TW202120560A - Recombinant polypeptides and uses thereof - Google Patents

Abstract

Disclosed herein is a recombinant polypeptide comprising a first polypeptide, and a second polypeptide operably linked to the N- or C- terminus of the first polypeptide, wherein the first polypeptide has an amino acid sequence at least 85％ identical to IL-15, and the second polypeptide has an amino acid sequence at least 85％ identical to albumin binding domain (ABD) or albumin binding peptide (ABP). Furthermore, the present disclosure is related to pharmaceutical compositions comprising the present recombinant polypeptide for preventing and/or treating a cancer in a subject in need thereof. Also encompassed within the scope of the present disclosure are methods for preventing and/or treating cancer using said pharmaceutical composition.

Description

Recombinant polypeptide and its use

The present disclosure as a whole is about the technical field of cancer treatment. More specifically, the present disclosure relates to a recombinant polypeptide (recombinant polypeptide) and its use in the treatment of cancer.

Cancer is a disease that involves abnormal cell growth and has the potential to invade or spread to other parts of the body. Over the years, cancer has become a serious problem faced by all human beings and has caused a heavy burden on the health care system of a society.

Therefore, many treatment options have been developed to treat cancer, including surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy, and palliative care. However, such conventional methods are usually accompanied by severe side effects, such as damage to normal tissues or cytotoxicity. In recent years, in research and preclinical trials, some related alternative therapies with lower side effects for the treatment of cancer are being tested. Among these treatments, immunotherapies using biological macromolecules (for example, polypeptides, nucleic acids, carbohydrates, etc.) are the most promising therapies, because these therapies eliminate tumors by regulating the immune system function of the affected individual.

One of the drugs used to treat cancer is IL-15. IL-15 is a member of the common γ chain receptor (γc) cytokine family. It is used to regulate the activation and proliferation of T cells, and promote the expansion and development of natural killer (NK) cells. In some preclinical models, IL-15 has been shown to enhance ^{the immunity of CD8 +} T cells against tumors (Klebanoff CA et al., PNAS 101 (7): 1969–74; Teague RM et al., Nature Medicine 12 (3): 335–41). However, it is still difficult to prepare a recombinant IL-15 with an appropriate pharmacokinetic profile, which greatly limits the clinical use of IL-15 in the treatment of cancer. Some studies have shown that IL-15 itself is quickly metabolized in the blood circulation. In addition, compared with natural IL-15 or recombinant IL-15 prepared from E. coli, the biological activity of IL-15 fusion protein is usually compromised. The IL-15 fusion protein is A protein isolated from mammalian cells by fusing IL-15 with an additional functional domain.

In view of this, the related field urgently needs an improved method for prolonging the half-life of IL-15 in the blood circulation so that it will not interfere with the biological activity of IL-15 and can have an optimized pharmacokinetic profile. According to this, IL-15 can be used to the greatest extent in the treatment of cancer.

The following presents a brief summary of the disclosure to facilitate readers to have a basic understanding of the disclosure. This summary is not a comprehensive overview of the content of this disclosure, nor is it used to identify the key/decisive elements of the content of this disclosure, or outline the scope of the content of this disclosure. Its sole purpose is to present certain concepts of the present disclosure in a simplified form as a prelude to the subsequent presentation of more detailed descriptions.

As implemented and broadly described herein, one aspect of the present disclosure relates to a recombinant polypeptide comprising, A first polypeptide having an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO:1; and A second polypeptide which is operably linked to the N- or C-terminus of the first polypeptide, wherein the second polypeptide has a sequence of at least 85% of SEQ ID NO: 2 or SEQ ID NO: 3 Amino acid sequence of similarity.

According to certain embodiments of the present disclosure, The first polypeptide has an amino acid sequence of SEQ ID NO: 1; and The second polypeptide has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

According to some optional embodiments of the present disclosure, The N-terminal of the recombinant polypeptide is acetylated, formylated, methylated, carbamylated, pegylated, phosphorylated , Or glycosylated; and/or The C-terminus of the recombinant polypeptide is amidated, glypiated, biotinylated, or glycosylated.

According to certain embodiments of the present disclosure, the recombinant polypeptide of the present disclosure further comprises a third polypeptide, which is operably linked to the N- or C-terminus of the first or the second polypeptide, wherein the The third polypeptide is: β-galactosidase (lacZ), chloramphenicol acetyltransferase (CAT), luciferase, green fluorescent protein (green fluorescent protein) , GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), β-lactamase (β-lactamase), Aminoglycoside-3'-phosphotransferase (aminoglycoside-3'-phosphotransferase, APH(3')), orotidine-5'-phosphate decarboxylase (ODCase), several Chitin binding protein (chitin binding protein, CBP), maltose binding protein (maltose binding protein, MBP), streptomycin tag (Strep-tag), glutathione-S-transferase (glutathione-S-transferase, GST) ), thioredoxin (TRX), alkaline phosphatase (AP), biotin-carboxy carrier protein (BCCP), calmodulin binding peptide (calmodulin binding peptide, CBP), bacteriophage T7 epitope (bacteriophage T7 epitope, T7-tag (T7-tag)), poly-histidine tag, FLAG-tag (FLAG-tag), Myc-tag (Myc-tag) ), HA-tag (HA-tag), Spot-. Spot-tag, NE-tag, or a combination thereof.

According to some embodiments of the present disclosure, the recombinant polypeptide further comprises a linker, wherein the linker is used to connect any two polypeptides of the first, second, and third polypeptides.

Another aspect of the present disclosure relates to a pharmaceutical composition for preventing and/or treating cancer in an individual in need. The pharmaceutical composition comprises the recombinant polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

Optionally, the pharmaceutical composition of the present disclosure further includes a primary anti-PD-L1 antibody.

In another aspect, the present disclosure relates to a method for preventing and/or treating cancer in an individual in need. The method comprises administering to the individual a therapeutically effective amount of the above-mentioned pharmaceutical composition of the present disclosure.

According to certain embodiments of the present disclosure, cancers that can be treated with the methods of the present disclosure include, but are not limited to, bladder cancer, biliary cancer, bone cancer, and brain tumors. brain tumor, breast cancer, cervical cancer, colorectal cancer, colon cancer, esophageal cancer, epidermal carcinoma, gastric cancer cancer, gastrointestinal stromal tumor (GIST), glioma, hematopoietic tumors of lymphoid lineage, hepatic cancer, non-Hodgkin’s lymphoma (non-Hodgkin's lymphoma), Kaposi's sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, myelogenous leukemia (myeloid leukemia), pancreatic cancer, prostate cancer, retinoblastoma, ovary cancer, renal cell carcinoma, spleen cancer, Squamous cell carcinoma, thyroid cancer, or thyroid follicular cancer. In a preferred embodiment of the present disclosure, the cancer is colon cancer. In another preferred embodiment of the present disclosure, the cancer is melanoma. Generally, the cancer can be an in situ cancer or metastatic cancer.

Preferably, the individual is a human.

According to still other embodiments of the present disclosure, the method of the present disclosure further includes subjecting the individual to a surgery, a radiotherapy, a chemotherapy, an immunotherapy, a hormonal therapy, a target therapy, a heat therapy, or Before, at the same time or after the combination therapy, the step of administering the pharmaceutical composition of the present disclosure to the individual.

According to certain embodiments of the present disclosure, the immunotherapy of the methods of the present disclosure comprises administering a primary anti-PD-L1 antibody to the individual.

After referring to the following detailed description and accompanying drawings, many accompanying features and advantages of the present disclosure should be easily understood.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description for the implementation aspects and specific embodiments of the present invention, but this is not the only way to implement or use the specific embodiments of the present invention. The implementation manners cover the characteristics of multiple specific embodiments, and the method steps and sequences used to construct and operate these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and sequence of steps.

I. Definition

For convenience, specific terms used in the specification, embodiments, and the scope of the attached patent application are collected here. Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meaning as understood and used by those with ordinary knowledge in the technical field to which the present invention belongs. Moreover, as long as there is no conflict with the context, the singular nouns used in this specification cover the plural nouns, and the plural nouns used also cover the singular nouns. Specifically, in this specification and the scope of the patent application, the singular form "one" (a, an, and the) includes plural reference values, unless otherwise indicated based on the context. In addition, in this specification and the scope of the patent application, the expressions of "at least one" and "one or more" have the same meaning, and both of them mean that they include one, two, three or more. many. Unless otherwise indicated in this specification, the present disclosure actually uses conventional techniques in molecular biology, microbiology, recombinant DNA, and immunology in the technical field to which the present invention pertains. This type of technology has been described in detail in the open literature.

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate numerical values, the relevant numerical values in the specific embodiments are presented here as accurately as possible. However, any value inevitably contains the standard deviation caused by the individual test method. And, here, the term "about" usually refers to the actual value within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average value, depending on the consideration of a person with ordinary knowledge in the technical field of the present invention. Except for the examples, or unless otherwise clearly stated, all ranges, quantities, values and percentages used herein (for example, used to describe the amount of material, length of time, temperature, operating conditions, quantity ratios and other similar Those) have been modified by "about". Accordingly, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent scope are approximate values and can be changed according to requirements. At least, these numerical parameters should be understood as the indicated effective number of digits and the value obtained by applying the general carry method.

As used herein, "sequence similarity" (identical) means that two or more sequences or subsequences are the same when performing measurement comparison and alignment (align) to obtain the maximum correspondence (maximum correspondence). Or the amino acid residues with a certain percentage are the same. In order to confirm the percentage of sequence similarity, the sequence is aligned for the purpose of optimal comparison (for example, a gap can be introduced in the sequence of the first amino acid sequence to achieve the comparison with the second amino acid sequence). The optimized alignment result of the base acid sequence), and does not consider any conservative substitution as part of the sequence similarity. Then compare the amino acid residues at the corresponding amino acid positions. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecule is identical at that position. Various methods well known to those skilled in the art can be used for comparison to confirm the percentage of sequence similarity. For example, public computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) can be used for comparison. Correct. Those skilled in the art can determine the appropriate parameters for the measurement alignment, including any required algorithm to facilitate the alignment of the full-length sequence to achieve the maximum alignment value. For the purpose of this article, the sequence alignment between the two amino acid sequences is analyzed using the computer software Blastp (protein-protein BLAST) provided by the National Center for Biotechnology Information (NCBI) online. The percentage of sequence similarity between two sequences is a function, which is determined by the number of identical positions shared between the sequences (ie, sequence similarity% = number of identical positions /Number of all positions (for example, overlapping positions)×100). In some embodiments, the two sequences have the same length.

The term "linker" refers to a peptide that has natural or synthetic amino acid residues and is used to link two polypeptides. For example, the linker can be used to connect human IL-15 and albumin binding domain (ABD) or albumin binding peptide (ABP), or to connect ABD or ABP and 6His (ie, polyhistidine tag), according to To form the recombinant polypeptide of the present disclosure. Preferably, the linker is a peptide having a length of at least 5 amino acid residues, for example, 5 to 100 amino acid residues in length, and more preferably, the linker is a peptide having 10 to 30 amino acid residues. The length of the residue. In the recombinant polypeptide of the present disclosure, the linker is a peptide having a length of at least 5 amino acid residues, preferably a peptide having a length of 5 to 15 amino acid residues. Preferably, the linker includes a _{sequence of (G n} S) _m , where G is glycine, S is serine, and n and m are individually between 1 and 4. digital.

In the present disclosure, the terms "cancer" and "tumor" can be used interchangeably, and refer to a physiological condition in mammals (especially humans) where uncontrolled cell growth is usually feature. In this respect, cancer includes in situ cancer, metastases cancer, and/or drug-resistant cancer. Accordingly, cancers or tumors that can be treated by the present disclosure occur in the chest, lungs, colon, colon and rectum, spleen, kidney, liver, bladder, head and neck, ovaries, prostate, brain, pancreas, skin, bones Cancer or tumors in the blood, thymus, uterus, testicles, cervix and neurons.

The term "subject" or "patient" refers to an animal, including humans, that can be treated with the recombinant polypeptide, pharmaceutical composition and/or method of the present disclosure. The term "individual" or "patient" refers to both males and females, unless one gender is specifically indicated. Accordingly, the term "individual" or "patient" includes any mammal that can benefit from cancer treatment. Examples of "individuals" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry. Preferably, the individual is human.

The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition or carrier, for example, liquid or solid fillers, diluents, excipients, solvents Or encapsulating material, which involves carrying or delivering a subject agent from one organ or part of the body to another organ or part of the body. Various carriers must be "acceptable", in the sense that they are compatible with the other ingredients in the dosage form. The carrier can be in the form of a solid, semi-solid or liquid diluent, cream or capsule.

The term "a therapeutically effective amount" or "an effective amount" as used herein refers to a predetermined dose that can achieve the desired therapeutic effect, that is, within a necessary period (As described in the specific embodiment), it can be discouraged, combated, ameliorate, improved, prevented, inhibited, blocked, or reversed ( reverse) A condition, disease, or symptom that the patient does not want. For example, the "therapeutically effective amount" described in the implementation of "a method of treating" is a predetermined dose used to achieve the desired therapeutic effect, that is, it can block and resist , Alleviate or improve an unwanted condition, disease or symptom. For example, the "therapeutically effective amount" described in the implementation of "a method of preventing" is a predetermined dose used to achieve the desired therapeutic effect, that is, before it occurs, To prevent, inhibit, or hinder an unwanted condition, disease, or symptom. Therefore, the therapeutically effective amount can be a dose sufficient to make the patient necessarily exposed to the recombinant polypeptide of the present disclosure. Under appropriate circumstances, the expected activity of the method of the present disclosure includes two parts: medical treatment and/or prevention. The therapeutically effective amount of the recombinant polypeptide of the present disclosure generally refers to a dose that, when administered in a physiologically acceptable excipient composition, is sufficient to achieve an effective systemic concentration or locality in tissues. concentration.

The terms "administration" or "administration" (administered, administering, or administration) are used interchangeably herein, and refer to the direct administration of the recombinant polypeptide or pharmaceutical composition of the present disclosure, or the combination therapy described above (ie, a pharmaceutical package). group).

The term "treatment" (treatment or treating) as used herein can refer to a curative or palliative measure. Specifically, the term "treatment" as used herein refers to administering the recombinant polypeptide or pharmaceutical composition of the present disclosure to an individual, or including the aforementioned combined treatment (ie, a pharmaceutical kit), wherein the individual has cancer , Symptoms related to cancer, subsequent diseases or conditions of cancer, in order to achieve partial or complete alleviate, ameliorate, relieve, delay onset, and inhibit the course of the symptoms or symptoms of the one or more cancers , Reduce the severity, and/or reduce the incidence, etc.

As used herein, "synergistic effect" (synergistic effect) or its synonymous terms refers to and includes a cooperative action (cooperative action), which is usually encountered in a combination of two or more active ingredients, wherein the two or more The activity of the active ingredients when used in combination will exceed the sum of the activities of the individual active ingredients when used alone.

II. Detailed description of the invention

The present disclosure is based at least in part on the inventor’s discovery that when ABD or ABP polypeptide is linked to the N- or C-terminus of natural IL-15 (ie wild-type IL-15), the circulation of the natural IL-15 in the blood can be greatly extended In the half-life. Previous studies have shown that in some preclinical models, IL-15 can have anti-cancer activity ^{by enhancing the ability of anti-tumor CD8 + T cells.} However, the difficulty of using IL-15 to treat cancer is that IL-15 is rapidly degraded in the blood circulation. In view of this, the purpose of the present disclosure is to prolong the half-life of IL-15 by connecting ABD or ABP to IL-5 to protect IL-15 from degradation, thereby increasing its half-life in the blood circulation, and achieve Desired pharmacokinetic profile (profiling).

1. Recombinant peptides

The first aspect of the present disclosure is about a recombinant polypeptide comprising A first polypeptide having an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO:1; and A second polypeptide which is operably linked to the N- or C-terminus of the first polypeptide, wherein the second polypeptide has a sequence of at least 85% of SEQ ID NO: 2 or SEQ ID NO: 3 Amino acid sequence of similarity.

According to some embodiments of the present disclosure, the first polypeptide has an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO:1, for example, 85%, 86%, 87% and SEQ ID NO: 1 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity. In a specific embodiment, the first polypeptide has an amino acid sequence of SEQ ID NO:1. According to some embodiments of the present disclosure, the second polypeptide has an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO: 2, for example, 85%, 86%, 87% of SEQ ID NO: 2 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity. In one embodiment, the recombinant polypeptide of the present disclosure has an amino acid sequence of SEQ ID NO: 2. According to some embodiments of the present disclosure, the second polypeptide has an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO: 3, for example, 85%, 86%, and 85% sequence similarity to SEQ ID NO: 3 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity. In a specific embodiment, the second polypeptide has an amino acid sequence of SEQ ID NO: 3.

According to certain embodiments of the present disclosure, the recombinant polypeptide of the present disclosure may have certain modifications at its N-terminus, C-terminus, or both ends. For example, the N-terminus of the recombinant polypeptide of the present disclosure may have acetylation, fortylation, methylation, carbamylation, pegylation, phosphorylation, or glycosylation modification; in addition or alternatively The C-terminus of the recombinant polypeptide of the present disclosure may be modified with amidation, glycosylphospholipid inositolization, biotinylation, or glycosylation.

Alternatively or in addition, for different purposes, the recombinant polypeptide of the present disclosure may further comprise a third polypeptide which is operably linked to the N of the first or the second polypeptide. -Or C-terminal. The third polypeptide may be a reporter, and in this case, the reporter is selected from: β-galactosidase (lacZ), chloramphenicol acetyltransferase (CAT), luciferase, Green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Alternatively, the third polypeptide may be a selection marker, and in this case, the selection marker is selected from: β-endoamidase, aminoglycoside-3'-phosphotransferase (APH (3')) and orotidine-5'-phosphate decarboxylase (ODCase). According to some alternative embodiments, the third polypeptide may be a tag for affinity purification, and in this case, the tag for affinity purification is selected from: chitin binding protein (CBP), maltose binding Protein (MBP), streptomycin tag (Strep-tag), glutathione-S-transferase (GST), thioredoxin (TRX), albumin-binding protein (ABP), alkali Phosphatase (AP), Biotin-Carboxy Carrier Protein (BCCP), Calcium Carrying Peptide (CBP), Phage T7 Epitope (T7-Tag), Polyhistidine Tag, FLAG-Tag, Myc-Tag , HA-tags, Spot-tags, NE-tags, and their combinations. In a specific embodiment of the present disclosure, the third polypeptide is a polyhistidine tag (ie, 6His).

In certain embodiments, the recombinant polypeptide of the present disclosure includes the first, the second, and the third polypeptide in sequence, wherein each polypeptide is directly linked to its neighboring polypeptide. Optionally, each of the first, the second, and the third polypeptides is individually connected to each other by an appropriate linker, for example, the linker is a form of (G _n S) _m , where G Is glycine, S is serine, and n and m are each an integer between 1 and 5. In one embodiment, the linker includes a (G ₄ S) ₁ sequence. In another embodiment, the linker includes a (G ₄ S) ₂ sequence. In another embodiment, the linker includes a (G ₄ S) ₃ sequence.

Accordingly, in certain embodiments of the present disclosure, the recombinant polypeptide of the present disclosure has an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO: 4, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity. In a specific embodiment, the recombinant polypeptide of the present disclosure has an amino acid sequence of SEQ ID NO: 4. In certain embodiments of the present disclosure, the recombinant polypeptide of the present disclosure has an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO: 5, for example, 85%, 86%, SEQ ID NO: 5 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity. In one embodiment, the recombinant polypeptide of the present disclosure has an amino acid sequence of SEQ ID NO: 5. In still other embodiments of the present disclosure, the recombinant polypeptide of the present disclosure has an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO: 6, for example, 85% and 86% of SEQ ID NO: 6 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity. In a specific embodiment, the recombinant polypeptide of the present disclosure has an amino acid sequence of SEQ ID NO: 6.

2. Pharmaceutical composition

According to another aspect of the present disclosure, the present disclosure relates to a pharmaceutical composition for preventing and/or treating cancer in an individual in need. The pharmaceutical composition comprises the recombinant polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

At the dosage and concentration used, the acceptable carrier should be non-toxic to the recipient. According to some embodiments of the present disclosure, the pharmaceutically acceptable carrier can be any liquid, gel, cream, ointment, lotion, suspension, emulsifier (emulsion) , Adhesive, amniotic membrane, skin substitute, artificial skin or skin equivalents. In a specific embodiment, the pharmaceutically acceptable carrier of the pharmaceutical composition of the present disclosure is a liquid (such as phosphate buffers (PBS)).

Optionally, the pharmaceutical composition may further include an antioxidant (for example, ascorbic acid or methionine); and a preservative (for example, stearyl dimethyl benzyl ammonium chloride). (octadecyldimethylbenzyl ammonium chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol (butyl or benzyl alcohol) ; Alkyl parabens (alkyl parabens) (for example, methyl or propyl paraben (methyl or propyl paraben)); catechol (catechol); resorcinol (resorcinol); Cyclohexanol (cyclohexanol); 3-pentanol (3-pentanol); benzoates, sorbate and m-cresol); a low molecular weight (ie, about less Within 10 residues) polypeptide; a protein (for example, serum albumin, gelatin or immunoglobulin); a hydrophilic polymer (for example, polyvinylpyrrolidone); an amino acid (for example, polyvinylpyrrolidone); For example, glycine, glutamine, aspartic acid, histidine, arginine, serine, alanine or lysine); monosaccharides, disaccharides, and other carbohydrates (such as , Glucose, mannose or dextrans); a chelating agent (for example, EDTA); a sugar (for example, sucrose, mannitol, trehalose or sorbitol); Salt-forming counter-ion (for example, sodium); a metal ion complex (for example, zinc-protein complex); a nonionic surfactant (for example, TWEEN™) , Poloxamers (PLURONIC™), polyethylene glycol (PEG); or a combination thereof.

The pharmaceutical composition of the present disclosure can be made into a unit dose form, for example, a tablet, pill, capsule, powder, granule, gel, solution or suspension, or suppository, It is used to administer by oral, parenteral or rectal route, or by inhalation or inhalation, intrathecal or intracerebral route. In one embodiment, the pharmaceutical composition of the present disclosure is in the form of a solution.

According to certain embodiments of the present disclosure, the weight of the recombinant polypeptide of the present disclosure accounts for about 0.1% to 99% of the total weight of the pharmaceutical composition of the present disclosure. In some embodiments, the weight of the recombinant polypeptide of the present disclosure accounts for at least 1% of the total weight of the pharmaceutical composition of the present disclosure. In some embodiments, the weight of the recombinant polypeptide of the present disclosure accounts for at least 5% of the total weight of the pharmaceutical composition of the present disclosure. In still other embodiments, the weight of the recombinant polypeptide of the present disclosure accounts for at least 10% of the total weight of the pharmaceutical composition of the present disclosure. In still other embodiments, the weight of the recombinant polypeptide of the present disclosure accounts for at least 25% of the total weight of the pharmaceutical composition of the present disclosure.

The pharmaceutical composition of the present disclosure can be prepared by any method well known in the field of pharmacology. Generally, such preparation methods include bringing the recombinant polypeptide of the present disclosure (ie, the "active ingredient") into a carrier or excipient, and/or one or more other auxiliary ingredients, and then, as required The product is shaped and/or packaged to make a desired single-dose or multi-dose unit. A "unit dose" (unit dose) refers to a discrete pharmaceutical composition dose, which contains a predetermined dose of the active ingredient. The amount of the active ingredient (amount) is usually equal to the dose of the active ingredient administered to a body and/or a practical fraction of such a dose, for example, one-half or one-third of such a dose One.

Recombinant polypeptides of the present disclosure are usually prepared in a unit dosage form to facilitate the administration of dosage and maintain uniformity. However, it should be understood that the medical staff can determine the total daily dosage of the pharmaceutical composition of the present disclosure within the scope of reasonable medical judgment to achieve a certain medical effect. The specific therapeutically effective amount for a specific individual or organism will vary depending on various factors, including the disease to be treated and the severity of the disease; the activity of the specific active ingredient used; the composition used; the species and age of the individual , Weight, general health, gender and eating habits, severity of side effects or drug abnormalities; time of administration, route of administration, and rate of discharge of active ingredients; duration of treatment; recombinant polypeptide with the present disclosure and used in a pharmacy The identity of the specific active ingredient in the composition; and other factors known in the medical field.

Therefore, the present disclosure also covers the use of a recombinant polypeptide of the present disclosure in preparing a pharmaceutical composition, wherein the pharmaceutical composition is used to treat cancer in an individual in need.

Optionally, the pharmaceutical composition of the present disclosure may further include a primary anti-PD-L1 antibody. Exemplary anti-PD-L1 antibodies used in the pharmaceutical composition of the present disclosure include, but are not limited to, atezolizumab (atezolizumab; MPDL3280A, RO5541267, Tecentriq®), avelumab (avelumab; Bavenciq®) , Durvalumab (durvalumab, MEDI4736, Imfinzi®), anti-PD-L1 antibody (from the 10F.9G2 cell line), or a multi-strain antibody (ie antiserum) produced by the usual steps of preparing monoclonal antibodies The anti-PD-L1 antibody, or an anti-PD-L1 antibody prepared by total gene synthesis, by combining the desired DNA sequence (for example, VH, VL, Fc, signal peptide, IRES) , Linker, etc.) grafted or fused to the desired construct to prepare. In a preferred embodiment of the present disclosure, the anti-PD-L1 antibody system is derived from the 10F.9G2 cell line.

3. Methods to treat cancer

Another aspect of the present disclosure is to provide a method for preventing and/or treating cancer in an individual in need. The method comprises administering to the individual a therapeutically effective amount of the aforementioned recombinant polypeptide or a pharmaceutical composition comprising the recombinant polypeptide of the present disclosure.

The recombinant polypeptide or pharmaceutical composition of the present disclosure is helpful for preventing and/or treating cancer of an individual in need (for example, a human patient suffering from cancer, suspected of suffering from cancer, or at risk of suffering from the cancer). In certain embodiments, cancers that can be treated by the methods of the present disclosure include, but are not limited to, bladder cancer, bile duct cancer, bone cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, colon cancer, and esophageal cancer. , Epithelial cancer, gastric cancer, gastrointestinal stromal tumor (GIST), glioma, lymphoid system hematopoietic tumor, liver cancer, non-Hodgkin's lymphoma, Kaposi's sarcoma, blood cancer, lung cancer, lymphoma, small intestine Cancer, melanoma, myelogenous leukemia, pancreatic cancer, prostate cancer, retinoblastoma, ovarian cancer, renal cell carcinoma, spleen cancer, squamous cell carcinoma, thyroid cancer, or thyroid follicular cancer. In one embodiment of the present disclosure, the cancer is colon cancer. In another embodiment of the present disclosure, the cancer is melanoma. The cancer can be a carcinoma in situ or metastatic carcinoma.

In the methods of the present disclosure, the recombinant polypeptide or pharmaceutical composition of the present disclosure can be administered by appropriate methods known in the art, including: oral, intracranial, intraspinal, intrathecal, intramedullary, intracerebral, and intracerebroventricular , Intravenous, intraarterial, intracardiac, intradermal, subcutaneous, transdermal, intraperitoneal or intramuscular administration. In general, most of the appropriate route of administration will depend on various factors, including: the nature of the agent (for example, its stability in the blood circulation), and/or the individual's condition (for example, whether the individual is Can tolerate intraperitoneal or intravenous (iv.) administration of drugs) and so on.

In order to achieve the precise dosage of the effective amount of the recombinant polypeptide or pharmaceutical composition of the present disclosure, it will vary from individual to individual, depending on the above-mentioned related factors. A therapeutically effective dose can encompass a single dose (for example, a single intraperitoneal injection dose, or a single intravenous injection dose), or multiple doses (for example, multiple intraperitoneal injection doses, or multiple intravenous injections). Injection volume). In certain embodiments, when multiple doses are administered to an individual, the frequency of administering the multiple doses to the individual is three doses a day, two doses a day, one dose a day, one dose every two days, and every three days. One dose, one dose per week, one dose every two weeks, one dose per month, or one dose every two months. In certain embodiments, the frequency of administration of the multiple doses to the individual is one dose per day. In certain embodiments, the frequency of administration of the multiple doses to the individual is one dose every two days (or three doses per week). In certain embodiments, the frequency of administration of the multiple doses to the individual is one dose every three days (or two doses per week). In some embodiments, when multiple doses are administered to an individual, the period between the first and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks , One month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, two Ten years, or the individual's life. In certain embodiments, the period between the first and last dose of the multiple dose is three months, six months, or one year. In certain embodiments, the period between the first and last dose of the multiple dose is the individual's lifetime. In a specific embodiment, the period between the first and last dose of the multiple doses is three weeks.

In some embodiments, one dose described herein (for example, a single dose or any one of multiple doses) individually contains a dose between about 8 nanograms and 80 micrograms, and the dose includes Including the recombinant polypeptide or pharmaceutical composition of the present disclosure, for example, the dose is about 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 nanograms, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 micrograms. In some embodiments, one of the doses described herein individually comprises a dose between about 40 nanograms and 8 micrograms, which dose includes the recombinant polypeptide or pharmaceutical composition of the present disclosure. In certain embodiments, one of the doses described herein individually comprises a dose between about 80 nanograms and 800 nanograms, which dose includes the recombinant polypeptide or pharmaceutical composition of the present disclosure. In certain embodiments, one of the doses described herein individually contains a dose between about 200 nanograms and 500 nanograms, which dose includes the recombinant polypeptide or pharmaceutical composition of the present disclosure. In certain embodiments, one of the doses described herein individually contains a dose between about 270 nanograms and 410 nanograms, which dose includes the recombinant polypeptide or pharmaceutical composition of the present disclosure.

The methods of the present disclosure may further comprise administering one or more additional therapies to the individual before, at the same time or after administering the recombinant polypeptide or pharmaceutical composition of the present disclosure to the individual. The additional therapy is helpful to prevent and/or treat cancer, for example, a surgery, a radiotherapy, a chemotherapy, an immunotherapy, a hormonal therapy, a target therapy, a heat therapy, or a combination therapy . In some embodiments, the combined use of the pharmaceutical composition of the present disclosure and the additional therapy will exhibit a synergistic effect for cancer treatment. When this type of combination therapy is administered during the current treatment period, different therapies can be administered to the cancer patient via different routes at different time intervals, depending on the dose and/or schedule of the therapy. For example, certain immunotherapies (eg, antibodies, interferons, and interleukins) are usually given at intervals of one, two, three, or four weeks. For example, the anti-PD-L1 antibody (from the 10F.9G2 clone) is given once every three days (or two doses per week), intraperitoneally, while the pharmaceutical composition of the present disclosure is usually once every two days. (Or three doses a week), intraperitoneal administration.

Non-limiting examples of chemotherapeutic agents include, but are not limited to, actinomycin D, aminoglutethimide, amsacrin, anastrozole, anthracycline ), bexaroten (bexaroten), bleomycin (bleomycin), buserelin (buselerin), butacaine sulfate (busulfan), camptothecin derivatives (camptothecin derivates), capecitabine (capecitabine) ), carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine (cytarabine or cytosinarabinoside), dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, Estramustine, etoposide, exemestane, fludarabine, fluorouracil, formestane, gemcitabine, gosere Goselerin, topotecan (hycamtin), idarubicin, ifosfamide, imatinib, irinotecan, Letrozole, leuprorelin, lomustine, melphalan, mercaptopurine, methotrexate, miltefosin , Mitomycin, mitoxantrone, nimustine, oxaliplatin, paclitaxel, pentostatin, procarbazine (procarbazine), temozolomide (temozolomide), teniposide (teniposide), testosterone ( testolactone), thiotepa, thioguanine, treosulfan, tretinoin, triptorelin, trofosfamide, vinblastine ( vinblastine, vincristine, vindesine, and vinorelbine.

Examples of non-limiting immunotherapeutics include, but are not limited to, anti-PD-1 antibodies (e.g., pembrolizumab (Keytruda®), nivolumab (nivolumab; Opdivo®)); anti-PD-L1 antibodies ( For example, atezolizumab (atezolizumab; MPDL3280A, RO5541267, Tecentriq®), avilumab (avelumab; Bavenciq®), devaluzumab (durvalumab; MEDI4736, Imfinzi®), antibodies from 10F.9G2 clones ); anti-CTLA-4 antibodies (for example, ipilimumab (ipilimumab; Yervoy®), tremelimumab (tremelimumab; CP-675,206)), riluzole, trigriluzole , IFN-γ, IL-2, IL-15, IL-23, M-CSF, GM-CSF, tumor necrosis factor (TNF), lipid A, CpG, CD80, CD86, and ICAM-1. In a preferred embodiment of the present disclosure, the immunotherapeutic agent is an anti-PD-L1 antibody (for example, an antibody from a 10F.9G2 clone).

Examples of non-limiting hormonal therapeutics include, but are not limited to, prednisone, methylprednisolone (Solumedrol®), dexamethasone (Decadron®), tamoxifen (tamoxifen ; Nolvadex®), leuprolide (Leuplin®), and cyproterone acetate (Androcur®).

Non-limiting examples of targeted therapeutic agents include, but are not limited to, gefitinib (gefitinib; ZD1839), erlotinib HCl (Tarceva™), CL-387,785, EKB-569, HKI-272, Trastuzumab (trastuzumab; Herceptin®), pyrazolopyridine, benzoxepin, adenine derivatives, and 2-carboxamide cyclic aminourea derivatives ( 2-carboxamide cycloamino urea derivatives).

According to certain embodiments of the present disclosure, the individual that can be treated by the pharmaceutical composition of the present disclosure is a mammal. In one embodiment, the individual is a mouse. In another embodiment, the individual is a human.

The present disclosure also covers a kit for treating any type of cancer in the present disclosure. The kit of the present disclosure may comprise a recombinant polypeptide or pharmaceutical composition of the present disclosure. Optionally, the kit may further include one or more additional pharmaceutical agents described in this disclosure.

A number of embodiments are presented below to illustrate certain aspects of the present disclosure, so as to facilitate those skilled in the art to which the present invention belongs to practice the present invention. These embodiments should not be regarded as limiting the scope of the present invention. It is believed that those with ordinary knowledge in the art to which the present invention pertains can fully utilize and practice the content of this disclosure without excessive interpretation after reading the description presented here. All publications cited herein are hereby incorporated by reference in their entirety.

Example

Materials and methods

1. Cells and animals

The mouse T-cell lymphoma cell line CTLL-2 and the mouse colon cancer cell line CT26 were cultured in the basic medium of Roswell Park Memorial Institute (RPMI)-1640, and the mouse melanin was added The tumor cell line B16F10 was cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal medium. All basal media are supplemented with 10% fetal bovine serum (FBS), 2 milli volume molar concentration of L-glutamine (L-glutamine), 100 units/ml penicillin and 100 micrograms/ml Streptomycin, sodium pyruvate at a molar concentration of 1 millivolume, and 10% T-STIM containing Concanavalin A (Con A), and the cells were cultured at 37°C with 5% CO ₂ In a wet incubator.

Male BALB/c and C57BL/6J mice about 5 or 6 weeks old were used in this study. All animals are kept in animal facilities, and control temperature (20-24°C), humidity (50-80%) and 12 hours/12 hours light/dark cycle, and provide food and water that can be eaten at will .

2. Construct the performance carrier of IL-15, IL-15-ABD and IL-15-ABP

The nucleic acid sequences used to express human IL-15-6His (SEQ ID NO: 4), human IL-15-ABD (SEQ ID: 5), and human IL-15-ABP (SEQ ID: 6) recombinant polypeptides were inserted into an E. coli (Escherichia coli, E. coli) in the expression plasmid pET56. These constructs were then sent to cells of the Escherichia coli expression strain Rosetta gami B (DE3). Then, the colonies of Escherichia coli that can successfully express the recombinant polypeptides are selected and amplified by standard Escherichia coli culture procedures to obtain a large amount of recombinant polypeptides. The steps of recovering proteins from E. coli inclusion bodies are carried out according to methods well known in the art, including the purification of inclusion bodies and the refolding of proteins. In all examples of the present disclosure, human IL-15-6His is used as the IL-15 control group. Therefore, for convenience, the terms "IL-15", "IL-15-ABD" and "IL-15-ABP" are individually used to represent human IL-15-6His, human IL-15-ABD and human IL-15-ABP.

3. Pharmacokinetics of IL-15, IL-15-ABD and IL-15-ABP

The pharmacokinetics of the recombinant polypeptides of the present disclosure are confirmed using mouse models in vivo. The BALB/c mice (n=9) were equally divided into 3 groups. IL-15 (3.33 micrograms/dose), IL-15-ABD (5 micrograms/dose) and IL-15-ABP (4.5 micrograms/dose) were injected intraperitoneally into mice, and the mice were injected at 0, Mouse blood was collected at 15, 30, 45, 60, 90, 120, 240, 480, 1,440, 2,160 and 3,120 minutes. The IL-15 paired enzyme immunoassay kit (IL-15 Duoset ELISA kit, R&D system) was used to quantify the IL-15 concentration in the serum.

4. In vitro binding test of recombinant polypeptide to mammalian albumin

Enzyme immunosorbent assay (ELISA) was used to confirm the binding of recombinant polypeptides (including IL-15, IL-15-ABD, and IL-15-ABP) of the present disclosure to human, dog, and mouse albumin. Spread albumin of a specific species in a 96-well microplate, in which the albumin is dissolved in ELISA coating buffer (coating buffer; 137 millivol NaCl with a molar concentration, 2.7 millivol KCl with a molar concentration, 10 millivol Na ₂ HPO ₄ at a molar concentration and KH ₂ PO _{4 at a} molar concentration of 1.8 milliliters were prepared to a final concentration of 300 ng/ml, and reacted overnight at 4°C. Each well was washed, and then blocked with 3% skimmed milk (dissolved in PBS) at 25°C for 2 hours. After washing, a specific concentration of the recombinant polypeptide of the present disclosure was added to each well and reacted at 25°C for 2 hours. Then wash the microplate, and react with an anti-6His antibody complexed with HRP (1:2000 dilution) at 25°C for 1 hour. Use 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) to detect HRP activity and terminate _{it with 0.5 volume molar concentration of H 2} SO ₄ reaction. Measure the absorbance of the microplate at 450nm with a microplate analyzer.

5. CTLL-2 proliferation test

The mouse cytotoxic T lymphocyte CTLL-2 cells that had been starved in IL-2 for 2 hours were cultured in a 96-well microplate, and serially diluted human IL-2 or IL15-ABD (dissolved in complete medium in RPMI) was added to the microplate. The cells were reacted at 37°C for 15 minutes, and then immediately fixed with 1.5% formaldehyde at room temperature (RT) for 10 minutes, and then permeabilized with ice-cold 100% methanol at 4°C for 30 minutes. The cells were washed twice with FACS buffer (0.8% bovine serum albumin (dissolved in PBS, pH 7.2)), and then washed with anti-STAT5 pY694 antibody (ThermoFisher) (1:50 diluted in FACS buffer) at RT React for 2 hours. Wash the cells, and then add a goat anti-rabbit Fc antibody (Jackson) compounded with FITC to the cells. Use a flow cytometer (CytoFLEX, Beckman-Coulter) to confirm the mean fluorescence intensity of the cells. , MFI).

6. Animal mode

Before establishing the animal model of melanoma, first establish a melanoma cell line with bioluminescence (named B16F10/luc2). For this purpose, a vector containing CMV-luciferase 2 (CMV-luciferase2, pGL4.50[luc2/CMV]) is used. B16F10 cells were transfected with 5 micrograms of the above vector. After one day of reaction, 200 μg/ml of hygromycin B (hygromycin B) was added to the petri dish and cultured for another two weeks to obtain cells expressing Luc2 (ie, B16F10/luc2).

During the surgical operation, the animals were anesthetized with 1-2% isoflurane. In the animal model of melanoma or colon cancer, 5×10 ⁴ B16F10/luc2 or CT26 cells were injected subcutaneously (sc.) into the right thigh of C57BL/6 or BALB/c mice (n=18; 20 -25 grams). Mice were injected intraperitoneally with PBS, wild-type IL-15 (600,000 international units (IU)) or IL-15-ABP (600,000 international units); or injection control group (0.1% DMSO dissolved in H ₂ O) , IL-15 (5 micrograms/time) or IL-15-ABD (5 micrograms/time), twice a week for 3 weeks. The tumor size was measured with calipers three times a week, and the tumor volume was calculated with the following formula: (0.523)×L×W ² .

In the part of the animal model of metastatic colon cancer, BALB/c mice (n=9) were equally divided into 3 groups. On day 0, 5×10 ⁵ CT26 cells were injected intravenously into mice via the tail vein. PBS, IL-15 (3.33 micrograms), and IL-15-ABD (5 micrograms) were administered to mice by intraperitoneal injection on day 1, and subcutaneous injection on days 3, 6, and 9. On day 19, the mice were sacrificed and tumor metastasis in the lungs was observed. All mouse lungs were removed, fixed with formaldehyde, and photographed. At the same time, count the number of tumor masses in the lungs.

In the combined treatment of IL-15-ABD and anti-PD-L1 antibody, B16F10/luc2 and CT26 cells were respectively inoculated into mice and allowed to grow for about 2 weeks. When the tumor reached 50-80 cubic millimeters, the mice were then randomly divided into four groups. The mice were given the following treatments: (1) control group (0.1% DMSO dissolved in H ₂ O, intraperitoneal injection); (2) IL-15-ABD group (IL-15-ABD (5 micrograms) three times a week) /Time), intraperitoneal injection); (3) Anti-PD-L1 group (anti-PD-L1 antibody (from 10F.9G2 strain) (100 micrograms/time), intraperitoneal injection twice a week); and ( 4) Combined treatment group (IL-15-ABD (5 micrograms/time), plus anti-PD-L1 antibody (from 10F.9G2 clone; 100 micrograms/time), each dosage is as described above). On the 21st day, the tumors in each group were removed and photographed. The tumor volume was assessed with calipers 3 times a week.

7. Assess immunosuppressive cells (T _reg cells and bone marrow-derived suppressor cells (MDSC)) in mice

On the 21st day, the tumor-draining lymph node (TDLN), spleen and femoral bone marrow of the mice were taken out, and red blood cells (RBC) were dissolved with ACK buffer. The cell populations containing T _reg cells (CD4 ⁺ /CD25 ⁺ /FOXP3 ⁺ ) or MDSC (CD11b ⁺ /GR-1 ⁺ ) were treated with anti-FOXP3-Alexa Fluor 488/anti-CD4-PerCP-Cy™5.5/anti-CD25, respectively -PE antibody, or anti-CD11b-FITC/anti-Gr-1-PE antibody (BD Bioscience) for staining, and then use flow cytometry (NovoExpress®) to confirm _{the percentage of T reg} cells or MDSC in the cell population . Use FlowJo software (Tree Star) to analyze the obtained data.

8. Evaluation of immune cells (CD8 ⁺ T cells and NK cells) in mice

To evaluate CD8 ⁺ T cells, proceed as follows. Three to four weeks after tumor inoculation, the mice were euthanized, and their tumors, ascites, TDLN and spleen were collected. In the part where the white blood cells are separated from the tumor, the tumor tissue is placed in 2.5 mg/ml collagenase IV (collagenase IV; Worthington) for 20 minutes, and then a silica gel particle (percoll) coated with vinylpyrrolidone is used for discontinuous density Separated by gradient centrifugation (GE Healthcare). Use anti-CD8 microbeads (Miltenyi Biotec) and a MACS column to separate CD8 ⁺ T cells in white blood cells. In the white blood cells, CD8 ⁺ T cells account for a percentage of >5%. Anti-CD8-FITC staining was used to confirm CD8 ⁺ T cells in TDLN and spleen. To evaluate the ^{expression level of IFN-γ or IL-2 in CD8 +} T cells, the cells were further stained with anti-IL-2-PerCP/Cy5.5 or anti-IFN-γ-PE antibodies (BD Bioscience). Use flow cytometry to detect the expression levels of IL-2 and IFN-γ in the cells to confirm the activation of ^{CD8 + T cells.} The total number of infiltrating CD8+ T cells per gram of tumor is ^{calculated by multiplying the percentage of CD8+} T cells by the total number of lymphocytes, and then dividing the number by 100 and the weight of the tumor.

To evaluate NK cells, follow the steps below. The mouse spleens were collected on the 21st day after tumor inoculation, and spleen cells were lysed and purified with ACK buffer. In the BALB/c part, it was stained with anti-CD3-FITC/anti-CD49b-PE/anti-CD335-PerCP-Cy5.5 antibody, and in the C57BL/6 part, it was stained with anti-CD3-FITC/anti-CD49b- PE/anti-NK1.1-PerCP-Cy5.5 antibody was stained to confirm the NK cells in the spleen. Has ^{CD3 - / CD49b +, CD3 -} / CD335 +, CD3 - / CD49b + / CD335 +, and ^{CD3 - / CD49b +, CD3 -} /NK1.1 +, CD3 - / CD49b + /NK1.1 + is labeled reporter NK cell population. Analyze by flow cytometry (NovoExpress®) and analyze relevant data by FlowJo software to obtain the percentage of these cell types.

9. Bioluminescence imaging

10 minutes before the contrast, each group of mice bearing B16F10/luc2 tumors (n=10) were injected intraperitoneally with 200 microliters of 150 mg/kg D-luciferin (D-luciferin; dissolved in PBS ), and use 1-2% isoflurane to anesthetize it. The mouse was then placed on the imaging platform and exposed to 1-2% isoflurane continuously during the entire image capture time. Once a week, a bioluminescence imaging system (IVIS50 Imaging System; Xenogen) was used to collect the luc2 signal, and the time to capture the image was 1 minute. Regions of interest (ROI) is approximately near the tumor and is quantified by Living Image (unit: photons/s/cm²/steradian (photons/s/cm2/sr)) .

10. Immunohistochemistry (IHC) staining

The mouse tissues fixed with formalin and embedded in paraffin were subjected to IHC staining as follows. The paraffin-embedded tumor tissue sections were placed on a glass slide, deparaffinized with xylene, and rehydrated with ethanol (0, 30, 70, 100% H2O) with successively reduced concentrations, and then 3 React in %H ₂ O ₂ for 10 minutes. After washing, the slides were blocked with 5% normal goat serum for 5 minutes, and then specific primary antibodies (dilution ratio 1:100-500) were added to react overnight at 4°C. Finally, the slides were stained with hematoxylin for comparison. Take at least three slides for IHC staining in each group. An optical microscope (Nikon ECLIPSE Ti-U) was used to obtain an image of the slide under 200× magnification, and the image was quantified with ImageJ software.

11. Assess the amount of secreted protein in the serum

Collect mouse serum, and use enzyme immunoassay to detect secreted proteins IL-15 and VEGF according to the instructions of the individual enzyme immunoassay detection kit (Elabscience). A microplate analyzer (SpectraMax iD3 microplate reader; Molecular Devices) was used to measure the OD450 reading of the enzyme immunoassay.

12. Statistical analysis

Use Excel 2017 software (Microsoft) for statistical analysis. The experimental data was expressed as the mean±standard deviation (mean±SD), and the data between the two groups were analyzed and compared by single-factor analysis of variance (ANOVA) and independent Stuton's t test method. The experiment was repeated at least three times. P<0.05 and P<0.01 were regarded as statistically significant. a1: p<0.05, compared with control group; a2: p<0.01, compared with control group; b1: p<0.05, compared with single treatment; b2: p<0.01, compared with single treatment.

Example 1 Confirm the recombinant polypeptide of the present disclosure

1.1 The pharmacokinetics of recombinant peptides in this disclosure

The structure of the recombinant polypeptides of the present disclosure (including IL-15, IL-15-ABD and IL-15-ABP) are shown in Figure 1A; and the pharmacokinetic results of each recombinant polypeptide are shown in Figures 1B and 1C. The results of the experiment found that IL-15-ABD can last for about 3,000 minutes in blood circulation, while the concentration of IL-15 at 3,000 minutes is about 10,000 picograms/ml (Figure 1B). Based on this calculation, the overall half-life of IL-15-ABD is approximately 552.7 minutes. As for IL-15-ABP and IL-15, they can last about 1,440 minutes and 240 minutes in the blood circulation respectively (Figure 1C). Therefore, the overall half-lives of IL-15-ABP and IL-15 are calculated individually to be approximately 560 minutes and 44 minutes. All pharmacokinetic data are shown in Table 1.

Table 1 Pharmacokinetics of recombinant polypeptides of the present disclosure Half-life (minutes) AUC (μg/ml/min) IL-15 44 1.021 IL-15-ABD 552.7 48.73 IL-15-ABP 560 8.505

1.2 Biological activity of the recombinant polypeptide of Example 1.1

In order to study the binding ability of the recombinant polypeptides of Example 1.1 (including IL-15, IL-15-ABD and IL-15-ABP) to serum albumin, each recombinant polypeptide was cultured in albumin containing human, dog or mouse In the serum, the experimental results are shown in Figures 2A to 2C. Compared with IL-15, IL-15-ABD and IL-15-ABP both show binding activity to serum albumin, but the degree of binding is different. On the whole, compared with IL-15-ABP, different concentrations of IL-15-ABD will continue to show a greater degree of binding ability to serum albumin. For example, IL-15-ABD at a concentration of 100, 200, or 300 nanomolar (nM) has a higher binding capacity to human serum albumin than IL-15-ABP at the same concentration. _{The dissociation constant (K d} ) of IL-15-ABD for human serum albumin is about 3.040 nanomolar concentration, while IL-15-ABP is about 169.3 nanomolar concentration. Similar experimental results can be found in the experimental results of dog serum albumin and mouse serum albumin. The dissociation constant of IL-15-ABD for dog serum albumin is about 2.252 nanomolar concentration, while IL-15-ABP is about 78.44 nanomolar concentration. In addition, the dissociation constant of IL-15-ABD for mouse serum albumin is about 2.793 nanomolar concentration, while IL-15-ABP is about 33.73 nanomolar concentration. The combined data is shown in Table 2.

Table 2 Binding data of the recombinant polypeptide of Example 1.1 with mammalian serum albumin Human albumin Nai volume molar concentration IL-15 IL-15-ABD IL-15-ABP Maximum binding value (Bmax) -0.002 1.615 0.961 Dissociation constant (Kd) 0.0579 3.040 169.3 Dog albumin Nai volume molar concentration IL-15 IL-15-ABD IL-15-ABP Maximum binding value (Bmax) 0.049 1.275 0.599 Dissociation constant (Kd) 0.024 2.252 78.44 Mouse albumin Nai volume molar concentration IL-15 IL-15-ABD IL-15-ABP Maximum binding value (Bmax) ^* N/D 1.401 0.718 Dissociation constant (Kd) ^* N/D 2.793 33.73 *N/D: Not detected.

In order to study whether the recombinant polypeptide of Example 1.1 still retains the biological activity of its natural counterpart, the ability of the recombinant polypeptide of Example 1.1 to stimulate cell growth was tested. As shown in Figure 2D, IL-2 is used here as a positive stimulus to induce cell growth, and the relationship between different concentrations of IL-2 and the corresponding cell growth (expressed in OD450 values) is plotted as one Standard curve for reference. According to the standard curve of IL-2, the ability of IL-15 (5 ng/ml) and IL-15-ABP (5 ng/ml) to stimulate cell growth corresponds to 8.1 units/ng (U/ng) And 5.2 units/nanogram, which means that IL-15-ABP is similar to its natural counterpart and can stimulate cell growth.

Another biological activity is to detect whether the recombinant polypeptide can phosphorylate STAT-5. The experimental results are shown in Figure 2E. In CTLL-2, the phosphorylation of STAT-5 caused by IL-15-ABD showed a dose-dependent manner. In other words, the experimental results found that the cells had a higher concentration of IL. -15-ABD will make STAT-5 have a higher degree of phosphorylation.

In short, the data of this example confirms that the recombinant polypeptide of Example 1.1 has the effect of prolonging the half-life, while maintaining its binding specificity and related biological activities.

Example 2 The efficacy of the recombinant polypeptide of Example 1.1 on tumor growth and tumor metastasis

In this example, two animal models of melanoma and colon cancer were used to study the ability of the recombinant polypeptide of Example 1.1 to inhibit tumor growth and tumor metastasis.

2.1 The recombinant polypeptide of Example 1.1 can inhibit the growth of melanoma and tumor metastasis

In the melanoma model, compared to the PBS control group and IL-15 (Figure 3A), IL-15-ABP showed a significant inhibition of tumor growth, and the tumor size in the IL-15-ABP group was about 500 mm3 The tumor size of both the PBS group and the IL-15 group was about 1,000 cubic millimeters. That is, the tumor size in the IL-15-ABP group was about half of that of the PBS and IL-15 groups.

In another batch of experiments, the study included the PBS control group, IL-15 (60,000 international units), IL-15 (200,000 international units), IL-15-ABP (60,000 international units) and IL-15-ABP (200,000 International Units) of therapeutic efficacy. As shown in Figure 3B, compared with the PBS control group, IL-15 (both doses of 60,000 IU and 200,000 IU, respectively) showed partial inhibition of tumor growth; while the high dose (200,000 IU) of IL- 15-ABP showed a higher degree of inhibition of tumor growth.

A live imaging system was used to record the metastasis of melanoma to the small intestine of mice. The experimental results are provided in Figure 3C. The photon count in the abdomen of the mouse indicates that the metastatic melanoma is located in the small intestine of the mouse. On the 12th day (D12), compared with the IL-15-ABP group, the metastatic tumors in the PBS group were more obvious. Similar experimental results can be seen on D19 and D28. At D28, all mice in the PBS group had severe tumors in the intestines, but in the IL-15-ABP group, only very few tumors were detected in the intestines of the mice.

On D28, the mice were sacrificed, and the tumor weight of the tumor in the intestine was measured. As shown in Figure 3D, the tumor weight in the PBS group was about 6 grams, while the tumor weight in the IL-15-ABP group was about 0.5 grams. In this experiment, the body weight (BW) and tumor weight (TW) of the animals to be tested were recorded, and the TW/BW ratio of each group was calculated (Table 3). The mice in the PBS group had larger tumors as a whole, with a TW/BW ratio ranging from 21.21 to 31.03, except for one mouse with a smaller tumor (its TW/BW ratio was about 7.66). In contrast, mice in the IL-15-ABP group had smaller tumors, in which their TW/BW ratio fell within the range of 0.00-0.21, except for one mouse whose TW/BW ratio was about 10.07, but still It is significantly lower than the TW/BW ratio of the PBS group. In summary, the recombinant polypeptide of Example 1.1 can inhibit the growth of melanoma and tumor metastasis.

Table 3 Body weight and tumor weight of the animals to be tested treatment Weight (g) Tumor weight (g) TW/BW(%) PBS 27.77 5.89 21.21 24.90 6.96 27.95 26.65 8.27 31.03 24.68 1.89 7.66 IL-15-ABP 23.94 2.41 10.07 21.60 0 0.00 24.22 0 0.00 23.90 0.05 0.21

2.2 The recombinant polypeptide of Example 1.1 inhibits the growth and metastasis of colon cancer

This experiment also tested the ability of the recombinant polypeptide of Example 1.1 to inhibit colon cancer growth and tumor metastasis. In order to evaluate the effect of the recombinant polypeptide of Example 1.1 on the growth of colon cancer, mice carrying CT26 tumor cells were given specific treatment. The experimental results showed that the tumor volume in the IL-15-ABD group was smaller than that on the 9th day after treatment. Tumor volume in the control group or IL-15 group (Figure 4A). In addition, the tumor weight was measured on the 18th day after treatment, and it was shown that mice in the IL-15-ABD group had the lowest tumor weight, which was about one-third or less of the tumor weight in the control group or IL-15 group One-half (Figure 4B).

Furthermore, this experiment also examined the ability of the recombinant polypeptide of Example 1.1 to inhibit colon cancer tumor metastasis. The number of tumor clumps in the lungs of the mice that were given a specific treatment was counted. The results of this experiment are shown in Figure 4C. The number of tumor clumps in the IL-15-ABD group was significantly lower than that of PBS and IL- 15 groups. In addition, in this experiment, the number of tumor masses in the lung, colon, and liver of the mice were counted. The results of the experiment are shown in Figure 4D. Similar to the experimental results in Figure 4C, the results in Figure 4D also confirmed that the number of tumor clumps in the IL-15-ABD group was significantly lower than that in the PBS or IL-15 group. In summary, these data show that IL-15-ABD can inhibit colon cancer growth and tumor metastasis more effectively than IL-15.

2.3 Changes in immune cell population after treatment

In order to analyze the mechanism behind the recombinant polypeptide of Example 1.1 exerting its tumor growth inhibitory effect, especially for the part that undergoes immune regulation, this experiment focuses on the study of changes in immune cell populations.

First, use flow cytometry to detect one of the immunosuppressive cells, MDSC, labeled CD11b ⁺ /GR-1 ⁺ . As shown in Figures 5A to 5B, compared to the control group and the IL-15 group, the IL-15-ABD group in the bone marrow and the spleen had a decrease in the percentage ^{of CD11b +} /GR-1 ^{+ cell populations.} This experiment also detects another type of immunosuppressive cell T _reg cells, the flag is labeled CD4 ⁺ /CD25 ⁺ /FOXP3 ⁺ . Compared with the control group and IL-15 group, in the IL-15-ABD group of TDLN and spleen, the CD4 ⁺ /CD25 ⁺ /FOXP3 ⁺ cell populations of the two were significantly reduced (Figures 5C to 5D). Furthermore, it has been noticed that the _{amount of T reg} cell population in the IL-15-ABD group is reduced to one-half of the amount in the control group, which is also lower than the amount in the IL-15 group.

On the other hand, this experiment also detects two kinds of immune cells, including CD8 ⁺ T cells and NK cells, which represent adaptive immunity and innate immunity, respectively. In ^{the part of CD8 +} T cells, it was found that in the IL-15-ABD group of TDLN and spleen, the ^{total population of CD8 +} T cells of both increased, and the increase was about twice that of the control group (5E To Figure 5F). In addition, the IL-15-ABD group is also effective to elicit NK cell population (which is labeled as ^{CD3 - / CD49b +, CD3 -} / CD49b + / CD335 + - / CD335 + and CD3) action; compared to the control group or IL-15 group, CD3 IL--ABD 15 in the group ^{- / CD49b +, CD3 - /} CD335 + and CD3 ^- / CD49b ⁺ / CD335 ⁺ cell population number increased substantially all (FIG section 5G). It was found that the weight of mice in the IL-15 group decreased (Figure 5H), which means that long-term use of IL-15 treatment may have harmful effects on normal physiology.

In summary, these data show that the inhibitory ability of IL-15-ABD on tumor growth and tumor metastasis can be achieved by regulating the host's immune mechanism, including recruiting immune cells and suppressing immunosuppressive cells.

Example 3 Combined treatment can show improvement effect on cancer treatment

3.1 Combined treatment can inhibit tumor growth

This example intends to investigate whether the efficacy of the recombinant polypeptide of Example 1.1 on an individual can be improved by co-administering an additional therapy (for example, immunotherapy) to the individual. For this purpose, an anti-PD-L1 antibody and the recombinant polypeptide of Example 1.1 were used in the animal model of colon cancer and melanoma in this experiment. The experimental results are provided in Figures 6A to 6J.

The data in Figures 6A to 6B show that in both the colon cancer model (Figure 6A) and the melanoma model (Figure 6B), compared with the control group, IL-15-ABD or anti-PDL1 antibody will be administered Effectively inhibit tumor growth. This experiment has noticed that in the two animal models, the tumor growth in the combined treatment group (ie, animals that received both IL-15-ABD and anti-PD-L1 antibody) was almost completely inhibited (Figures 6A to 6B) ). A consistent experimental result can be observed by measuring the tumor weight on the 18th day. The co-administration of IL-15-ABD and anti-PD-L1 antibody can almost completely inhibit tumor growth (Figures 6C to 6D).

In addition, this embodiment analyzes the tumor growth in mice by bioluminescence imaging. A live imaging system is used to detect melanomas that emit Luc2 signals in the tumor area. In the combined treatment group, only very few signals (indicated by total photon flux) were detected (Figure 6E), indicating that compared with single-agent treatment, combined treatment has a certain degree of inhibiting tumor growth. Synergistic effect.

This example also studies the general toxicity of various treatments used in colon cancer and melanoma models. The experimental results are provided in Figures 6F to 6G. In the different treatment groups, no significant weight changes were found in the mice, which means that the treatment used in this experiment does not cause obvious general toxicity in the treatment of cancer.

In order to explore the molecular mechanisms that lead to the inhibition of tumor growth, the effects of various treatments on apoptosis-related molecules (for example, BAX and cleaved apoptotic protease 3) and cell proliferation-related molecules (for example, Ki-67) are studied below. The experimental results indicated that in the colon cancer and melanoma models, the protein expression levels of BAX and cleaved apoptotic protease 3 increased, and the highest expression levels were detectable in the combined treatment group (Figures 6H to 6I) ). On the other hand, in the colon cancer and melanoma models, the expression of Ki-67 decreased, and the lowest expression was detectable in the combined treatment group (Figure 6J).

In summary, the above data shows that the combined treatment of cancer patients (ie, the co-administration of IL-15-ABD recombinant polypeptide and anti-PD-L1 antibody of the present disclosure) will produce a synergistic inhibitory effect on tumor growth. , This effect comes from inhibiting cell proliferation and triggering cell apoptosis.

3.2 Immune response related to inhibiting tumor growth

In order to study the possible immune response caused by the recombinant polypeptide of Example 1.1 or the combined treatment in tumor suppression, this example studied the immune cells (for example, CD8 ⁺ T cells and NK cells) and immunosuppression Changes in the population of sex cells (e.g., T _{reg cells and MDSC).}

3.2.1 CD8 ⁺ T cells

First, use flow cytometry to detect a functional CD8 ⁺ T cell population, which will express IFN-γ and IL-2 (labeled as CD8 ⁺ /IFN-γ ⁺ or CD8 ⁺ /IL-2, respectively) ⁺ cell). Cells were extracted from the TDLN and spleen of mice treated with specific treatments, and then flow cytometry was used to analyze the percentage of CD8 ⁺ /IFN-γ ⁺ or CD8 ⁺ /IL-2 ⁺ cell populations in the sample. ^{In the two models of colon cancer and melanoma, the percentages of CD8 +} /IFN-γ ⁺ T cells in the mouse TDLN and spleen in the IL-15-ABD group were higher than those in the control group. The ^{percentage of CD8 +} /IFN-γ ⁺ T cells was higher than that of the control group (Figures 7A to 7B). ^{Consistent with the above results, the percentages of CD8 +} /IL-2 ⁺ T cells in both TDLN and spleen of mice in the IL-15-ABD group were higher than those in the control group, and the CD8 ⁺ /IL in the combined treatment group The ^{percentage of -2 +} T cells was higher than that of the control group (Figures 7C to 7D). Another key indicator is cytotoxic T cells (CD8 ⁺ ) and granzyme B, which are confirmed by IHC staining. The experimental results show that in the two models of colon cancer and melanoma, after IL-15-ABD treatment, the expression of CD8 and granzyme B increased, and IL-15-ABD and anti-PD-L1 antibody were co-administered It further enhances the expression of the above two markers (Figures 7E to 7F).

In summary, these data confirm that the synergistic effect of IL-15-ABD and anti-PD-L1 antibody on tumor suppression can be partly due to the promotion of functional CD8 ⁺ T cells in the tumor area, which in turn triggers cell-mediated Cytotoxicity prevents tumor growth.

3.2.2 NK cells

This example also examines the percentage of NK cell populations in mice treated with specific treatments in two models of colon cancer and melanoma.

In colon cancer model, the experimental study of NK cell marker ^{(i.e., CD3 - / CD49b +, CD3} - / CD335 + and ^{CD3 - / CD49b + / CD335 +} ) in the amount of expression, as in the first experimental results shown in FIG 8A. Compared with the control group, IL-15-ABD treatment significantly triggered the NK cell population in the mouse spleen, while co-administration of IL-15-ABD and anti-PD-L1 antibody further enhanced IL-15-ABD The effect of inducing NK cell population (Figure 8A).

In melanoma model, the experimental study of NK cell marker ^{(i.e., CD3 - / CD49b +, CD3} - /NK1.1 + and CD3 ^- / CD49b ⁺ /NK1.1 ⁺⁾ expression amount, and Figure 8B provides Related experimental results. FIG 8A is similar to the first found, compared to the control group, IL-15-ABD will increase in the therapeutic mouse spleen ^{CD3 - / CD49b +, CD3 -} /NK1.1 + and CD3 ^- / CD49b ⁺ / NK1 .1 ^{+ the} number of NK cells, and co-administration of IL-15-ABD and anti-PD-L1 antibody will further enhance the efficacy of IL-15-ABD in inducing NK cell population (Figure 8B).

In addition, this experiment measures the IFN-γ expression level of the functional NK cells to test the functionality of the NK cells. The experimental results are provided in Figures 6A to 6J. This study found that the combined treatment in group of two ^{modes, CD3 - / CD335 + / IFN} -γ + and ^{CD3 - /NK1.1 + / IFN-γ} + cells will increase significantly the amount, namely, in the combination therapy group described The activity of IFN-γ will be significantly triggered (Figures 8C to 8D). In addition, this experiment further confirmed the expression levels of CD49b and IFN-γ proteins in both colon cancer and melanoma by IHC staining. According to Figures 8E to 8F, the protein expression levels of CD49b and IFN-γ increased in all treatment groups.

To summarize the above, the data in Figures 8A to 8F show that the synergistic effect of IL-15-ABD and anti-PD-L1 antibody on tumor suppression can be partly due to the increase in the number and activity of NK cells in the tumor area, and then Causes the inhibitory effect of tumor growth.

Furthermore, the following experiments reveal other beneficial immune responses that can help individuals fight tumors. For this purpose, the secretion of VEGF (as an inflammatory indicator) was confirmed. As shown in the experimental results in Figure 8G, in the combined treatment group, the amount of VEGF secretion in the serum of the mice was greatly reduced, which means that the combined treatment can help reduce inflammation in the mice. More importantly, the combined treatment will effectively trigger the secretion of IL-15. (Figure 8H). These experimental results confirmed that the combined treatment of IL-15-ABD and anti-PD-L1 antibody not only recruits immune cells (for example, CD8 ⁺ T cells and NK cells) to the tumor area, but also develops a positive regulation Immune response to help individuals fight tumors.

3.2.3 T _reg cells

Next, this experiment confirmed the T _reg cell populations (labeled CD4 ⁺ /CD25 ⁺ /FOXP3 ⁺ cells) in the two tumor models. According to flow cytometry analysis of these cells from the TDLN and spleen of mice treated with specific treatments, it was found that the _{amount of T reg} cells in the combined treatment group was significantly reduced, which was about 5 to 10 times lower than that of the control group ( Figures 9A to 9B).

3.2.4 MDSC

In this experiment, the mouse bone marrow and spleen MDSC (labeled CD11b ⁺ /Gr-1 ⁺ cells) populations in the two tumor models were analyzed by flow cytometry. The experimental results show that compared with the control group, in the IL-15-ABD or anti-PD-L1 antibody monotherapy group, the number of MDSCs in both bone marrow and spleen decreased, and in the combined treatment group, MDSC The number is the lowest (Figures 9C to 9D).

Based on the above findings, IHC staining was used to further verify the expression levels of FOXP3 and IDO in the two tumors. The above two markers are known key immunosuppressive factors that can guide tumor cells to avoid immunosurveillance. As shown in Figures 9E to 9F, compared with the control group, in the combined treatment group, the expression of the two proteins was reduced by 10 to 30%. In summary, these experimental results prove that the combination of IL-15-ABD and anti-PD-L1 antibody can prevent tumor cells from evading immune surveillance in cancer treatment.

In summary, the recombinant polypeptides of the present disclosure exhibit an inhibitory effect on tumor growth, and this effect can be further synergistically enhanced by co-administering an additional therapy (for example, administering a primary anti-PD-L1 antibody) .

It should be understood that the above descriptions of the embodiments are only exemplary embodiments, and those with ordinary knowledge in the technical field to which the present invention pertains can make various changes and modifications. The above specification, examples and experimental data provide a complete description of the structure and usage of the present disclosure as an exemplary embodiment. Although the various embodiments in the present disclosure have been described above as having certain characteristics, or with reference to one or more individual embodiments, those with ordinary knowledge in the art to which the present invention pertains can still do not depart from the spirit of the present disclosure. Under the circumstances of scope and scope, many modifications have been made to the disclosed implementations.

no

After referring to the following detailed description, the scope of patent application and accompanying drawings, the content of this disclosure and other features, aspects and advantages will be more obvious and understandable, among which:

Figures 1A-1C illustrate the recombinant polypeptide of the present disclosure and its pharmacokinetic results in vivo according to an embodiment of the present disclosure. Figure 1A is a schematic diagram illustrating the construct of the recombinant polypeptide of the present disclosure; Figure 1B is the pharmacokinetic results of IL-15-albumin binding domain (ABD); Figure 1C is IL-15- The pharmacokinetic results of albumin binding peptide (ABP) included IL-15 as a benchmark for comparison.

Figures 2A-2E illustrate the experimental results of the biological activity of the recombinant polypeptide of the present disclosure. Figures 2A-2C The binding activity of recombinant polypeptides of the present disclosure on serum albumin of humans (Figure 2A), dogs (Figure 2B) and mice (Figure 2C); Figure 2D shows the recombinant polypeptides of the present disclosure ( 5 ng/ml) induced proliferation of CTLL-2 cells; Figure 2E is the result of the disclosure of recombinant polypeptides inducing STAT-5 phosphorylation (STAT-5-p) in CTLL-2 cells.

Figures 3A-3D illustrate the therapeutic efficacy of the recombinant polypeptide of the present disclosure on melanoma. Figure 3A shows the tumor volume of mice treated with specific treatments; Figure 3B shows the tumor weight of mice treated with specific treatments; Figure 3C shows the results of tumor metastasis in the intestines of mice, using in vivo imaging The monitoring results obtained by the system; Figure 3D shows the tumor weight in metastatic lesions on the 28th day, where the tumor was taken from the intestines of mice treated with PBS or IL15-ABP.

Figures 4A-4D illustrate the therapeutic efficacy of recombinant polypeptides of the present disclosure on colon cancer. Figure 4A shows the tumor volume of mice treated with a specific treatment; Figure 4B shows the tumor weight of a mouse treated with a specific treatment on day 18; Figure 4C shows a mouse treated with a specific treatment. The number of tumor lumps in the lungs; Figure 4D shows the number of tumor lumps in the lungs, colon and liver of mice treated with specific treatments. a1: p<0.05, compared to the control group; a2: p<0.01, compared to the control group; b1: p<0.05, compared to IL-15; b2: p<0.01, compared to IL-15.

Figures 5A-5H illustrate the influence of the recombinant polypeptide of the present disclosure on the change of immune cell population, wherein the immune cell population is taken from mice carrying colon cancer and treated with specific treatments. ^{Figures 5A-5B show the percentage (%) of CD11b +} /GR-1 ⁺ myeloid-derived suppressor cells (MDSC) population from bone marrow (Figure 5A) and spleen (Figure 5B); ^{Figures 5C-5D are CD4 +} /CD25 ⁺ /FOXP3 ⁺ regulatory T cells (regulatory T, T _reg ) from tumor-draining lymph node (TDLN) (Figure 5C) and spleen (Figure 5D) percentage (%) of the group; of 5E-5F and percentage graph TDLN from CD8 ⁺ T cell population in the spleen (FIG. 5F section) (first FIG. 5E) (%); 5G second graph from the spleen CD3 ^- / CD49b ^+, CD3 ^- / CD335 ⁺ and CD3 ^- / CD49b ⁺ percentage / CD335 ⁺ population of NK cells (%); weight during the course of the measurement result in the first graph 5H mice. a1: p<0.05, compared to the control group; a2: p<0.01, compared to the control group; b1: p<0.05, compared to IL-15; b2: p<0.01, compared to IL-15.

Figures 6A-6J are based on an embodiment of the present disclosure, illustrating the therapeutic effect of combined treatment on tumor growth. Figure 6A shows the tumor volume of mice carrying colon cancer (CT26) after specific treatment; Figure 6B shows the tumor volume of mice carrying melanoma (B16F10) after specific treatment; Figure 6C-6D shows Tumor weights of colon cancer (Figure 6C) and melanoma (Figure 6D) measured on day 18; Figure 6E is a quantitative graph of bioluminescence images to measure mice treated with specific treatments The volume of the B16F10/luc2 tumor in the body; Figures 6F-6G are measured during the course of treatment in mice with colon cancer (CT26) (Figure 6F) or mice with melanoma (B16F10) (Figure 6G) Weight results; Figures 6H-6J show the use of immunohistochemistry (IHC) staining to measure protein expression in colon cancer or melanoma: in colon cancer (Figure 6H) and melanoma (Figure 6I) BAX and cleaved caspase 3 (cleaved caspase 3), and KI-67 in both colon cancer and melanoma (Figure 6J). a1: p<0.05, compared with control group; a2: p<0.01, compared with control group; b1: p<0.05, compared with IL-15-ABD and anti-PD-L1 antibody group; b2: p<0.01, compared with IL-15 -ABD and anti-PD-L1 antibody group.

Figures 7A-7F are based on an embodiment of the present disclosure, illustrating ^{the efficacy of combination therapy in inducing CD8 +} T cell activation. ^{Figures 7A-7B show the percentage (%) of CD8 +} /IFN-γ ⁺ T cell population in TDLN (Figure 7A) or spleen (Figure 7B), where the cell line was taken from a tumor-carrying tumor and treated with specific treatment ^{Treated mice; Figure 7C-7D shows the percentage (%) of CD8 +} /IL-2 ⁺ T cell population in TDLN (Figure 7C) or spleen (Figure 7D), where the cell line is taken from the carrier Tumors and mice treated with specific treatments; Figures 7E-7F use IHC staining to detect protein expression in colon cancer or melanoma: in colon cancer (Figure 7E) and melanoma (Figure 7F) Granzyme B (Granzyme B) and CD8. a1: p<0.05, compared with control group; a2: p<0.01, compared with control group; b1: p<0.05, compared with IL-15-ABD and anti-PD-L1 antibody group; b2: p<0.01, compared with IL-15 -ABD and anti-PD-L1 antibody group.

Figures 8A-8H are based on an embodiment of the present disclosure, illustrating the efficacy of combination therapy in inducing NK cell accumulation and functioning. 8A from the graph of spleen ^{CD3 - / CD49b +, CD3 -} / CD335 + and CD3 ^- / CD49b ⁺ / CD335 ⁺ NK cell population percentage (%), which is derived from the spleen carrying colon (of CT26), and the specific- treatment of mice after treatment; 8B second graph from the spleen CD3 ^{- / CD49b +, CD3 - /NK1.1} + and CD3 ^- / CD49b ⁺ /NK1.1 ⁺ NK cell population percentage (%), which is the spleen from carrying melanoma (B16F10), and specific therapeutic mice after treatment; 8C second graph from the spleen CD3 ^{- / CD335 + / IFN-γ} + NK population percentage (%) cell, wherein the spleen is carried from the colon cancer (of CT26), and by the mice after a particular treatment process; 8D second graph from the spleen CD3 ^{- /NK1.1 + / IFN-γ +} NK population percentage (%) cell, wherein the spleen is carried from melanoma (B16F10), and treated mice with specific treatments; Figures 8E-8F are the use of IHC staining to detect protein expression in colon cancer or melanoma: in colon cancer (Figure 8E) and melanoma (Figure 8E) Figure 8F) CD49b and IFN-γ; Figure 8G-8H uses enzyme-linked immunosorbent assay (ELISA) to detect protein expression in serum: secreted VEGF (Figure 8G) and secretion Type IL-15 (Figure 8H). a1: p<0.05, compared with control group; a2: p<0.01, compared with control group; b1: p<0.05, compared with IL-15-ABD and anti-PD-L1 antibody group; b2: p<0.01, compared with IL-15 -ABD and anti-PD-L1 antibody group.

Figures 9A-9F are based on an embodiment of the present disclosure, illustrating the efficacy of the combination therapy for suppressing the accumulation of immunosuppressive cells. Figures 9A-9B show the percentage (%) ^{of CD4 +} /CD25 ⁺ /FOXP3 ⁺ T _reg cell populations in TDLN (Figure 9A) and spleen (Figure 9B). Mice treated with specific treatment; Figure 9C-9D shows the ^{percentage (%) of CD11b +} /GR-1 ⁺ MDSC population in bone marrow (Figure 9C) and spleen (Figure 9D), where the cell line is taken from Mice carrying tumors and treated with specific treatments; Figures 9E-9F use IHC staining to detect protein expression in colon cancer or melanoma: in colon cancer (Figure 9E) and melanoma (Figure 9F) ) In FOXP3 and IDO. a1: p<0.05, compared with control group; a2: p<0.01, compared with control group; b1: p<0.05, compared with IL-15-ABD and anti-PD-L1 antibody group; b2: p<0.01, compared with IL-15 -ABD and anti-PD-L1 antibody group.

Claims (15)

A recombinant polypeptide comprising: A first polypeptide having an amino acid sequence with a sequence similarity of at least 85% to SEQ ID NO:1; and A second polypeptide, which is operably linked to the N- or C-terminus of the first polypeptide, wherein the second polypeptide has a sequence of at least 85% of SEQ ID NO: 2 or SEQ ID NO: 3 Amino acid sequence of similarity. The recombinant polypeptide according to claim 1, wherein The first polypeptide has an amino acid sequence of SEQ ID NO: 1; and The second polypeptide has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. The recombinant polypeptide according to claim 1, wherein The N-terminal of the recombinant polypeptide is acetylated, formylated, methylated, carbamylated, pegylated, phosphorylated , Or glycosylated; and/or The C-terminus of the recombinant polypeptide is amidated, glypiated, biotinylated, or glycosylated. The recombinant polypeptide according to claim 1, further comprising a third polypeptide which is operably linked to the N- or C-terminus of the first or the second polypeptide, wherein the third polypeptide is Choose from: β-galactosidase (lacZ), chloramphenicol acetyltransferase (CAT), luciferase, green fluorescent protein (GFP) , Red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), β-lactamase, aminoglycoside -3'-phosphotransferase (aminoglycoside-3'-phosphotransferase, APH(3')), orotidine-5'-phosphate decarboxylase (ODCase), chitin binding Protein (chitin binding protein, CBP), maltose binding protein (maltose binding protein, MBP), streptomycin tag (Strep-tag), glutathione-S-transferase (glutathione-S-transferase, GST), sulfur Thioredoxin (TRX), alkaline phosphatase (AP), biotin-carboxy carrier protein (BCCP), calmodulin binding peptide (CBP), Phage T7 epitope (bacteriophage T7 epitope, T7-tag (T7-tag)), poly-histidine tag, FLAG-tag (FLAG-tag), Myc-tag (Myc-tag), HA -A group consisting of HA-tag, Spot-tag, NE-tag, and combinations thereof. The recombinant polypeptide according to claim 4, further comprising a linker, which is used to connect any two of the first, second and third polypeptides. A pharmaceutical composition for preventing and/or treating cancer of an individual in need, wherein the pharmaceutical composition comprises the recombinant polypeptide according to any one of claims 1-4, and a pharmaceutically acceptable Accepted carrier. The pharmaceutical composition according to claim 6, further comprising a primary anti-PD-L1 antibody. A method for preventing and/or treating cancer in an individual in need thereof comprises administering to the individual a therapeutically effective amount of the pharmaceutical composition according to any one of claims 5-6. The method according to claim 8, wherein the cancer is bladder cancer, biliary cancer, bone cancer, brain tumor, breast cancer, and cervical cancer (cervical cancer), colorectal cancer (colorectal cancer), colon cancer (colon cancer), esophageal cancer (esophageal cancer), epithelial cancer (epidermal carcinoma), gastric cancer (gastric cancer), gastrointestinal stromal tumor (gastrointestinal stromal tumor, GIST), glioma, hematopoietic tumors of lymphoid lineage, hepatic cancer, non-Hodgkin's lymphoma, Kaposi's sarcoma ( Kaposi's sarcoma, leukemia, lung cancer, lymphoma, intestinal cancer, melanoma, myeloid leukemia, pancreatic cancer, prostate Cancer (prostate cancer), retinoblastoma (retinoblastoma), ovary cancer (ovary cancer), renal cell carcinoma (renal cell carcinoma), spleen cancer (spleen cancer), squamous cell carcinoma (squamous cell carcinoma), thyroid cancer ( thyroid cancer, or thyroid follicular cancer. The method according to claim 9, wherein the cancer is colon cancer. The method according to claim 9, wherein the cancer is melanoma. The method according to claim 8, wherein the cancer is an in situ cancer or a metastatic cancer. The method according to claim 8, wherein the individual is a human. The method according to claim 8, further comprising subjecting the individual to a surgical operation, a radiotherapy, a chemotherapy, an immunotherapy, a hormonal therapy, a target therapy, a heat therapy, or a combination therapy before , Simultaneously or afterwards, administer the pharmaceutical composition according to any one of Claims 5-6 to the individual. The method of claim 14, wherein the immunotherapy comprises administering a primary anti-PD-L1 antibody to the individual.

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