Cytokines Activated in the Tumor Microenvironment and Their Uses

Abstract

The present disclosure provides an IFN-α cytokine activated in the tumor microenvironment and its uses. After mutating INF-α with cysteine, combining it with a cleavage substrate and a mask (R) distinguishable by a substance highly expressed in tumors can form a biomolecular complex. The biomolecular complex has its INF-α activity inhibited in normal tissues, causing no toxicity to normal tissues, while having its INF-α activity activated in the tumor environment, resulting in effective tumor suppression.

Description

Technical Field

[0001] This application was filed on July 18, 2022, claims the priority of the patent application with the invention title "Cytokine Activated in Tumor Microenvironment and Its Use" and application number CN 202210844222.6, and incorporates all its contents into the text.

[0002] This disclosure belongs to the field of biochemistry; more specifically, this disclosure relates to cytokines activated in the tumor microenvironment and their uses.

Background Art

[0003] Cytokines are molecular messengers that enable cells of the immune system to communicate with each other and mount a coordinated, robust, and self-limiting response against a target antigen. Many forms of communication in the immune system occur through direct cell-cell interactions, but cytokine secretion rapidly transmits immune signals in a multi-faceted and effective manner.

[0004] In recent years, many cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21, have entered clinical trials for patients with advanced cancer. Based on the potent antitumor activity of some pro-inflammatory cytokines discovered in animal models, there is an urgent need to improve the therapeutic effect, and clinical studies on treating various malignancies using recombinant interferon-α and interleukin-2 have been approved.

[0005] Interferons are classified by their ability to bind to specific receptors called type I, type II, and the recently described type III IFN receptors. When providing adjuvant IFNα to melanoma patients, large amounts of IFNα are the most commonly used option, but there is a great need for therapies with better therapeutic indices, and currently, various other options in this context are being studied. The experience with the use of IFN has already identified and formed established guidelines for managing toxicity and side effects. The toxic characteristics of IFNα are usually dose-related, and many side effects can be controlled without discontinuing treatment. Systemic symptoms such as fever, fatigue, headache, gastrointestinal symptoms, and myalgia are common and can occur in more than 80% of patients. IFNα can increase liver enzymes in some patients, especially during high-dose intravenous administration, and at this time, patients must always be monitored, and treatment must be maintained or the dose reduced for patients with elevated liver enzymes during treatment. Therefore, reducing the dose of IFNα and enhancing its administration effect are urgent problems to be solved in this field.

[0006] [Summary of the Invention] The object of the present disclosure is to provide an IFNα cytokine activated in the tumor microenvironment and its uses.

[0007] In a first aspect of the present disclosure, there is provided an IFNα cytokine biomolecular complex having the following structure: R1-AAN-PABC-R3-S-Cys-IFNα wherein Cys represents a cysteine residue with a single point mutation on the surface of IFNα; S represents a sulfur atom in the cysteine residue; IFNα has the amino acid sequence shown in SEQ ID NO: 1; R1 is a group that inhibits the binding of an IFNα variant to its ligand or receptor; AAN represents a tripeptide, A is alanine, N is asparagine, and PABC is p-aminobenzylcarbamoyl; R3 is a spacer arm formed by covalently coupling to the sulfur atom of a cysteine resulting from a single-point mutation of IFNα, having an EMC group. Its role is that even after AAN is cleaved, the R3 structure remains bound to the cytokine, and the affinity with the IFNα receptor may be enhanced whether R3-S-Cys-IFNα partially recovers or completely recovers.

[0008] In one or more embodiments, R1 of the biomolecular complex is polyethylene glycol having a molecular weight of 44 to 132,000, such as polyethylene glycol of 1,000 to 50,000, 3,000 to 80,000, or 10,000 to 60,000, but is not limited thereto.

[0009] In one or more embodiments, the IFNα variant is a variant obtained by single-point mutation of INF-α, and its amino acid sequence is based on SEQ ID NO: 1, and the residues of Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162 are mutated to Cys.

[0010] In one or more embodiments, R1 of the biomolecular complex is selected from the following: TIFF2025523889000001.tif193170TIFF2025523889000002.tif176170

[0011] In one or more embodiments, R3 of the biomolecular complex is selected from the following, but is not limited thereto: TIFF2025523889000003.tif144170

[0012] In one or more embodiments, the structure of R1-AAN-PABC-R3 of the biomolecular complex is selected from the following: TIFF2025523889000004.tif212170

[0013] In another aspect of the present disclosure, there is provided a use of the biomolecular complex, which is the preparation of a composition or kit for suppressing tumors.

[0014] In another aspect of the present disclosure, there is provided a use of the biomolecular complex, which is the preparation of a composition or kit for suppressing tumors in combination with an anti-tumor antibody.

[0015] In another aspect of the present disclosure, there is provided a composition or kit for suppressing tumors, which contains the above-mentioned biomolecular complex; preferably, the composition further contains a pharmaceutically acceptable carrier.

[0016] In one or more embodiments, the composition further contains an anti-tumor antibody.

[0017] In one or more embodiments, the kit further includes an anti-tumor antibody or a composition containing an anti-tumor antibody.

[0018] In one or more embodiments, the anti-tumor antibody is an anti-PD-1 antibody.

[0019] In one or more embodiments, by mass ratio, the biomolecular complex and the anti-PD-1 antibody are 1:(0.05 - 50) (for example, 1:0.1, 1:0.15, 1:0.2, 1:0.4, 1:0.5, 1:0.6, 1:0.8, 1:1, 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:20, 1:30, 1:40); preferably 1:(0.2 - 8); more preferably 1:(0.4 - 5).

[0020] In one or more embodiments, the dosage form of the composition includes, but is not limited to, injections, infusions, powders, tablets, capsules; preferably an injection.

[0021] In one or more embodiments, the kit includes a container, and the INF-α variant and / or the anti-tumor antibody or a composition containing them are placed in the container.

[0022] In one or more embodiments, the container includes, but is not limited to, a syringe.

[0023] In one or more embodiments, the kit further includes an instruction manual that describes a method for suppressing tumors.

[0024] In another aspect of the present disclosure, a method for preparing INF-α activated in a tumor microenvironment is provided, which includes single-point mutating INF-α and mutating specific residues of its amino acid sequence to Cys.

[0025] In another aspect of the present disclosure, an INF-α variant, which is a mutant obtained by single-point mutating INF-α, is provided, and its amino acid sequence is based on SEQ ID NO: 1 and mutates the Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162 residues to Cys.

[0026] In another aspect of the present disclosure, a separated polynucleotide is provided, and the above polynucleotide encodes the above INF-α variant.

[0027] In another aspect of the present disclosure, an expression vector containing the above polynucleotide is provided.

[0028] In another aspect of the present disclosure, a genetically engineered host cell is provided, and the host cell contains the above vector or the above polynucleotide is integrated into the genome of the host cell.

[0029] In another aspect of the present disclosure, a method for producing the above INF-α variant is provided, which includes the following steps: (1) culturing the above host cell to obtain a culture; and (2) separating the above INF-α variant from the culture.

[0030] In another aspect of the present disclosure, there is provided the aforementioned use of the INF-α variant, which is to couple with R1-AAN-PABC-R3 to form a biomolecular complex that suppresses tumors; provided that R1 is a group that inhibits the binding of the IFNα variant to its ligand or receptor; AAN represents a tripeptide, A is alanine, N is asparagine, PABC is p-aminobenzylcarbamoyl; R3 is a spacer arm having an EMC group and covalently coupled to the sulfur atom of a cysteine formed by a single point mutation of IFNα, and its role is that even after AAN is cleaved, the R3 structure remains bound to the cytokine, and R3-S-Cys-IFNα may partially recover, completely recover, or even enhance the affinity with the IFNα receptor.

[0031] In one or more embodiments, the aforementioned suppression of tumors includes preventing, alleviating and / or treating tumors.

[0032] In one or more embodiments, the aforementioned anti-tumor antibody is a monoclonal antibody or a polyclonal antibody.

[0033] In one or more embodiments, the aforementioned anti-PD-1 antibody is an optional antibody that specifically neutralizes / binds to PD-1.

[0034] In one or more embodiments, the aforementioned tumors include melanoma, NSCLC, head and neck squamous cell carcinoma, urothelial carcinoma, classical Hodgkin lymphoma, gastric cancer, esophagogastric junction cancer, cervical cancer, B-cell lymphoma, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, primary liver cancer, small cell lung cancer.

[0035] Based on other contents of the present disclosure, for those skilled in the art, other aspects of the present invention are self-evident.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2

Figure 3

[0037] Specific Embodiments As a result of intensive research, the inventor has disclosed an optimized and modified INF-α. When it is combined with a cleavage substrate and a mask (R) that can be identified by a substance highly expressed in tumors, a biomolecular complex can be formed. This biomolecular complex inhibits INF-α activity in normal tissues without causing toxicity to normal tissues, and activates INF-α activity in the tumor environment, resulting in effective tumor suppression. The complex of the present disclosure highly concentrates the inhibitory activity of INF-α in the tumor environment, and can achieve a very ideal tumor suppression effect.

[0038] Terms Unless otherwise indicated, as used in this specification, "IFNα mutant" and "mutant IFNα" are used interchangeably and refer to a product formed by mutating wild-type IFNα; preferably, it refers to a protein formed after mutating the amino acid residues at the following positions with respect to wild-type IFNα (such as SEQ ID NO: 1): Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162; preferably, the residues of Arg149, Ala145, His57, Gln61, Phe64 or Ser68 are mutated to Cys.

[0039] As used in this specification, when representing wild-type IFNα, it is called the protein with the amino acid sequence shown in WT, SEQ ID NO: 1.

[0040] As used herein, "Recombinant" refers to proteins, genetic engineering vectors, cells, etc. obtained (or mass-produced) by genetic engineering means.

[0041] As used herein, "antibody" includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, provided that they exhibit the desired biological activity.

[0042] As used herein, the terms "comprising" or "including" indicate that each component may be applied to the disclosed mixtures or compositions. The terms "consisting essentially of" and "consisting of" are included in the terms "comprising", "including", "having". The sum of all weight % or volume % should be 100%.

[0043] As used herein, terms such as "suppress", "down-regulate", "attenuate", "decrease" refer to statistically significant "suppression", "down-regulation", "attenuation", "decrease". It refers to obvious "suppression", "down-regulation", "attenuation", "decrease" compared to the control group; more specifically, for example, "suppression", "down-regulation", "attenuation", "decrease" of 20% or more, preferably 50% or more, more preferably 80% or more. In the present disclosure, unless otherwise indicated, suppressing the tumor includes preventing, alleviating and / or treating the tumor.

[0044] As used herein, the anti-PD-1 antibody is an optional antibody that specifically neutralizes / binds to PD-1.

[0045] In the present disclosure, "pharmaceutically acceptable" components are those that are applicable to humans and / or animals and have no excessive adverse side effects (e.g., toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0046] As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable solvent, suspending agent, or excipient for administering the active ingredients of the present disclosure to animals or humans. The "pharmaceutically acceptable carrier" may be liquid or solid.

[0047] As used herein, "effective amount" indicates that the amount of the component is sufficient to cause the necessary reaction.

[0048] INF-α variant The present disclosure provides an INF-α variant, wherein the Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162 residues of its amino acid sequence are mutated to Cys; preferably, the Arg149 residue is mutated to Cys.

[0049] The INF-α variant of the present disclosure may be a chemically synthesized product, or may be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) by recombinant techniques.

[0050] The present disclosure further includes fragments, derivatives, and analogs of the INF-α variant. As used herein, the terms "fragment", "derivative", and "analog" refer to proteins that substantially retain the same biological function or activity as the native INF-α variant of the present disclosure. The protein fragments, derivatives or analogs of the present disclosure may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a protein having substituents on one or more amino acid residues; or (iii) a protein formed by fusing an additional amino acid sequence to this protein sequence (for example, a leader sequence or a secretion sequence, or a sequence for purifying this protein or a protein original sequence, or a fusion protein); However, according to the definitions herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art. However, the conditions to be satisfied are as follows: at least one of the mutations specifically pointed out in the present disclosure must be present in the amino acid sequences of the above INF-α variant and its fragments, derivatives and analogs; preferably, the mutation is to mutate the Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162 residues to Cys in the amino acid sequence shown in SEQ ID NO: 1; preferably, it is to mutate the Arg149 residue to Cys.

[0051] In the present disclosure, the term "INF-α variant" includes, but is not limited to, deletions, insertions and / or substitutions of several (generally, 1 to 20, more preferably 1 to 10, most preferably 1 to 8, 1 to 5, 1 to 3, or 1 to 2) amino acids, and addition or deletion of one or more (generally, 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and / or N-terminus. For example, in this field, substitution by amino acids with similar properties generally does not change the function of the protein. Also, for example, addition or deletion of one or more amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the INF-α variant. However, there must be mutations as described above in the present disclosure among these variant forms; preferably, in the amino acid sequence shown in SEQ ID NO: 1, the mutation is to mutate the residues of Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162 to Cys; preferably, it is to mutate the Arg149 residue to Cys.

[0052] In the present disclosure, the term "INF-α variant" includes, but is not limited to, derivative proteins having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, such as 98% or more, 99% or more with the amino acid sequence of the above-mentioned INF-α variant and retaining its protein activity. Similarly, there must be mutations as described above in the present disclosure among these derivative proteins; preferably, in the amino acid sequence shown in SEQ ID NO: 1, the mutation is to mutate the residues of Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162 to Cys; preferably, it is to mutate the Arg149 residue to Cys.

[0053] The present disclosure further provides analogs of the INF-α variant. The differences between these analogs and the INF-α variant may be differences in amino acid sequences, differences in modified forms that do not affect the sequence, or both. These polypeptides include naturally occurring or induced genetic variants. Induced variants can be obtained by various techniques such as techniques that induce random mutations by exposing to radiation or mutagens, site-directed mutagenesis methods, and other known molecular biology techniques. Analogs include analogs having residues different from natural L-amino acids (e.g., D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the polypeptides of the present disclosure are not limited to the representative polypeptides listed above. Modification forms (generally, those that do not change the primary structure) include chemically induced forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, e.g., polypeptides produced by glycosylation modifications during the synthesis and processing of polypeptides or further processing steps. This modification can be achieved by exposing the polypeptide to enzymes that glycosylate the polypeptide (e.g., mammalian glycosylation enzymes or deglycosylation enzymes). Modification forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Polypeptides whose anti-proteolytic performance is improved or whose solubility performance is optimized by modification are also included.

[0054] The present disclosure further provides a polynucleotide sequence encoding the INF-α variant or a conservative mutant protein thereof of the present disclosure.

[0055] The polynucleotide of the present disclosure may be in DNA form or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0056] The polynucleotide encoding the mature protein of the mutant includes: a coding sequence encoding only the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence of the mature protein (and any additional coding sequences) and non-coding sequences.

[0057] The "polynucleotide encoding a protein" may include the polynucleotide encoding the protein, and may also include polynucleotides of additional coding and / or non-coding sequences.

[0058] The present disclosure also relates to a vector containing the polynucleotide of the present disclosure, a host cell genetically engineered using the vector of the present disclosure or the coding sequence of the INF-α mutant, and a method for producing the mutant enzyme described in the present disclosure by recombinant techniques.

[0059] By conventional recombinant DNA techniques, recombinant INF-α mutants can be expressed or produced using the polynucleotide sequences of the present disclosure. Generally, the following steps are involved: (1) transforming or transducing an appropriate host cell with the polynucleotide (or mutant) encoding the INF-α mutant of the present disclosure or with a recombinant expression vector containing the polynucleotide; (2) culturing the host cell in an appropriate medium; (3) isolating and purifying the protein from the medium or the cells.

[0060] In the present disclosure, the polynucleotide sequence of the INF-α variant can be inserted into a recombinant expression vector. Any plasmid and vector can be used as long as it is replicable and stable within the host. One of the important features of an expression vector is usually to contain an origin of replication, a promoter, a marker gene, and translation control elements. Using methods well known to those skilled in the art, an expression vector containing a DNA sequence encoding the INF-α variant and appropriate transcription / translation control signals can be constructed. The DNA sequence can be operably linked to an appropriate promoter within the expression vector to direct mRNA synthesis. The expression vector further contains a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for the selection of transformed host cells.

[0061] A vector containing the appropriate DNA sequence as described above together with an appropriate promoter or control sequence is used for the transformation of appropriate host cells so as to be able to express the protein.

[0062] In the present disclosure, the above-mentioned host cells may be prokaryotic cells such as bacterial cells (preferably Escherichia coli); or lower eukaryotic cells such as fungal cells and yeast cells; or higher eukaryotic cells such as plant cells. Representative examples are Escherichia coli, Bacillus subtilis, Streptomyces, Agrobacterium; eukaryotic cells such as yeast and plant cells.

[0063] Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells. The recombinant cells (host cells) established in the present disclosure can be cultured by conventional methods to express the polypeptide encoded by the gene of the present disclosure. Depending on the host cells used, the medium used for culturing can be selected from various normal media. Culture under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (for example, temperature conversion or chemical induction), and the cells are recultured for a while.

[0064] Upon expression, the INF-α variants of the present disclosure can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, various separation methods can be used to separate and purify the recombinant protein by utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, normal refolding treatment, treatment with protein precipitants (salting-out method), centrifugation, cell disruption by osmosis, sonication, high-speed centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and other liquid chromatography techniques and combinations of these methods.

[0065] The optimized INF-α variants of the present disclosure have various uses related to INF-α properties, including use as an active molecule for tumor suppression. It should be understood that host cells expressing this variant or its expression products (such as degradation products or secreted products) also have this use.

[0066] In a preferred embodiment, the above INF-α variant is coupled with R1-AAN-PABC-R3 and used to form a biomolecular complex that suppresses tumors.

[0067] Biomolecular complex The INF-α variants described in the present disclosure are prepared into biomolecular complexes having the following structure: R1-L-IFNα variant R1 is a protecting group for the IFNα variant and may be selected from any group that can inhibit the binding of the IFNα variant to its ligand or receptor, and can prevent interference from other molecules. For example, it is a molecule that inhibits the binding to the ligand or receptor before reaching the pathological microenvironment (such as the tumor microenvironment). Suitable R1s are selected from polyethylene glycol, polyethylene glycol-C1-5 alkylcarbonyl, naphthylcarbonyl, quinolinolylcarbonyl, fluorenylcarbonyl, and adamantylcarbonyl. TIFF2025523889000005.tif78170

[0068] In the above structural formula, each R group is independently a C1-4 alkyl group; each n is independently an integer in the range of 1 to 30000, such as an integer of 1 to 15000, 1 to 5000, 1 to 2000, 1 to 300, 1 to 150, 1 to 50, 1 to 20 or 3 to 12; polyethylene glycol or Peg m represents polyethylene glycol with a molecular weight of 44 to 132000, such as polyethylene glycol of 1000 to 50000 or 10000 to 30000; m represents the molecular weight of polyethylene glycol; the wavy line indicates the position of R1 linked to the said L.

[0069] In some embodiments, R1 is selected from the following: TIFF2025523889000006.tif36170TIFF2025523889000007.tif245170TIFF2025523889000008.tif35170

[0070] In the present disclosure, L is a linker moiety; preferably, L comprises L1 and L2; preferably, L1 is AA-PABC and L2 is R3; provided that AA is a dipeptide or tripeptide fragment (i.e., a fragment in which 2-3 amino acids are linked by peptide bonds), preferably Ala-Ala-Asn (glycine-glycine-asparagine), and PABC is p-aminobenzylcarbamoyl.

[0071] In the present disclosure, L2 may be a cleavable spacer arm. It may also be a peptide that can be activated by a proteolytic enzyme, protease, or peptidase. In the present disclosure, the proteolytic enzyme, protease, or peptidase may be various proteolytic enzymes, proteases, or peptidases present in the pathological microenvironment. For example, the protease may be a cysteine protease, aspartic protease, glutamic protease, threonine protease, gelatinase, metalloprotease, or asparagine peptidase. In some embodiments, L2 can be cleaved by at least one selected from asparagine endopeptidase (Legumain), cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stellate collagenase, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, peptidase (pepsm), matriptase, legumain, plasmepsm, nepenthesin, metal exopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin 1β converting enzyme, thrombin, FAP (FAP-a), meprin, granzyme, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).In a preferred embodiment, the content of the present disclosure relates to asparagine endopeptidase mainly expressed and secreted by tumor cells in the tumor microenvironment. Due to the expression of asparagine endopeptidase, tumor-associated macrophages (M2 type) are also different from monocytes and inflammatory macrophages (M1 type). In the present disclosure, the polypeptide may be a substrate of a proteolytic enzyme and may be recognized and cleaved by a proteolytic enzyme. L2 of the present disclosure is represented by -L2a-, -L2b-, -L2a-N-, -L2a-D-, -L2a-AAN-, -L2a-AAD- or -L2a-L2b-; provided that L2a is a peptide cleaved at the amide bond by one or more proteolytic enzymes; L2b is a peptide that forms a carbamate with PABC by the amino group in the side chain, and the carbamate can be cleaved by one or more proteolytic enzymes; A is alanine; N is asparagine, and the amino group in its side chain forms a carbamate with the neighboring group, and the carbamate can be cleaved by asparagine endopeptidase; D is aspartic acid, and the amino group in its side chain forms a carbamate with PABC and can be decomposed by granzyme B. L2a and L2b can be linked by the formation of an amide bond. After asparagine endopeptidase, granzyme B degrades the bond (such as carbamate) between L2 and PABC, and PABC can be rapidly and automatically released.

[0072] In some embodiments, a suitable polypeptide that can be activated by a proteolytic enzyme may be a tripeptide. Any substrate peptide that is recognized and cleaved (activated) by a proteolytic enzyme in a pathophysiological microenvironment well known in the art can be used as L2 disclosed herein. Such peptides have the structures disclosed in WO 2016 / 026458, the content of which is incorporated herein by reference. In some embodiments, in the tripeptide structure suitable for the present disclosure, the amino acid residue linked to R1 may be selected from Ala, Thr, Val and Ile, the middle amino acid residue may be selected from Ala, Thr, Val and Asn, and the third amino acid residue may be selected from Asn and Asp. Usually, L is linked to R1 by an amide, ester, carbamate, urea or hydrazone linkage through the amino group of its amino acid residue, and is linked to PABC by an amide, ester, carbamate, urea or hydrazone linkage through the carboxy group of its amino acid residue. In some preferred embodiments of the present disclosure, the tripeptide is a tripeptide selected from Ala-Ala-Asn and Ala-Ala-Asp. Ala-Ala-Asn is recognized and cleaved by asparagine endopeptidase, and Ala-Ala-Asp is recognized and cleaved by granzyme.

[0073] Suitable R3 is represented by the following formula: TIFF2025523889000009.tif27170

[0074] wherein Rc is C 1-12 alkyl group, C 1-12 oxyalkyl group-C 1-12 alkyl group, C 1-12 alkyl group-C 3-8 cycloalkyl group, (C 1-4 alkyl group-O) p -C 1-12 alkyl group, C 1-12 alkylcarbonylamino group-(C 1-4 alkyl-O)p-C 1-12 alkyl group, phenyl group-C1-12 an alkyl group, C 3-8 a cycloalkyl group, C 1-12 alkyl group-C 3-8 cycloalkyl group-C 1-12 alkyl group and C 1-12 alkyl group-phenyl group-C 1-12 selected from alkyl groups, provided that the phenyl group may be one or two optionally substituted phenyl groups, and the substituent may be a halogen atom; In some embodiments of the present disclosure, the specific structure of R3 is selected from the following: TIFF2025523889000010.tif102170

[0075] In the complex of the present disclosure, R3 can form a covalent bond with the S of cysteine at a specific position of the IFNα variant. R1 is cleaved from R1-AAN-PABC-R3-S-Cys-IFNα by a proteolytic enzyme to release PABC-R3-S-Cys-IFNα. Thereafter, PABC automatically drops off to release the R3-S-Cys-IFNα variant.

[0076] In the present disclosure, it should be understood that the wavy line used in each of the formulas shown indicates the connection position between the part containing the wavy line and the other part.

[0077] In the complex described in the present disclosure, for example, R1-AAN-PABC-R3, R1-AAN-PABC-R3-S-Cys-IFNα, etc. may be synthesized by methods known in the art.

[0078] Composition The present disclosure further includes a composition (e.g., a drug composition) containing the biomolecular complex, which includes the complex described herein. The drug composition may further include a pharmaceutically acceptable carrier. The carrier may be any pharmaceutically acceptable carrier or excipient, and may vary depending on the dosage form and the mode of administration. Pharmaceutically acceptable carriers are usually safe and non-toxic, and include known substances used in the pharmaceutical industry to prepare drug compositions, such as fillers, diluents, aggregating agents, binders, lubricants, glidants, stabilizers, coloring agents, wetting agents, disintegrants, etc. Suitable pharmaceutically acceptable carriers can include sugars such as lactose, sucrose, mannitol, and sorbitol; cellulose preparations, and / or calcium phosphates such as tricalcium phosphate or calcium hydrogen phosphate; starches including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; silica, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate; and / or polyethylene glycol, etc. When selecting a pharmaceutically acceptable carrier, the mode of administration of the drug dosage form is mainly considered. This is well known in the art.

[0079] The drug composition may contain a therapeutically or prophylactically effective amount of the complex. The specific effective amount depends on various factors, such as the specific disease to be treated, the physical condition of the patient such as body weight, age, gender, the duration of treatment, the treatment by co-administration (if any), and the specific formulation used. Usually, the "effective amount" described herein is a normal amount of the biomolecule. However, in some embodiments, the therapeutically or prophylactically effective amount of the complex contained in the drug composition of the present disclosure may be less than the normal biomolecule amount, but a better therapeutic or prophylactic effect can be obtained, because the biomolecule is protected by a protecting group before reaching the pathological microenvironment and binding to its ligand or receptor.

[0080] The pharmaceutical composition of the present disclosure can be formulated into various suitable dosage forms such as tablets, capsules, injections, etc., and can be administered by any suitable method to achieve the purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally or externally. The dosage of the drug depends on factors such as the patient's age, health status and weight, concurrent treatment, frequency of treatment, etc. The pharmaceutical composition of the present disclosure can be applied to any subject having this need, for example, mammals, especially humans.

[0081] Among tumor patients, tumor cells or antigen-presenting cells (APCs) having tumor antigens bind to T cells, thereby partially or completely suppressing the immune killing of the host's tumor. In some embodiments, the conjugate of the present disclosure is activated and released via a proteolytic enzyme, particularly asparagin endopeptidase or granzyme, or under acidic conditions, in a pathological microenvironment.

[0082] Therefore, the conjugate of the present disclosure can effectively break through the individual's immune barrier, reach the pathological microenvironment, and be activated and released in the pathological microenvironment. As a result, by selectively promoting the proliferation and killing effects of T cells and the like in the tumor microenvironment, low autoimmunity and high efficacy can be achieved.

[0083] The biomolecule of the present disclosure may be used for the treatment of tumors, or may be used as an active ingredient for preparing a drug for treating tumors. The tumors described herein may be any tumors known to be treated by the IFNα described herein, including but not limited to those listed above.

[0084] The present disclosure further includes a method for treating or preventing tumors, which includes administering a therapeutically or prophylactically effective amount of the conjugate or its pharmaceutical composition described herein to a subject in need thereof. The method can be used in combination with known radiotherapy or immunotherapy.

[0085] Accordingly, the present disclosure constructs a cytokine prodrug, which loses the activity of the cytokine in normal tissues and reduces the toxicity of the cytokine by chemically coupling a mask (R) and a mutated cytokine via a chemical linker. At the same time, the cytokine prodrug is further coupled with an antibody to further improve the performance of the drug (further optimize the targeting, half-life, and expression level), and after being activated by enzyme cleavage or acidic conditions near the target tissue, it can return to its original or better activity to exert the medicinal effect.

[0086] Combined use of a biological conjugate and an antibody The present disclosure provides a method of combined administration, including a method of combined administration using the above-mentioned biomolecular conjugate with an anti-tumor antibody.

[0087] The anti-tumor antibody mentioned above includes, for example, anti-PD-1 antibody, anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-TNFα antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-CD40 antibody or anti-ICOS antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD3 antibody, anti-CD22 antibody, anti-CCR4 antibody, anti-CD38 antibody, anti-CD52 antibody, anti-complement C5 antibody, anti-F protein of RSV, anti-GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib / Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 antibody, anti-integrin α4 antibody, anti-lgE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR2 antibody, anti-VISTA antibody or a functional fragment thereof.

[0088] In a preferred embodiment of the present disclosure, the anti-tumor antibody is an anti-PD-1 antibody. In a specific example of the present invention, when combined with the biomolecular conjugate of the present disclosure, a very significant synergistic effect is shown.

[0089] The present disclosure provides the use of the biomolecular complex and an anti-tumor antibody (such as an anti-PD-1 antibody) for the preparation of a mixture, a pharmaceutical composition or a kit for suppressing tumors.

[0090] When administering, the biomolecular complex and the anti-tumor antibody may be administered separately or simultaneously. It should be understood that various administration methods are included in the present disclosure.

[0091] The anti-tumor antibody is derived from mouse, human, humanized, chimeric, or other species. The antibody may be of any type (such as IgG, IgE, IgM, IgD and IgA), category (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subcategory of immunoglobulin molecules. The antibody may be derived from any species. In some specific embodiments, the antibody is derived from human, mouse, or rabbit.

[0092] Antibody fragments or binding portions of the antibody can also be applied to the technical solution of the present disclosure. The antibody fragment or binding portion of the antibody includes a part of the full-length antibody and usually includes the antigen-binding region or variable region of the antibody. Preferably, the antibody fragment is a functional fragment that retains the antigen-binding ability of the complete antibody. Examples of antibody fragments or functional fragments include Fab, Fab’, F(ab’)2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (scFv); and the like.

[0093] The present disclosure provides a mixture containing the biomolecular complex and the anti-tumor antibody as an active ingredient. Preferably, in the above mixture, the mass ratio of the biomolecular complex to the anti-tumor antibody is 1:(0.05 - 50) (such as 1:0.1, 1:0.15, 1:0.2, 1:0.4, 1:0.5, 1:0.6, 1:0.8, 1:1, 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:20, 1:30, 1:40).

[0094] The present disclosure provides a drug composition comprising: (a) an effective amount of the biomolecular complex; (b) an effective amount of an anti-tumor antibody; and (c) a pharmaceutically acceptable carrier or excipient. Preferably, in the said composition, the mass ratio of the biomolecular complex to the anti-tumor antibody is 1:(0.05 - 50) (for example, 1:0.1, 1:0.15, 1:0.2, 1:0.4, 1:0.5, 1:0.6, 1:0.8, 1:1, 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:20, 1:30, 1:40).

[0095] The drug composition or mixture of the present disclosure can be made into any ordinary dosage form by ordinary methods. The dosage form may be various as long as it can efficiently reach the active ingredient into the body of a mammal. For example, it is selected from injections, infusions, tablets, capsules, and pills. The active ingredient therein can be present in a suitable solid or liquid carrier or diluent. The pharmaceutically acceptable carrier applied is the same as described above.

[0096] The mixture or drug composition of the biomolecular complex and the anti-tumor antibody of the present disclosure can also be stored in a sterilized device suitable for injection or drip. Generally, in the drug composition of the present invention, as the active ingredients, the said biomolecular complex and anti-tumor antibody account for 0.0001 - 20% of the total weight of the drug composition, and the rest is a pharmaceutically acceptable carrier.

[0097] The effective dosage of the said biomolecular complex and anti-tumor antibody used varies depending on the administration pattern and the severity of the disease to be treated. If necessary, the said biomolecular complex can also be administered in combination with other active ingredients or drugs.

[0098] When the said biomolecular complex and anti-tumor antibody are used in combination, the present disclosure further provides a kit for treating tumors, which contains container 1 and the biomolecular complex contained in container 1; and container 2 and the anti-tumor antibody contained in container 2.

[0099] The kit may contain a mixture of the biomolecular complex and the anti-tumor antibody, wherein the contents of the biomolecular complex and the anti-tumor antibody are as described above.

[0100] Furthermore, the kit may also include materials for assisting administration, such as injection needles.

[0101] Furthermore, the kit may include an instruction manual for explaining a method of suppressing tumors using the combined administration method disclosed herein.

[0102] In the present disclosure, for the first time, it is disclosed that the combined use of the biomolecular complex and the anti-tumor antibody has an excellent tumor-suppressing effect and can achieve a very remarkable synergistic effect, far exceeding the expectations of those skilled in the art / physicians.

[0103] Hereinafter, the present disclosure will be further described with reference to specific examples. It should be understood that these examples do not limit the scope of the present disclosure and are merely illustrative of the present disclosure. The experimental methods that do not specify specific conditions in the following examples are generally carried out according to the normal conditions described in J. Sambrook et al., Guide to Molecular Cloning, Third Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0104] INF-alpha amino acid sequence (SEQ ID NO: 1): Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu PheSer Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu The biomolecular complex formed with the IFNα protein has the following structure.

[0105] R1-AAN-PABC-R3-S-Cys-IFNα However, Cys represents the cysteine residue contained in IFNα; S represents the sulfur atom in the cysteine residue; R1 is a group that inhibits the binding of the IFNα protein to its ligand or receptor; AAN represents a tripeptide, where A is alanine and N is asparagine; PABC is p-aminobenzylcarbamoyl; R3 is a group that can form a covalent bond with the sulfur atom in the cysteine residue and contains an EMC group, and its role is to maintain or improve the binding ability of IFNα to its antigen after AAN is cleaved.

[0106] In the following examples, the R1 used is shown in Table 1.

[0107] TIFF2025523889000011.tif42170

[0108] In the experiments of the present disclosure, several linkers R1-AAN-PABC-R3 were selected for research. The synthesized linker compounds were uniformly dissolved and diluted 10-fold with water to 1 mg / mL. Under the conditions of 37°C for 2 hours, 1 mg / mL of the sample compound was added to 100 micrograms of legumain. The enzyme cleaved the linker to release R3, and the decrease of the linker and the increase of R3 were detected by HPLC, and the activation efficiency of the enzyme for the linker (the ratio of R3 cleaved and released by the enzyme to the original compound) could be compared. As shown in Table 2, when R1 = peg5000, the linker and its activation efficiency are shown in Table 2.

[0109] TIFF2025523889000012.tif195170

[0110] When R1 = peg20000, the preferred linker R1-AAN-PABC-R3 and its activation efficiency are shown in Table 3: TIFF2025523889000013.tif186170

[0111] From the above results, in the comprehensive evaluation of stability and activation efficiency, both linker 1 and linker 3 showed very high activation efficiency, and the activation efficiency of linker 10 was also high.

[0112] Example 1, the situation of coupling with the R1-AAN-PABC-R3 compound after mutating the amino acids on the surface of the INF-α protein The linker R1-AAN-PABC-R3 was adopted, R1 is peg5000, and the linker is linker 3 in Table 2.

[0113] When any amino acid on the surface of the INF-α protein is mutated to Cys, it can be coupled with the R1-AAN-PABC-R3 compound, but the coupling efficiency varies. Single-site amino acid mutations at positions 2 - 165 were performed on the amino acid sequence of the INF-α protein. Based on the amino acid sequence and its DNA sequence of INF-α containing the mutation site, a plasmid (GeneWiz) capable of expressing His*6-INF-α in mammalian cells was constructed. The expression host is HEK293 cells (Life Technologies). Before transfection, HEK293 cells were cultured at 37°C with 5% CO2 in complete medium (Gibico) containing 10% FBS. One day before transfection, the cells were inoculated into a 15 cm culture dish at an appropriate density, and the medium was replaced with low IgG FBS. Six hours or two days after transfection, the medium was replaced with Freestyle293 (Gibico). On the day of transfection, when the cells reached a certain confluence, the plasmid expressing the target protein was co-transfected into 293T cells using Lipofectamine 2000 (Life Technologies) and PEI (Sigma). On the 4th and 6th days after transfection, the culture supernatants were replaced respectively. The protein expression and activity were detected, and the protein was purified.

[0114] The purified variant was incubated in 20 mM phosphate buffer (pH 7.4) containing 5 mM EDTA at a concentration of 0.3 mg / mL. A TCEP solution was added to the variant at a molar ratio of 100:1, and the resulting mixture was gently stirred while incubating at 4 °C for 4 hours. Then, the mixture was dialyzed against 20 mM phosphate buffer (pH 7.4) containing 150 mM NaCl at 4 °C for 2 hours. Immediately thereafter, R1-AAN-PABC-R3 was added to the mixture at a molar ratio of 20:1, and the resulting mixture was gently stirred at 25 °C for 16 hours. The reaction was stopped, and the remaining R1-AAN-PABC-R3 compound was removed. Samples of equal volume were taken and subjected to SDS-PAGE analysis, and the coupling efficiency was obtained by scanning the gray scale of the bands (scanning the ratio of the gray scale, i.e., the coupling efficiency: gray scale of the protein band after coupling / (gray scale of the uncoupled protein band + gray scale of the protein band after coupling)). Table 4 shows the efficiency of coupling with the R1-AAN-PABC-R3 compound after mutating a single-site amino acid on the surface of the INF-α protein to Cys.

[0115] TIFF2025523889000014.tif253170TIFF2025523889000015.tif255170TIFF2025523889000016.tif103170

[0116] Example 2. Changes in receptor binding ability after mutating the amino acids on the surface of the INF-α protein and then coupling with the R1-AAN-PABC-R3 compound Single-site amino acid mutations at positions 2 - 165 were made to the amino acid sequence of the INF-α protein. After coupling each mutant with a point mutation, the binding activity was detected by ELISA in comparison with wild-type INF-α. Specifically, after incubating a 96-well plate overnight with INFR1 or INFR2, it was blocked with 1% BSA blocker (ThermoFisher) at 37°C for 2 hours and washed three times with PBST. The corresponding antibody or mutant was added and allowed to bind at 37°C for 1 hour, followed by three washes with PBST. HRP enzyme coupled to anti-human INF-α 2b was added and allowed to bind at 37°C for 1 hour, followed by three washes with PBST. TMB substrate (Solarbio, Inc) was added, and the absorbance was detected at 450 nm. The effect on the mutant binding strength after coupling was calculated as EC50 after coupling / wild-type EC50, that is, (binding ability after coupling / binding ability of wild type) * 100% = 1 / (EC50 after coupling / wild-type EC50).

[0117] After mutating the single-site amino acid on the surface of the INF-α protein to Cys, Table 5 shows the changes in receptor binding ability after coupling with the R1-AAN-PABC-R3 compound. In R1-AAN-PABC-R3, R1 is peg5000, and the linker is linker 3 in Table 2.

[0118] TIFF2025523889000017.tif255170TIFF2025523889000018.tif255170TIFF2025523889000019.tif103170

[0119] Example 3. Changes in receptor binding after mutating the amino acids on the surface of the INF-α protein and activating by coupling with the R1-AAN-PABC-R3 compound Single-site amino acid mutations at positions 2 - 165 were made to the amino acid sequence of the INF-α protein, and an activation test was conducted. In the activation test, 1 mg / ml of the mutant after coupling was placed in an acidic environment with pH = 6 and reacted overnight at 37°C. The receptor binding status after activation was detected by ELISA.

[0120] Table 6 shows the changes in receptor binding after mutating a single-site amino acid on the surface of the INF-α protein to Cys and then coupling it with the R1-AAN-PABC-R3 compound for activation. In R1-AAN-PABC-R3, R1 is peg5000, and the linker is linker 3 in Table 2.

[0121] TIFF2025523889000020.tif255170TIFF2025523889000021.tif255170TIFF2025523889000022.tif103170

[0122] Example 4, Changes in binding activity after coupling mutated INF-α with different R1-AAN-PABC-R3 compounds Single-site amino acid mutations at positions 2 - 165 were made to the amino acid sequence of the INF-α protein. Based on the amino acid sequence and DNA sequence of INF-α and the INF-α amino acid sequence containing the mutation site, a plasmid (GeneWiz) capable of expressing His*6-INF-α in mammalian cells was constructed. The expression host was HEK293 cells (Life Technologies). Before transfection, HEK293 cells were cultured at 37°C with 5% CO2 in complete medium (Gibico) containing 10% FBS. One day before transfection, the cells were seeded in a 15 cm culture dish at an appropriate density, and the medium was replaced with low-IgG FBS. Six hours or two days after transfection, the medium was replaced with Freestyle293 (Gibico). On the day of transfection, when the cells reached a certain confluence, the plasmid expressing the target protein was co-transfected into 293T cells using Lipofectamine 2000 (Life Technologies) and PEI (Sigma). On the 4th and 6th days after transfection, the culture supernatants were replaced respectively. The expression and activity of the protein were detected, and the protein was purified.

[0123] The purified variant was incubated in 20 mM phosphate buffer (pH 7.4) containing 5 mM EDTA at a concentration of 0.3 mg / mL. The TCEP solution was added to the variant at a molar ratio of 100:1, and the resulting mixture was gently stirred while incubating at 4 °C for 4 hours. Then, the mixture was dialyzed against 20 mM phosphate buffer (pH 7.4) containing 150 mM NaCl at 4 °C for 2 hours. Immediately thereafter, R1-AAN-PABC-R3 was added to the mixture at a molar ratio of 20:1, and the resulting mixture was gently stirred at 25 °C for 16 hours. The reaction was stopped, and the remaining R1-AAN-PABC-R3 compound was removed.

[0124] In the present disclosure, for each variant having a point mutation compared to wild-type INF-α, the binding activity after coupling was detected by ELISA. Specifically, the 96-well plate was incubated overnight with INFR1 or INFR2, then blocked with 1% BSA blocker (ThermoFisher) at 37 °C for 2 hours, and washed 3 times with PBST. The corresponding antibody or variant was added, allowed to bind at 37 °C for 1 hour, and then washed 3 times with PBST. The HRP enzyme that couples with anti-human INF-α 2b was added, allowed to bind at 37 °C for 1 hour, and then washed 3 times with PBST. The TMB substrate (Solarbio, Inc) was added, and the absorbance was detected at 450 nm. The effect on the variant binding strength after coupling was calculated as EC50 after coupling / wild-type EC50, that is, (binding ability after coupling / binding ability of wild-type) * 100% = 1 / (EC50 after coupling / wild-type EC50).

[0125] By the above method, the changes in binding activity after coupling of variants mutated at different sites with different R1-AAN-PABC-R3 compounds were detected.

[0126] Table 7 shows the changes in the binding activity after coupling of mutated INF-α with R.

[0127] TIFF2025523889000023.tif205170

[0128] Therefore, for the mutation sites that suppress the binding to INFR1, after mutating His57, Gln61, Phe64, and Ser68 to Cys respectively and then coupling with a part of R1-AAN-PABC-R3, their abilities to bind to INFR1 all decreased to 10% or less of the wild type. For the mutation sites that suppress the binding to INFR2, after mutating Ala145 and Arg149 to Cys respectively and then coupling with some R1-AAN-PABC-R3, their abilities to bind to INFR2 all decreased to 1% or less of the wild type, and thus less than 0.1%. Phe27, Ser152, and Arg162 also decreased to some extent.

[0129] Example 5, Change in binding activity after coupling mutated INF-α with R1-AAN-PABC-R3 and activation In the activation test, 10 μg of the activating Legumain enzyme (capable of cleaving at the AAN site) was added to the mutant after coupling at 1 mg / ml, and the reaction was carried out at 37°C for 1 hour, or the mutant after coupling at 1 mg / ml was placed in an acidic environment with pH = 6 and reacted at 37°C for 4 hours. The binding status after activation was detected by ELISA.

[0130] The change in binding activity after coupling mutated INF-α with R1-AAN-PABC-R3 and activation is shown in Table 8.

[0131] TIFF2025523889000024.tif148170

[0132] As a result, for the mutation sites that suppress the binding of INFR1, except for Phe64 and Ser68 whose side chains are similar in structure to R3, when mutants corresponding to His57 with a relatively large structural difference from R3 and Gln61 with a charged side chain are coupled and reactivated, it was found that their ability to bind to INFR1 can mostly be restored to over 200% of the wild type. Since INF-α has a high binding activity with INFR2, any variation on INF-α may cause a decrease in the binding activity with INFR2. Therefore, the key to screening for mutation sites of INFR2 is the recovery of the binding activity after coupling and activation. For the mutation sites that suppress INFR2 binding, except for Phe27, Ala145, and Ser152 whose side chains are similar in structure to R3, the mutant corresponding to Arg149 (R149, that is, the mutation at position 149 from Arg to Cys) had its activity decreased to 0.27% of the original after coupling, and after reactivation, its ability to bind to INFR2 was restored to nearly 100% of the wild type.

[0133] Example 6, Influence of Mutated INF-α on Cell Signaling after Coupling with R1-AAN-PABC-R3 and after Activation A 280000 cells / ml HEK-Blue IFN-a / b cell suspension was prepared in a medium containing 10% heat-inactivated FBS. 20 μl of different gradients of INF-α, mutated INF-α + R1-AAN-PABC-R3 (where R1 is peg20000), and the active form were added to a 96-well cell plate, and 180 μl of the cell suspension was added to each well. The 96-well cell plate was placed in a 37 °C carbon dioxide incubator and cultured for 20 - 24 hours.

[0134] According to the product manual, prepare QUANTI-Blue TM and add 180 μl of QUANTI-Blue to each well of a new 96-well cell plate. TM Then, 20 μl of the above HEK-Blue IFN-a / b cell supernatant was added and incubated at 37 °C for 1 hour. The SEAP level was detected at a wavelength of 620 - 055 nm.

[0135] After the mutated INF-α is coupled with R1-AAN-PABC-R3, and the influence on the activated cell signal transduction is shown in Table 9.

[0136] TIFF2025523889000025.tif82170

[0137] *(Cell signal transduction after coupling / Cell signal transduction of wild type)*100%.

[0138] From the results in Table 6, it was found that the samples after INF-α R149 + R1-AAN-PABC-R3 coupling and the samples after activation had the most ideal influence effect on cell signal transduction.

[0139] The influence on cell signal transduction after INF-α R149 coupling and after activation is shown in Figure 1. As a result, after multiple mutant forms of INF-α were coupled with R1-AAN-PABC-R3, the cell signal transduction became extremely weak or disappeared, but showed a significant recovery after activation. Among them, the sample after INF-α R149 + R1-AAN-PABC-R3 coupling can completely suppress the corresponding cell signal transduction, but the activation form after activation can return to the same cell signal transduction level as wild-type INF-α.

[0140] Example 7, the situation where INF-α activated in the tumor microenvironment inhibits the growth of MC38 tumors MC38 cells were subcutaneously inoculated into C57BL / 6 mice (Shanghai Model Organisms Research Center), and for each mouse, 2×10 6Cells were inoculated. One week later, mice transplanted with MC38 tumors were randomly grouped. For the first group G1, a solvent was injected for control, administered once a week, and the remaining groups were each injected with 100 μg of mutated INF-α + R1-AAN-PABC-R3 and administered once a week. The administration period was 3 weeks. The tumor volume of the mice was recorded three times a week. Table 10 shows the situation of TGI (Tumor Growth Inhibition) after different INF-α + R1-AAN-PABC-R3 administrations.

[0141] TIFF2025523889000026.tif77170

[0142] The results of this table showed that after multiple mutant forms of INF-α were coupled with r1-AaN-PABC-r3, they had the effect of inhibiting tumor growth. Among them, INF-α R149 + R1-AAN-PABC-R3 had the most significant effect on inhibiting tumor growth.

[0143] Example 8, Tumor Suppression Effect of INF-α Whose Complex Is Activated in the Tumor Microenvironment MC38 cells were subcutaneously inoculated into C57BL / 6 mice (Shanghai Model Organisms Research Center), and for each mouse, 2×10 6 cells were inoculated. One week later, the mice transplanted with MC38 tumors were randomly divided into 4 groups.

[0144] The group situation is as follows: The first group: G1, injected with 100 μg of INF-α and administered three times a week; The second group: G2, injected with 300 μg of INF-α R149 + R1-AAN-PABC-R3 and administered once a week; The third group: G3, injected with 100 μg of INF-α R149 + R1-AAN-PABC-R3 and administered once a week; The fourth group: G4, injected with a solvent for control and administered once a week.

[0145] The administration cycle is 3 weeks. The tumor volume of the mice was recorded three times a week. The results of the tumor volume after the administration are shown in Figure 2.

[0146] However, R1-AAN-PABC-R3 is specifically linker 10 in Table 3.

[0147] As a result, it was found that 300 μg INF-α R149 + R1-AAN-PABC-R3 could effectively suppress the growth of MC38 tumors, cure one mouse, and have a significant inhibitory effect of more than 80% on five mice. Administering 100 μg INF-α R149 + R1-AAN-PABC-R3 once a week has a higher tumor suppression effect than administering 100 μg INF-α three times a week.

[0148] From these results, it was found that the INF-α complex can enhance the activity of effector T cells and promote the concentration of INF-α in the tumor microenvironment, so the complex shows a higher effect than the original INF-α.

[0149] Example 9, Tumor suppression effect of combining the INF-α complex with a PD-1 antibody CT26 cells were subcutaneously inoculated into Balb / c mice (Shanghai Model Organisms Research Center), and 2×10 6 cells were inoculated per mouse. One week later, the mice transplanted with CT26 tumors were randomly divided into 4 groups: Group 1 (G1): Injected with 100 μg INF-α R149 + R1-AAN-PABC-R3 and administered once a week; Group 2 (G2): Injected with 100 μg of anti-PD-1 mouse antibody (Sino Biological; Cat: 50124-RP02) and administered twice a week; Group 3 (G3): Injected with 100 μg INF-α R149 + R1-AAN-PABC-R3 and administered once a week; at the same time, injected with 100 μg of anti-PD-1 mouse antibody and administered twice a week; Group 4 (G4): Injected with a solvent for control and administered once a week.

[0150] However, R1-AAN-PABC-R is specifically Linker 10 in Table 3.

[0151] The administration cycle is 3 weeks. The tumor volume of the mice was recorded three times a week. The results of the tumor volume after the administration are shown in Figure 3. From the results, it was found that the combination of 100 μg INF-α R149 + R1-AAN-PABC-R3 and the anti-PD-1 antibody could effectively inhibit the growth of CT26 tumors, cured 4 mice, and had a significant inhibitory effect on all mice. The decrease in tumor volume was extremely significant, indicating that the combination of the two achieved a synergistic effect.

[0152] Therefore, the combination of INF-α R149 activated in the tumor microenvironment and the PD-1 antibody exhibits higher efficacy and cure rate.

[0153] The embodiments described above show some embodiments of the present disclosure, and the description is more specific and detailed, but it cannot be used to understand the limitation of the patent scope of the present disclosure. For those skilled in the art, some modifications and improvements can be made without departing from the concept of the present disclosure, and these belong to the protection scope of the present disclosure. Therefore, the patent protection scope of the present disclosure shall be subject to the appended claims. At the same time, all references mentioned in the present disclosure are incorporated herein by reference and cited in this application as if each were individually cited by reference.

Claims

1. An IFNα cytokine biomolecular complex having the following structure: R1 - AAN - PABC - R3 - S - Cys - IFNα However, Cys represents a cysteine residue with a single point mutation on the surface of IFNα; S represents a sulfur atom in the cysteine residue; IFNα has the amino acid sequence shown in SEQ ID NO: 1; R1 is a group that inhibits the binding of an IFNα variant to its ligand or receptor; AAN represents a tripeptide, where A is alanine, N is asparagine, and PABC is p - aminobenzylcarbamoyl; R3 is a spacer arm that covalently binds to and couples with the sulfur atom of a cysteine with a single point mutation of IFNα having an EMC group. Its role is that even after AAN is cleaved, the R3 structure remains bound to the cytokine, and R3 - S - Cys - IFNα may partially or completely recover, or even enhance the affinity with the IFNα receptor.

2. R1 is selected from polyethylene glycols with a molecular weight of 44 - 132000, such as polyethylene glycols of 1000 - 50000, 3000 - 80000, or 10000 - 60000; or The IFNα variant is a variant obtained by a single point mutation of INF - α, and its amino acid sequence is based on SEQ ID NO: 1, where the Arg149, Ala145, His57, Gln61, Phe64, or Ser68, Phe27, Ser152, Arg162 residues are mutated to Cys. The biomolecular complex according to claim 1, characterized in that.

3. R1 is The biomolecular complex according to claim 2, characterized in that it is selected from.

4. R3 is The biomolecular complex according to claim 1, characterized in that it is selected from.

5. The structure of R1 - AAN - PABC - R3 is The biomolecular complex according to claim 1, characterized in that it is selected from.

6. Use of the biomolecular complex according to any one of claims 1 - 5 in the preparation of a composition or kit for suppressing tumors.

7. Use of the biomolecular complex according to any one of claims 1 - 5 in the preparation of a composition or kit for suppressing tumors in combination with an anti - tumor antibody.

8. The composition or kit contains the biomolecular complex according to any one of claims 1 to 5; preferably, the composition further contains a pharmaceutically acceptable carrier, and is a composition or kit for suppressing tumors.

9. The composition further contains an anti-tumor antibody; or The kit further contains an anti-tumor antibody or a composition containing an anti-tumor antibody The composition or kit according to claim 8, characterized in that.

10. The anti-tumor antibody is an anti-PD-1 antibody, and the composition or kit according to claim 9 is characterized in that.

11. In terms of mass ratio, the biomolecular complex and the anti-PD-1 antibody are 1:(0.05 - 50); preferably, 1:(0.2 - 8); more preferably, 1:(0.4 - 5), and the composition or kit according to claim 8 is characterized in that.

12. A method for preparing INF-α activated in a tumor microenvironment, which includes single-point mutating INF-α and mutating specific residues of its amino acid sequence to Cys.

13. A mutant obtained by single-point mutating INF-α, and its amino acid sequence is based on SEQ ID NO: 1, and the residues of Arg149, Ala145, His57, Gln61, Phe64 or Ser68, Phe27, Ser152, Arg162 are mutated to Cys, and it is an INF-α mutant characterized in that.

14. An isolated polynucleotide, characterized in that the polynucleotide encodes the INF-α mutant according to claim 13.

15. The use of the INF-α mutant according to claim 13 for forming a biomolecular complex for suppressing tumors by coupling with R1 - AAN - PABC - R3; However, R1 is a group that inhibits the binding of the IFNα mutant to its ligand or receptor; AAN represents a tripeptide, A is alanine, N is asparagine, and PABC is p-aminobenzylcarbamoyl; R3 is a spacer arm formed by covalently coupling with the sulfur atom of cysteine obtained by single-point mutating IFNα and having an EMC group. Its role is that even after AAN is cleaved, the R3 structure remains bound to the cytokine, and R3 - S - Cys - IFNα may partially or completely recover, or even enhance the affinity with the IFNα receptor.

16. The tumor according to any one of claims 1 to 15, characterized in that it includes melanoma, NSCLC, head and neck squamous cell carcinoma, urothelial carcinoma, classical Hodgkin lymphoma, gastric cancer, esophagogastric junction cancer, cervical cancer, B-cell lymphoma, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, primary liver cancer, and small cell lung cancer.

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