E, and Y) and/or are hydrophilic and may undergo potential O/N glycosylation modification amino acid substitutions. Specifically, the mutant of the present invention contains T/S and N-X-S/T (X can be any amino acid except proline (P)) in the region, with enhanced hydrophilicity and potential glycosylation modification protection as strategies to improve the stability of fusion proteins.

Compared with the fusion protein containing wild-type TGFBR2-ECD, the fusion protein of the present invention contains an improved TGFBR2-ECD mutant, making the fusion protein of the present invention less prone to breakage and degradation during the production process and showing higher stability. . Compared with fusion proteins containing truncated TGFBR2-ECD, the TGFBR2-ECD mutant in the fusion protein of the invention is closer to wild-type TGFBR2-ECD, which may help reduce potential risks in the in vivo environment. Furthermore, the fusion protein of the present invention (such as mutant anti-PD-L1/TGF-β Trap) has higher affinity activity for TGF-β1 and PD-L1. Therefore, the TGFBR2-ECD mutant of the present invention and the fusion protein containing it are useful supplements for the drug development of TGF-β Trap.

Example

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

Unless otherwise stated, the reagents and instruments used in the Examples section are commercially available.

Example 1 Construction and expression of anti-PD-L1/TGF-β Trap

Mass spectrometry revealed that the breakpoint of wild-type anti-PD-L1/TGFBR2-ECD is mainly located at the N-terminus of TGFBR2-ECD (the structure is shown in Figure 1A), and its sequence is "IPPHV QKS V NNDMI VT DNNG AVKFPQL". Verified by mass spectrometry, the degradation sites of wild-type TGFBR2-ECD are mainly located in three regions, namely the first region QKS, the second region N-NDM-I (where "-" indicates the break point), and the third region "DNNG" .

As can be seen from Figure 1A, the antibody terminal structure of the main degradation products generated by fragmentation is intact. 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 drug development of the molecule. The inventors found that when mutations are mainly targeted at the first region (such as Mu1 and Mu3), the main degradation point will shift to the second region; and when only the first and second regions are modified (such as Mu2 and Mu4), the main degradation point will shift to the second region. The sequence "DNNG" in the three regions will become a new degradation site. Therefore, simultaneous targeted and effective mutation optimization of three regions or three regions and their upstream and downstream sequences is an inevitable choice to obtain high-quality fusion proteins.

In this example, anti-PD-L1 antibodies were used as the targeting moiety, and TGFBR2-ECD wild type or mutants were used as TGF-β Trap to construct several bifunctional molecules containing TGF-β Trap and anti-PD-L1 antibodies ( Anti-PD-L1/TGF-β Trap). The anti-PD-L1 antibody used in the examples is the B11 antibody described in WO 2019/233462, the relevant content of which is incorporated herein by reference in its entirety. Anti-PD-L1 antibody B11 includes two heavy chains and two light chains, each of the two heavy chains includes the amino acid sequence of SEQ ID NO: 43, and the two light chains each includes the amino acid sequence of SEQ ID NO: 42. . The anti-PD-L1/TGF-β Trap contains two fusion polypeptides and two light chains, in which the C-terminus of each antibody heavy chain (SEQ ID NO: 43) of antibody B11 is fused with a TGFBR2-ECD (wild type or mutant The N terminus of the antibody) is connected through a linker (G ₄ S) ₄ G to form a fusion polypeptide, and the light chain of the anti-PD-L1/TGF-β Trap is the light chain of antibody B11 (SEQ ID NO: 42).

The fusion protein Mu1-11 containing the TGFBR2-ECD mutant is also known as the mutant anti-PD-L1/TGF-β Trap. The TGFBR2-ECD mutants included in Mu1-4 respectively have the amino acid sequences of SEQ ID NO: 44-47, and the fusion polypeptides respectively have the amino acid sequences of SEQ ID NO: 48-51. The TGFBR2-ECD mutants included in Mu5-11 have the amino acid sequences of SEQ ID NO: 2-8 respectively, and the fusion polypeptide of the TGFBR2-ECD mutant and the antibody B11 heavy chain has the amino acid sequences of SEQ ID NO: 9-15 respectively. acid sequence. Compared to Mu5-11, Mu1-4 only contains amino acid mutations in the first and/or second of the three regions (see Table 1).

A wild-type fusion protein (WT) containing TGFBR2-ECD wild type (SEQ ID NO: 1) was also constructed as a reference, also known as wild-type anti-PD-L1/TGF-β Trap (see Table 1).

It can be seen from the results in Table 2 that simultaneous mutation and transformation of the three regions or the three regions and their upstream and downstream regions is the key to improving the stability of the fusion protein. Mu5-9 are mutations targeting only three regions: Mu5 contains only one amino acid "S" that is hydrophilic and can undergo potential O-glycosylation modification in each region; Mu6 contains only one amino acid "S" in each region Two hydrophilic amino acids "S/T" that can undergo potential O-glycosylation modification; Mu7, based on Mu5, also contains a hydrophilic and potential N-glycosylation modification in the first region Amino acid "N", and forms a potential N-glycosylation site "N-A-S"; Mu8, based on Mu7, also contains a hydrophilic amine group in the second region that can undergo potential N-glycosylation modification Acid "N", and forms a potential N-glycosylation site "N-A-S"; Mu9, based on Mu8, also contains a hydrophilic amino acid in the third region that can undergo potential N-glycosylation modification " N", and forms a potential N-glycosylation site "N-A-S". Mu10 and Mu11 introduce a wide range of hydrophilic amino acids in the three regions and their upstream and downstream regions, especially amino acids "S/T" that can undergo potential O-sugar modification.

linker為接頭(G₄S)₄G，每個重鏈的C末端通過其與一個突變體的N末端連接形成融合多肽；Mu1-11中的TGFBR2-ECD突變體中包含的胺基酸突變以加粗字體示出。

The linker is (G ₄ S) ₄ G, and the C-terminus of each heavy chain is connected to the N-terminus of a mutant to form a fusion polypeptide; the amino acid mutations contained in the TGFBR2-ECD mutant in Mu1-11 are as follows: Shown in bold font.

A fusion protein Fc-TGFBR2-ECD (SEQ ID NO: 52) was also constructed, in which the N-terminus of the TGFBR2-ECD mutant (SEQ ID NO: 2) and the C-terminus of the Fc fragment of human IgG1 (SEQ ID NO: 53) Connection via connector (G ₄ S) ₄ G.

Plasmids expressing wild-type and mutant anti-PD-L1/TGF-β Trap or fusion protein Fc-TGFBR2-ECD were transiently transfected into Expi-CHO cells (Thermo Fisher). After about 14 days of serum-free cell culture, the culture supernatant was harvested. The antibody expression level in the culture supernatant is detected, and the culture supernatant is purified with Protein A medium to obtain the target fusion protein and perform subsequent detection.

Example 2 Stability detection of anti-PD-L1/TGF-β Trap

After the wild-type and mutant anti-PD-L1/TGF-β Trap were purified in one step through Protein A medium as described in Example 1, the degradation product content was detected through reducing protein electrophoresis, SEC-HPLC and CE-SDS.

Reduced protein electrophoresis: Take a sample and add an appropriate amount of Loading buffer, and add DTT to one sample for reduction. After the reduced sample is heated in a water bath at 70°C for 10 minutes, 2 μg is loaded and separated by 10% protein gel electrophoresis. The experimental results are shown in Figure 1B. Lane 1 is anti-PD-L1 antibody, lane 2 is wild-type anti-PD-L1/TGF-β Trap, and lane 3 is Mu5. It can be seen from the results that wild-type anti-PD-L1/TGF-β Trap has significant fusion protein-HC degradation products (TGFBR2-ECD deletion) near 53kDa, and the degradation of Mu5 molecules is effectively controlled.

SEC-HPLC: Use 100mM PB 200mM Arg (pH 6.8) as the mobile phase, 1mL/min flow rate, use Waters BEH SEC 200A 7.8x300mm 3.5μm chromatographic column for analysis, and inject 50μg of each sample.

CE-SDS: Take two samples and add an appropriate amount of sample buffer. Add β-ME (β-mercaptoethanol) to one of them for reduction. The non-reduced sample is bathed in 70°C water for 5 minutes, and the reduced sample is bathed in 70°C water for 10 minutes. Maurice was used for CE-SDS analysis, non-reduced samples (nrCE-SDS) were separated for 40 minutes, and reduced samples (rCE-SDS) were separated for 35 minutes.

The stability analysis of wild-type anti-PD-L1/TGF-β Trap (WT) and Mu5 by SEC-HPLC is shown in Figure 2: the proportion of monomers in WT is 85.2%, and the proportion of degradation products is 14%, while The proportion of monomers in Mu5 is 97%.

The electrophoresis pattern of wild-type anti-PD-L1/TGF-β Trap (WT) and Mu5 stability analyzed by nrCE-SDS is shown in Figure 3: the proportion of intact protein in WT is 86.4%, and the proportion of protein fragments is as high as 13.6%, while The proportion of complete protein in Mu5 is as high as 94.7%, and the proportion of protein fragments is as low as 5.3%.

The electrophoresis patterns of wild-type anti-PD-L1/TGF-β Trap (WT) and Mu5 stability analyzed by rCE-SDS are shown in Figure 4: the proportion of protein fragments in WT is as high as 8.3%, while the proportion of protein fragments in Mu5 is as low as 1.8%.

The SEC-HPLC and CE-SDS data of wild type (WT) and mutant anti-PD-L1/TGF-β Trap (Mu1-11) are summarized in Table 2.

The SEC-HPLC results in Table 2 show that the proportion of monomer (Mono) in the wild-type anti-PD-L1/TGF-β Trap is 85.2%, and the proportion of degraded products (LMW) is as high as 14%. The mutant anti-PD-β Trap of the present invention The proportion of monomers in L1/TGF-β Trap (Mu5-11) can reach more than 95%, and the proportion of degraded products can be as low as less than 3.5%, indicating that the mutant anti-PD-L1/TGF-β Trap of the present invention is more stable. high.

The nrCE-SDS results in Table 2 show that the proportion of intact protein (Intact) in wild-type anti-PD-L1/TGF-β Trap is 86.4%, and the proportion of protein fragments (Fragment) is as high as 13.6%. The mutant anti-PD-β Trap of the present invention The proportion of intact protein in L1/TGF-β Trap (Mu5-11) is as high as 94.7% or higher, and the proportion of protein fragments is as low as about 5%.

The rCE-SDS results in Table 2 show that wild-type anti-PD-L1/TGF-β Trap protein The fragment ratio is as high as 8.3%, and the protein fragment ratio of the mutant anti-PD-L1/TGF-β Trap (Mu5-11) of the present invention is as low as less than 2%. These results indicate that the mutant anti-PD-L1/TGF-β Trap of the present invention is more stable.

It can be seen from this table that for the easily broken regions of the wild-type anti-PD-L1/TGF-β Trap (the first region QKS (amino acids at positions 6-8), the second region NNDMI (amino acids at positions 10-14) ) and random mutations in the third region DNNG (amino acids 17-20)) may not necessarily have a significant quality improvement effect. Comparing Mu5-11 with wild-type anti-PD-L1/TGF-β Trap, it can be seen that replacing the amino acids in these three regions with hydrophilic amino acids and/or N-X-S/T motifs can improve the anti-PD-L1/TGF-β Trap. Stability of TGF-β Trap. Mu5, which carries out effective mutations in three regions at the same time, and Mu6-8, which continues to introduce the N-X-S/T motif that is hydrophilic and can undergo N-glycosylation modification, have better quality improvements. In addition, Mu10/11 introduces a wide range of hydrophilic amino acids into the easily broken region and the amino acid residues near it, especially the extensive introduction of amino acid T/S that can undergo potential O-glycosylation modification. Promotes the stability of anti-PD-L1/TGF-β Trap.

In addition, compared with Mu5-11, which contains amino acid mutations in all three regions, Mu1-4 only contains amino acid mutations in one or two of the three regions. The mutation does not significantly improve the stability of anti-PD-L1/TGF-β Trap and may even aggravate protein degradation. It can be seen that to improve the stability of anti-PD-L1/TGF-β Trap, the region of amino acid mutation also has certain selectivity.

Table 2 Quality detection of wild-type and mutant anti-PD-L1/TGF-β Trap

Example 3 Analysis of specific recognition activity of anti-PD-L1/TGF-β Trap

In this example, ELISA was used to determine the binding of anti-PD-L1/TGF-β Trap to the target proteins human PD-L1 (hPD-L1) and human TGF-β1 (hTGF-β1).

Human PD-L1 (R&D Company) or TGF-β1 (Acro Company) was diluted to 1 μg/ml with PBS and coated on a 96-well plate. After incubating overnight at 4°C, discard the coating solution and wash with PBST the next day. Then add 200 μl BSA blocking solution to each well and incubate at room temperature for 1 h. Wash three times with PBST, dilute wild-type anti-PD-L1/TGF-β Trap, anti-PD-L1/TGF-β Trap and PD-L1 antibody of the present invention with 1% BSA PBST to a maximum concentration of 14 nM, and proceed for 4 Doubling the gradient dilution, make 8 concentration gradients, add 100 μl per well as the primary antibody to a 96-well plate, and incubate at room temperature for 2 hours. Wash three times with PBST, then add anti-human IgG-Fab-HRP (secondary antibody, Abcam) and incubate at room temperature for 1 h. Finally, each well was washed four times with PBST, TMB chromogenic solution (Beijing Tiangen Biochemical Technology Co., Ltd.) was added, and the color was developed at room temperature. After stopping the color development, measure the OD value with a microplate reader at 450 nm. The experimental results are shown in Figure 5. The EC50 of wild-type (WT) and mutant anti-PD-L1/TGF-β Trap (Mu5-11) binding to PD-L1 is between 0.1-0.2nM (Figure 5A), and The EC50 of TGF-β1 binding is between 0.1-0.2nM (Figure 5B), indicating that the in vitro binding abilities of WT and Mu5-11 to the two target proteins PD-L1 and TGF-β1 are similar.

Example 4 Affinity analysis of anti-PD-L1/TGF-β Trap

In this example, the Octet RED96 detection system was used to determine the affinity of the fusion protein to the target protein TGF-β1.

Coupling ligand: This method uses FAB2G (Anti-Human Fab-CH1 2nd Generation) wafer, soak the wafer in SD buffer for 10-15 minutes in advance, and load the ligand (wild type or mutant anti-PD-L1) at a concentration of 50 μg/ml. /TGF-β Trap), the preset time is 1500s, the preset value of ligand Loading is 2.5nm, infiltrate the wafer with running buffer (SD buffer) until the baseline is stable, and remove free ligands.

Kinetic experiment: TGF-β1 was diluted to 44.4nM, 22.2nM, 11.1nM, 5.55nM, 2.775nM and 1.39nM respectively with SD buffer. An affinity detection experiment was performed using TGF-β1 as the analyte and wild-type and mutant anti-PD-L1/TGF-β Trap as the ligand. The binding time (Association) of ligand and analyte is 240s, and the dissociation time (Dissociation) of analyte is 300s. SD buffer is used as a blank control for background subtraction.

FortéBio Data Analysis 9.0 was used to conduct analysis using a static fitting model. The results are shown in Figure 6. The K _D between wild-type anti-PD-L1/TGF-β Trap (WT) and TGF-β1 is 7.5×10 ^-9 M , the K _D between Mu5 and TGF-β1 is 3.6×10 ^-9 M, indicating that the binding affinities of WT and Mu5 to TGF-β1 are similar.

Example 5 Regulation of expression of EMT pathway genes by anti-PD-L1/TGF-β Trap

A549 human non-small cell lung cancer cells (Cell Bank of the Chinese Academy of Sciences, SCSP-503) were inoculated in a 6-well plate and cultured for 24-36 hours to reach 60-70% confluence. F-containing 2ng/ml TGF-β1 was added to the control group. 12K complete medium, the experimental group added 5 μg/ml wild-type anti-PD-L1/TGF-β Trap or Mu5 to the complete medium containing TGF-β1.

After co-culture for 48 hours, the cells were recovered, total cellular RNA was extracted, and reverse transcription reaction was performed to obtain cDNA. Prepare a real-time fluorescent PCR reaction system to detect whether anti-PD-L1/TGF-β Trap Key genes involved in inhibiting the downstream EMT (epithelial-mesenchymal transition) pathway induced by TGF-β. The key genes of related pathways mainly include: CDH2, MMP-2, MMP-3, MMP-9, TWIST2 and Vimentin, etc. The results are shown in Figure 7. Both the wild-type anti-PD-L1/TGF-β Trap (WT) and the anti-PD-L1/TGF-β Trap (Mu5) of the present invention can effectively inhibit the expression of EMT pathway genes induced by TGF-β1. .

Example 6 Validation of the efficacy of Mu5 on subcutaneous xenografts of colon cancer cells in MC38/hPD-L1 mice

In this example, Mu5 is used as the representative drug of the anti-PD-L1/TGF-β Trap fusion protein of the present invention to conduct drug efficacy verification on transplanted tumors. 1×10 ⁶ colon cancer MC38/hPD-L1 (human PD-L1 stably transduced MC38 cell line; constructed by the Animal Experiment Center) cells were injected into the armpits of mice. When the tumor grew to an average volume of about 50 mm ³ , the mice were randomly divided into 3 groups according to tumor volume: solvent control group (PBS), Mu5 1.22 mg/kg group, and Mu5 3.66 mg/kg group. There were 8 animals in each group. Each group was intraperitoneally injected with the corresponding concentration of the test substance at a dosage volume of 10 ml/kg. The dosage frequency was twice a week, and the tumor volume was weighed and measured twice a week. The first dose is day 1 (D1). The mice were weighed and tumor volume (TV) measured on days 1, 4, 7, 11, 15 and 18 (D18). On the 18th day, the mice were sacrificed, and the tumors were dissected and the tumor weight (TW) was measured. The relative tumor volume (RTV), relative tumor proliferation rate (T/C), and tumor weight inhibition rate (IR) were calculated based on the tumor volume and tumor weight. The results are shown in Table 3, Table 4 and Figure 8.

As shown in Table 3 and Figure 8, on day 18, compared with the RTV value of the solvent control group (PBS) of 41.96±2.89, the RTV values of the Mu5 1.22mg/kg group and the Mu5 3.66mg/kg group were 17.45± respectively. 2.81 and 12.27±3.42.

As shown in Table 4, on day 18, compared with the tumor weight of the solvent control group (PBS) of 2.4269±0.3936g, the tumor weights of the Mu5 1.22mg/kg group and the Mu5 3.66mg/kg group were 1.1161±0.0856g respectively. and 0.6751±0.2231g. The tumor weight inhibition rates (IR) of the Mu5 1.22mg/kg group and Mu5 3.66mg/kg group were 54.01% and 72.18% respectively.

As shown in Table 3, Table 4 and Figure 8, compared with the solvent control group, tumor growth was significantly inhibited in the Mu5 treatment group and showed a good dose-dependent effect.

<110> Hisun Biopharmaceutical Co., Ltd.

<120> TGFBR2-ECD mutants and fusion proteins containing them and their applications

<130> NI2021TC942TW01

<150> CN202011011927.7

<151> 2020-09-23

<160> 53

<170> PatentIn version 3.5

<210> 1

<211> 136

<212> PRT

<213> Artificial Sequence

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<223> Wild-type TGF- β R Ⅱ extracellular domain

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<213> Artificial Sequence

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<223> TGF- β R Ⅱ extracellular domain of Mu5

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<213> Artificial Sequence

<220>

<223> TGF- β RⅡ extracellular domain of Mu6

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<213> Artificial Sequence

<220>

<223> TGF- β R Ⅱ extracellular domain of Mu7

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<213> Artificial Sequence

<220>

<223> TGF- β RⅡ extracellular domain of Mu8

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<213> Artificial Sequence

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<223> TGF- β R Ⅱ extracellular domain of Mu9

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<213> Artificial Sequence

<220>

<223> TGF- β R Ⅱ extracellular domain of Mu10

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<211> 136

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<213> Artificial Sequence

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<223> TGF- β R Ⅱ extracellular domain of Mu11

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<211> 604

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<213> Artificial Sequence

<220>

<223> Fusion polypeptide of Mu5

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<211> 604

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<213> Artificial Sequence

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<223> Fusion polypeptide of Mu6

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<213> Artificial Sequence

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<223> Fusion polypeptide of Mu7

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<213> Artificial Sequence

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<223> Fusion polypeptide of Mu8

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<213> Artificial Sequence

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<223> Fusion polypeptide of Mu9

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<213> Artificial Sequence

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<223> Fusion polypeptide of Mu10

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<211> 604

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<213> Artificial Sequence

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<223> Fusion polypeptide of Mu11

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<213> Artificial Sequence

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<223> B10#HCDR1

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<213> Artificial Sequence

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<223> B10#HCDR2

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<213> Artificial Sequence

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<223> B10#HCDR3

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<213> Artificial Sequence

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<223> B10#VH

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<213> Artificial Sequence

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<223> B10#LCDR1

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<213> Artificial Sequence

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<223> B10#LCDR2

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<213> Artificial Sequence

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<223> B10#LCDR3

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<211> 107

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<213> Artificial Sequence

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<223> B10#VL

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<212> PRT

<213> Artificial Sequence

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<223> B11#HCDR1

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<213> Artificial Sequence

<220>

<223> B11#HCDR2

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<213> Artificial Sequence

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<223> B11#HCDR3

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<213> Artificial Sequence

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<223> B11#VH

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<213> Artificial Sequence

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<223> B11#LCDR1

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<211> 7

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<213> Artificial Sequence

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<223> B11#LCDR2

<400> 29

<210> 30

<211> 9

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<213> Artificial Sequence

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<223> B11#LCDR3

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<210> 31

<211> 107

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<213> Artificial Sequence

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<223> B11#VL

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<213> Artificial Sequence

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<223> H12#HCDR1

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<211> 17

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<213> Artificial Sequence

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<223> H12#HCDR2

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<213> Artificial Sequence

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<223> H12#HCDR3

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<213> Artificial Sequence

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<223> H12#VH

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<213> Artificial Sequence

<220>

<223> H12#LCDR1

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<213> Artificial Sequence

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<223> H12#LCDR2

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<211> 9

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<213> Artificial Sequence

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<223> H12#LCDR3

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<213> Artificial Sequence

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<223> H12#VL

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<213> Artificial Sequence

<220>

<223> Light chain constant region

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<213> Artificial Sequence

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<223> Heavy chain constant region

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<213> Artificial Sequence

<220>

<223> B11# light chain

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<213> Artificial Sequence

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<223> B11# heavy chain

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<213> Artificial Sequence

<220>

<223> TGF- β R Ⅱ extracellular domain of Mu1

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<213> Artificial Sequence

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<223> TGF- β RⅡ extracellular domain of Mu2

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<213> Artificial Sequence

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<223> TGF- β R Ⅱ extracellular domain of Mu3

<400> 46

<210> 47

<211> 136

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<213> Artificial Sequence

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<223> TGF- β RⅡ extracellular domain of Mu4

<400> 47

<210> 48

<211> 604

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion polypeptide of Mu1

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<211> 604

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<213> Artificial Sequence

<220>

<223> Fusion polypeptide of Mu2

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<211> 604

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<213> Artificial Sequence

<220>

<223> Fusion polypeptide of Mu3

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<211> 604

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<213> Artificial Sequence

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<223> Fusion polypeptide of Mu4

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<211> 383

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<213> Artificial Sequence

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<223> Fc-TGFBR2-ECD

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<211> 226

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<223> Human IgG1 Fc fragment

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Claims (31)

A mutant of the extracellular region of TGF-βRII, which has an amino acid sequence selected from SEQ ID NO: 2-8. A targeting molecule comprising the mutant of claim 1 and a targeting moiety, wherein the targeting moiety is covalently connected to the N-terminus of the mutant. The targeting molecule of claim 2, wherein the molecules targeted by the targeting moiety are selected from the group consisting of: growth factors and their receptors, cytokines and their receptors, cell surface antigens, immune checkpoint molecules and hormones. The targeting molecule of claim 2, wherein the molecule targeted by the targeting moiety is selected from the group consisting of CD13, CD19, CD22, CD25, CD27, CD30/TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117/KIT, CD123, CD138, CD142, CD174, CD227/MUC1, CD352, CLDN18.2, DLL3, CN33, GPNMB, ENPP3, Nectin-4, HER2/ErbB2, EGFR, FGFR, VEGFR, HGFR, PDGFR, VEGF, HGF, FGF, FGF-2, PDGF, Galectin-3, EGFSLC44A4/AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, SLITRK6, SLAMF7/CS1, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, CA6, Integrin αV, TDGF1, Ephrin A4, PTK7, NOTCH3, C4.4A, FLT3, PD1, CTLA4, LAG-3, BTLA, TIM-2 , LAIR1, PD-L1, B7-DC, B7-H3, B7-H4, HVEM and TIM-4. The targeting molecule of claim 2, wherein the targeting portion is an Fc fragment, an antibody or an antigen-binding fragment thereof, a mimetic antibody, a cell surface antigen, a cytokine, a growth factor, a hormone, a T cell receptor, a kinase or ion channels. The targeting molecule of claim 5, wherein the cytokine is selected from: G-CSF, GM-CSF, eotaxin/CCL11, MCP-1/CCL2, MIP-1α/CCL4, RANTES/CCL5, 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, IL -23. INFα, INFβ, INFγ, TNFα and TNFβ; the growth factors are selected from: VEGF, HGF, FGF, FGF-2, PDGF, IGF, TGF, NGF, EPO and EGF; the hormones are selected from: peptides Hormones and steroid hormones. The targeting molecule of claim 2, wherein the targeting moiety is selected from the group consisting of synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, The group consisting of hybrid antibodies, intrabodies, single domain antibodies, Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, dsFv fragments, Fd fragments, Fd' fragments, scFv, scFab and diabodies . The targeting molecule according to claim 2, wherein the targeting moiety is an anti-PD-L1 antibody or an antigen-binding fragment thereof. A fusion polypeptide comprising the mutant of claim 1 and a first polypeptide, and the C-terminus of the first polypeptide is covalently connected to the N-terminus of the mutant. The fusion polypeptide of claim 9, wherein the first polypeptide comprises at least one heavy chain variable region of an antibody or at least one light chain variable region of an antibody. The fusion polypeptide of claim 10, wherein the antibody is an anti-PD-L1 antibody. A fusion protein comprising the fusion polypeptide of any one of claims 9-11 and a second polypeptide, wherein the second polypeptide forms a targeting portion when combined with the first polypeptide, and the targeting portion Partially as defined in any of claims 2-8. The fusion protein of claim 12, wherein The first polypeptide comprises at least one heavy chain variable region of an antibody, the second polypeptide comprises at least one light chain variable region of an antibody; or the first polypeptide comprises at least one light chain variable region of an antibody. region, the second polypeptide comprising at least one heavy chain variable region of the antibody. The fusion protein of claim 13, wherein the antibody is an anti-PD-L1 antibody. The fusion protein of claim 14, which includes: (1-1) a fusion polypeptide, from the N terminus to the C terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region, the heavy chain The variable region includes HCDR1, which includes the amino acid sequence of SEQ ID NO: 16, HCDR2, which includes the amino acid sequence of SEQ ID NO: 17, HCDR3, which includes the amino acid sequence of SEQ ID NO: 18; and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (1-2) a second polypeptide comprising a light chain variable region, the light chain variable region comprising LCDR1, which includes the amino acid sequence of SEQ ID NO: 20, LCDR2, which includes the amino acid sequence of SEQ ID NO: 21, and LCDR3, which includes the amino acid sequence of SEQ ID NO: 22; or (2- 1) A fusion polypeptide, from the N terminus to the C terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 25, HCDR3 comprising the amino acid sequence of SEQ ID NO: 26; and (ii) a mutant comprising an amine selected from SEQ ID NO: 2-8 and (2-2) a second polypeptide comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, LCDR2 comprising The amino acid sequence of SEQ ID NO: 29, and LCDR3, which includes the amino acid sequence of SEQ ID NO: 30; or (3-1) a fusion polypeptide, from the N terminus to the C terminus, comprising: (i) the first A polypeptide comprising a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, HCDR3 , which comprises the amino acid sequence of SEQ ID NO: 34; and (ii) a mutant, which comprises the amino acid sequence selected from SEQ ID NO: 2-8; and (3-2) a second polypeptide, which Comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 36, LCDR2 comprising the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprising Amino acid sequence of SEQ ID NO: 38. The fusion protein of claim 14, which includes: (1-1) a fusion polypeptide, from the N terminus to the C terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region, the heavy chain The variable region comprises the amino acid sequence of SEQ ID NO: 19, and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (1-2) a second polypeptide comprising a light chain variable region, the light chain variable region comprising The amino acid sequence of SEQ ID NO: 23; or (2-1) a fusion polypeptide, from the N terminus to the C terminus, comprising: (i) a first polypeptide comprising a heavy chain variable region, the heavy chain can The variable region comprises the amino acid sequence of SEQ ID NO: 27, and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2-2) a second polypeptide, which Comprising a light chain variable region, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 31; or (3-1) a fusion polypeptide, from the N terminus to the C terminus, comprising: (i) a first polypeptide A peptide comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35, and (ii) a mutant comprising an amine group selected from the group consisting of SEQ ID NO: 2-8 and (3-2) a second polypeptide comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39. The fusion protein of claim 14, comprising: (1) a fusion polypeptide, from the N terminus to the C terminus, comprising: (i) a first polypeptide comprising a heavy chain, the heavy chain comprising SEQ ID NO: The amino acid sequence of 43, and (ii) a mutant comprising an amino acid sequence selected from SEQ ID NO: 2-8; and (2) A second polypeptide comprising a light chain comprising the amino acid sequence of SEQ ID NO: 42. The fusion protein of claim 15, further comprising a linker between the first polypeptide and the mutant. The fusion protein of claim 15, wherein the linker is (G ₄ S) _n G, where n is an integer from 2 to 6. The fusion protein of claim 15, which forms a dimer. A pharmaceutical composition comprising the targeting molecule of any one of claims 2-8, the fusion polypeptide of any one of claims 9-11, or the fusion protein of any one of claims 12-20, and a pharmaceutically acceptable Accepted carriers. A targeting molecule according to any one of claims 2-8, a fusion polypeptide according to any one of claims 9-11, a fusion protein according to any one of claims 12-20 or a pharmaceutical composition according to claim 21 is being prepared Use in medicines for the treatment and/or prevention of neoplastic diseases. The use as claimed in claim 22, wherein the neoplastic disease is selected from the group consisting of lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer Carcinoma, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal area cancer , testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, Adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem nerve Glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, asbestos-induced cancer, and combinations of said cancers. The use as described in claim 22, wherein the neoplastic disease is metastatic cancer expressing PD-L1. Use of the fusion protein of any one of claims 14-20 in the preparation of drugs for treating cancer. The use as described in claim 25, wherein the cancer is a PD-L1 positive cancer. The use as claimed in claim 25, wherein the cancer is selected from the group consisting of lung cancer, ovarian cancer, colorectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, and glioma , gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine corpus cancer, osteosarcoma, colorectal cancer and non-small cell lung cancer. A nucleic acid molecule encoding the mutant of claim 1, the targeting molecule of any one of claims 2-8, the fusion polypeptide of any one of claims 9-11, or the fusion polypeptide of claim 12-20 Any fusion protein. An expression vector comprising the nucleic acid molecule of claim 28. A host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29. A method for producing a mutant of claim 1, a targeting molecule of any one of claims 2-8, a fusion polypeptide of any one of claims 9-11, or a fusion protein of any one of claims 12-20 method, the method includes: (i) culturing the host cell of claim 30 under conditions suitable for expression of nucleic acid molecules or expression vectors; and (ii) isolating and purifying the mutant, targeting molecule, fusion polypeptide or fusion protein.