CN116375880A - Fusion protein and preparation method and application thereof - Google Patents

Abstract

The present invention relates to a fusion protein and its preparation method and application. The pairing domain of the fusion protein comprises the amino acid sequence of engineered HLA‑Iα3 and the amino acid sequence of engineered β2 microglobulin. The present invention also relates to the preparation method of fusion protein, nucleic acid encoding fusion protein, vector, host cell, pharmaceutical composition and conjugate. The invention also relates to therapeutic uses of the fusion proteins. The fusion protein provided by the invention performs engineering transformation on the MHC element according to the requirement of the biologically active peptide, and exerts the biological function of the biologically active peptide.

Description

Fusion protein and its preparation method and application

technical field

The invention relates to the technical field of molecular biology, in particular to a fusion protein and a preparation method thereof.

Background technique

Biologically active peptides are a general term for a class of different peptides consisting of amino acids in different compositions and arrangements, ranging from dipeptides to complex linear and circular structures, including cytokines or their receptors, CD molecules or their receptors, antibodies or Its fragments, etc., play different physiological functions in organisms. Some biologically active peptides, such as IL-15, need to bind to the sushi domain contained in the N-terminal extracellular region of its receptor IL-15Rα (IL-15Rα sushi domain) to exert biological functions. Some studies have shown that after IL-15 binds to IL-15Rα, it is presented to the surface of CD8+ T cells or natural killer (NK) cells expressing IL-2Rβ and γc through trans-presentation, thereby effectively promoting the proliferation of these cells. Proliferation and activation, play a role in the body's normal immune response. IL-15 is structurally similar to IL-2, but unlike IL-2, IL-15 does not trigger the proliferation of Treg cells. However, some biologically active peptides, including IL-15, have short half-lives in vivo, and systemic administration is likely to cause greater toxicity, which urgently needs to be improved.

Contents of the invention

The fusion protein provided by the invention adopts engineered MHC elements, which is beneficial for the biologically active peptides therein to exert the required biological functions.

In one aspect, the invention provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein

The first polypeptide comprises a first biologically active peptide, a first pairing domain operably linked to the first biologically active peptide, and a first pairing domain operably linked to the first biologically active peptide from the N-terminus to the C-terminus. Fc polypeptide, and

The second polypeptide comprises a second biologically active peptide and a second pairing domain operably linked to the second biologically active peptide from the N-terminus to the C-terminus,

Wherein, one of the first pairing domain and the second pairing domain includes the amino acid sequence of engineered HLA-I α3, and the other pairing domain includes the amino acid sequence of engineered β2 microglobulin;

The first pairing domain and the second pairing domain are capable of forming a dimer, and the first bioactive peptide and the second bioactive peptide are capable of forming a dimer;

The amino acid sequence of the engineered HLA-I α3 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, the amino acid sequence of the engineered β2 microglobulin has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

In some embodiments, the fusion protein comprises two of the first polypeptides and two of the second polypeptides, and the Fc polypeptides of the two first polypeptides associate to form an Fc domain.

In some embodiments, the first pairing domain comprises the amino acid sequence of engineered HLA-I α3 and the second pairing domain comprises the amino acid sequence of engineered β2 microglobulin.

In some embodiments, the first pairing domain comprises the amino acid sequence of engineered β2 microglobulin and the second pairing domain comprises the amino acid sequence of engineered HLA-I α3.

In some embodiments, the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO:3, and the engineered β2 microglobulin comprises the amino acid sequence shown in SEQ ID NO:5 or 6.

In some embodiments, the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO:4, and the engineered β2 microglobulin comprises the amino acid sequence shown in SEQ ID NO:5 or 6.

In some embodiments, the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO:7, and the engineered β2 microglobulin comprises the amino acid sequence shown in SEQ ID NO:9 or 10.

In some embodiments, the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO:8, and the engineered β2 microglobulin comprises the amino acid sequence shown in SEQ ID NO:9 or 10.

In one aspect, the invention provides an isolated nucleic acid encoding the fusion protein of the invention.

In one aspect, the invention provides a vector comprising the isolated nucleic acid of the invention.

In one aspect, the present invention provides a host cell comprising the isolated nucleic acid of the present invention, or the vector.

In another aspect, the present invention provides a method for preparing the fusion protein, which comprises culturing the host cell under the condition that the fusion protein is expressed, and isolating and purifying the expressed fusion protein.

In another aspect, the present invention provides a pharmaceutical composition, which comprises the fusion protein described in the present invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides the use of the fusion protein or the pharmaceutical composition described in the present invention in the preparation of medicines for treating diseases of subjects.

In another aspect, the present invention provides a method for treating a disease in a subject in need, comprising administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the present invention to the subject.

According to the type of the first bioactive peptide and the second bioactive peptide and the need to form a dimer, the fusion protein of the present invention can properly engineer the MHC element to ensure the activity of the bioactive peptide.

The fusion protein of the present invention has a longer half-life.

The fusion protein of the present invention has better stability.

Description of drawings

Figure 1 is the consensus sequence of the HLA-I-B molecule α chain constant region;

Figure 2 is a schematic diagram of the structure of ALT-803;

Fig. 3 is a structural schematic diagram of a specific example of the fusion protein provided by the present invention;

Fig. 4 is pro15MHCin of different concentrations and the proliferation effect of control on PBMC;

Figure 5 is the effect of different concentrations of pro15MHCin and the control on the proliferation of NK cells;

Figure 6 shows the effect of different concentrations of pro15MHCin and the control on the proliferation of CD8+ T cells.

Detailed ways

the term

Unless otherwise specified, the following terms used in the present invention have the following meanings. A specific term should not be regarded as indeterminate or unclear if there is no special definition, but should be understood according to the ordinary meaning in the art.

The term "biologically active peptide" is a biologically active polypeptide formed by covalently linking multiple amino acids, which plays an important role in the regulation of cell or body physiological and metabolic functions. Non-limiting examples of biologically active peptides include cytokines or their receptors, CD molecules or their receptors, antibodies or fragments thereof, antigens or antigenic epitopes thereof. In a specific example herein, the biologically active peptide is IL-15, IL-15Rα or IL-15Rα sushi domain.

The term "IL-15" may be any native interleukin 15 (IL-15) or variants thereof, non-limiting examples include human IL-15 or variants thereof, non-human mammalian IL-15 or variants thereof , or non-mammalian IL-15 or a variant thereof. IL-15 herein is preferably primate IL-15, more preferably human IL-15. Unless otherwise stated, "IL-15" herein refers to the mature molecule of IL-15. The "variant" of natural IL-15 refers to the increase or decrease in the affinity between IL-15 and its receptor obtained by one or more amino acid substitutions, addition or deletion mutations, or the increase or decrease in its activity of stimulating T cells or NK cells mutant molecules. As used herein, "IL-15 with a propeptide linked to its N-terminal" refers to IL-15 or its variants with a propeptide sequence linked to its N-terminal. The mature molecule and propeptide sequence of human IL-15 can be found in the database UniProtKB, accession number P40933, wherein amino acid residues 30 to 48 are the propeptide sequence, and amino acid residues 49 to 162 are the mature molecular sequence.

The term "IL-15Rα" may be interleukin 15 receptor alpha (IL-15Rα) or variants thereof, or IL-15Rα functional fragments or variants thereof, of any species, non-limiting examples include human IL-15Rα or A variant thereof, a non-human mammalian IL-15Rα or a variant thereof, or a non-mammalian IL-15Rα or a variant thereof. IL-15Rα contains a domain called “sushi domain” (Wei X Q, Orchardson M, Gracie J A, et al. The Sushi Domain of Soluble IL-15 Receptorα Is Essential for Binding IL-15and Inhibiting Inflammatory and Allogenic Responses In Vitro and In Vivo [J]. Journal of Immunology, 2001, 167(1): 277-82.). As used herein, "IL-15Rα sushi domain" comprises a domain that starts at the first cysteine residue (C1) after the signal peptide of IL-15Rα and ends at the fourth cysteine residue (C4) after the signal peptide. 61 amino acid residues (as amino acid residues 3 to 63 in the amino acid sequence shown in SEQID NO: 13), or a variant comprising the above 61 amino acid residues. Specifically, the "IL-15Rα sushi domain" used herein comprises an amino acid residue IT at the N-terminus of the above-mentioned 61 amino acid residues or variants thereof; The C-terminus of the body contains an IL-15Rα hinge region fragment; the "IL-15Rα hinge region fragment" can be 1 (such as I), 2 (such as IR), 3 (such as IRD), 4 (such as IRDP ), 5 (eg IRDPA), 6 (eg IRDPAL), 7 (IRDPALV), 8 (IRDPALVH), 9 (IRDPALVHQ), 10, 11, 12, 13 or 14 amino acid residues base fragment. According to some embodiments of the present invention, the amino acid sequence of the IL-15Rα sushi domain is shown in SEQ ID NO:13.

The term "operably linked" refers to the juxtaposition of two or more biological sequences of interest, whether or not a spacer or linker is present, in such a way that they are placed in a relationship that enables them to function in the intended manner. When applied to polypeptides, the term is intended to mean that polypeptide sequences are linked in such a way that the linked product has the desired biological function. The term can also be applied to nucleic acids, for example, when a nucleic acid encoding a polypeptide is operably linked to regulatory sequences (such as promoters, enhancers, silencer sequences, etc.), the term is intended to refer to the nucleic acid sequence to allow the described The polypeptides are linked in such a way as to regulate expression of the nucleic acid.

Major histocompatibility complex (MHC) is a collective term for a group of genes encoding animal major histocompatibility antigens. MHC in mice is called H-2 gene complex, MHC in humans is called HLA (human leukocyte antigen, HLA) gene complex, and its encoded products are called HLA molecules, HLA antigens or human leukocyte antigens. The HLA gene complex is located at 6p21.3, the short arm of human chromosome 6, and contains more than 220 genes with different functions. Many of these genes code for proteins in the immune system. MHC class I molecules are expressed on the surface of almost all nucleated cells and recognized by CD8+ T cells. MHC-II molecules are expressed on the surface of antigen-presenting cells and recognized by CD4+ T cells. MHC is polymorphic, and most people mainly include 6 classic MHC molecules: 3 classic MHC-I molecules (HLA-A, HLA-B, HLA-C) and 3 classic MHC-II molecules (HLA-DR, HLA-DP, HLA-DQ). Human MHC class I molecule (HLA-1) consists of heavy chain (α chain) and β2 microglobulin (β2m, or β chain), in which the extracellular segment of α chain has three domains (α1, α2 and α3) , the two domains α1 and α2 at the distal end of the membrane constitute the antigen-binding groove, and the domain α3 is homologous to the constant region of human immunoglobulin.

The terms "Fc domain", "Fc" or "Fc domain" are used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least part of the constant region. The term includes native sequence Fc and variant Fc. The C-terminal lysine (Lys447) of Fc may or may not be present. Unless otherwise stated, the numbering of amino acid residues in Fc is according to the EU numbering system, also known as the EU index, described in Kabat, E.A et al., Sequences of Proteins of Immunological Interest, 5th edition, PublicHealth Service, National Institutes of Health , Bethesda, MD (1991), NIH Publication 91-3242. One of the "Fc polypeptides" of an Fc domain as used herein refers to one of the two polypeptides forming a dimeric Fc domain. For example, the Fc polypeptide of an IgG Fc domain comprises IgG CH2 and IgG CH3 constant regions.

When the "first pairing domain" and "second pairing domain" refer to the description of amino acid positions herein, they refer to the sequence numbering according to the starting amino acid of the corresponding sequence as the first position, for example, R60C in SEQ ID NO:1 Substitution refers to sequence numbering starting with the first amino acid in SEQ ID NO: 1 as position 1, and the 60th position R in the sequence is replaced with C.

The term "isolated" refers to a compound of interest, such as a fusion protein, nucleic acid, etc., that has been separated from its natural environment.

The term " _EC50 " refers to the effective concentration, 50% of the maximal response of a certain compound molecule (eg, a fusion protein).

The term "pharmaceutically acceptable" refers to substances that do not eliminate the biological activity or properties of the fusion protein of the present invention, such as carriers or diluents. Such a substance is administered to an individual without causing an undesired biological effect or interacting in a deleterious manner with any component of the composition in which it is contained.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivery of an active ingredient such as a fusion protein, non-limiting examples include diluents, fillers, buffers, isotonic agents, or other reagent. Except for carriers incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

The term "treatment" refers to an attempt to alter the natural course of a disease in an individual, and may be clinical intervention for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, amelioration or palliation of the disease state, and regression or improved prognosis.

The term "therapeutically effective amount" refers to the amount of fusion protein or pharmaceutical composition or other administration necessary to provide a therapeutic benefit to a subject.

The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, ovines, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. Preferably, the subject according to the invention is a human. Unless indicated otherwise, the terms "patient" or "subject" are used interchangeably.

The term sequence "identity", also called identity. "Percent identity (%)" of an amino acid sequence refers to the alignment of the sequences to be aligned with the specific amino acid sequences shown herein and after introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to Where any conservative substitutions are considered as part of the sequence identity, the percentage of amino acid residues in the aligned sequences that are identical to the amino acid residues of the particular amino acid sequence shown herein. Alignment of amino acid sequences for identity can be performed by various means within the skill in the art, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Amino acid residues in proteins are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine Amide is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic acid is Asp or D; Glutamic acid is Glu or E; Cysteine is Cys or C; Tryptophan Acid is Trp or W; Arginine is Arg or R; Glycine is Gly or G. In the context of the present invention, amino acid substitutions are expressed as: original amino acid-position-substituted amino acid, using three-letter codes or single-letter codes, including codes Xaa and X for amino acid residues. Thus, for example, "M252Y" means substitution of amino acid M at position 252 with amino acid Y.

Various aspects of the invention are described in further detail in the following subsections.

fusion protein

MHC (major histocompatibility complex) is the basis of immune system recognition and participates in the body's adaptive immune process. The fusion protein designed based on the molecular composition of MHC has good compatibility with the human immune environment. Compared with MHC-II molecules, MHC-I molecules are more widely distributed, expressed on the surface of almost all nucleated cells, and recognized by CD8+ T cells. Among the HLA-I molecules, the number of human MHC-I molecules of the HLA-I-B class is the largest, so the construction of fusion protein structural elements based on the constant region of the human MHC-I molecule of the HLA-I-B class can reduce immunity. original risk. In addition, the constant region of the HLA-I-B-based human MHC-I molecule does not contain N-linked and O-linked glycosylation sites, has good developability and low immunogenicity.

The present invention provides a fusion protein comprising a first polypeptide and a second polypeptide, wherein

The first polypeptide comprises a first biologically active peptide, a first pairing domain operably linked to the first biologically active peptide, and a first pairing domain operably linked to the first biologically active peptide from the N-terminus to the C-terminus. Fc polypeptide, and

The second polypeptide comprises a second biologically active peptide and a second pairing domain operably linked to the second biologically active peptide from the N-terminus to the C-terminus,

Wherein, one of the first pairing domain and the second pairing domain includes the amino acid sequence of engineered HLA-Iα3, and the other pairing domain includes the amino acid sequence of engineered β2 microglobulin;

The first pairing domain and the second pairing domain are capable of forming a dimer, and the first bioactive peptide and the second bioactive peptide are capable of forming a dimer;

The amino acid sequence of the engineered HLA-I α3 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, the amino acid sequence of the engineered β2 microglobulin has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. The sequence shown in SEQ ID NO: 1 is derived from the consensus sequence generated by the constant region sequence alignment of multiple HLA-I-B molecules, and it has good versatility to use it or transform it to form a paired domain.

In some embodiments, the fusion protein comprises two of the first polypeptides and two of the second polypeptides, and the Fc polypeptides of the two first polypeptides associate to form an Fc domain. In a specific example, the fusion protein comprises two first polypeptides and two second polypeptides, wherein one first polypeptide and one second polypeptide form a dimer, and the other first The polypeptide and another second polypeptide form another dimer; the Fc polypeptides of the two first polypeptides associate to form an Fc domain such that two of said dimers form a tetramer.

In some embodiments, the first pairing domain comprises the amino acid sequence of engineered HLA-I α3 and the second pairing domain comprises the amino acid sequence of engineered β2 microglobulin.

In some embodiments, the first pairing domain comprises the amino acid sequence of engineered β2 microglobulin and the second pairing domain comprises the amino acid sequence of engineered HLA-I α3.

In some embodiments, at least one non-native interchain bond is capable of forming between the first pairing domain and the second pairing domain and the non-native interchain bond is capable of stabilizing the dimer. In some embodiments, the amino acid residue forming the non-native interchain bond is at the contact interface of the first pairing domain and the second pairing domain effective to form a bond. The term "contact interface" refers to specific regions on said polypeptides where said polypeptides interact/associate with each other. A contact interface comprises one or more amino acid residues capable of interacting with corresponding amino acid residues in contact or association when the interaction occurs. The amino acid residues in the contact interface may or may not be in contiguous sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface may be located separately at different positions on the linear sequence.

In some embodiments, the number of non-natural interchain bonds is 1-3, such as 1, 2 or 3, preferably 1. In one embodiment, the non-natural interchain linkage is a disulfide linkage.

In some embodiments, no non-native interchain linkage is formed between the first pairing domain and the second pairing domain. In some embodiments, no disulfide bond is formed between the first pairing domain and the second pairing domain. In some embodiments, the first pairing domain and the second pairing domain are combined through a non-covalent bond to form a dimer.

In some embodiments, the engineered HLA-I α3 comprises an amino acid sequence having amino acid substitutions in SEQ ID NO:1 and the engineered β2 microglobulin comprises an amino acid sequence having amino acid substitutions in SEQ ID NO:2 sequence.

Said amino acid substitutions in engineered HLA-I α3 and engineered β2 microglobulins comprise amino acid substitutions that form non-native interchain bonds (e.g. disulfide bonds) or/and enhance pairing domain formation of dimers or Amino acid substitution of the isoelectric point of the fusion protein.

In some embodiments, the amino acid substitutions in both the engineered HLA-I α3 and the engineered β2 microglobulin comprise cysteine residues that occur at the contact interface of the two and are capable of forming disulfide bonds with each other base substitution. In some embodiments, the cysteine residue substitution is one to at most pairs selected from the group consisting of: (1) R60C in SEQ ID NO:1 and Y26C in SEQ ID NO:2; (2) A62C in SEQ ID NO:1 and R12C in SEQ ID NO:2; and (3) G63C in SEQ ID NO:1 and Y67C in SEQ ID NO:2. In a specific embodiment, said cysteine residue is substituted with R60C in SEQ ID NO:1 and Y26C in SEQ ID NO:2. In a specific embodiment, said cysteine residue is substituted with A62C in SEQ ID NO:1 and R12C in SEQ ID NO:2. In a specific embodiment, said cysteine residue is substituted with G63C in SEQ ID NO:1 and Y67C in SEQ ID NO:2. In other specific embodiments, the cysteine residue substitution may be selected from two or three pairs of R60C/Y26C, A62C/R12C and G63C/Y67C.

In some embodiments, the amino acid substitutions in the engineered HLA-I α3 or/and engineered β2 microglobulin comprise amino acid substitutions that increase the isoelectric point of the dimer or fusion protein formed by the pairing domain. These amino acid substitutions can occur in both paired domains, or in any one of the paired domains. Generally, the isoelectric point of the dimer or fusion protein formed by the paired domain is increased to 6.5-9.0, so as to facilitate the development of the production process and improve the druggability. In some embodiments, the isoelectric point of the dimer or fusion protein formed by the pairing domain is increased to 6.5-8.5, 7.0-8.5, 7.0-9.0, 7.0-7.8, 7.0-8.0, 7.5-7.8, or 7.5-8.0 . In some specific embodiments, the isoelectric point of the dimer or fusion protein formed by the pairing domain is increased to 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.5 , 8.7 or 9.0. The isoelectric point can be determined experimentally or calculated theoretically. For example, the calculation of a theoretical isoelectric point can use the online calculation and analysis tool Expasy-Compute pI/Mw tool (http://web.expasy.org/compute_pi/).

In some embodiments, the amino acid substitution that increases the isoelectric point of the dimer or fusion protein formed by the pairing domain comprises: engineered HLA-I α3 at E3, D22, E48, D53, One or several positions in E90 and E101 were substituted with positively charged amino acids.

In some embodiments, the amino acid substitution that increases the isoelectric point of the dimer or fusion protein formed by the pairing domain comprises: E74, E47, E69, D34 in the sequence of SEQ ID NO: 2 in engineered β2 microglobulin One or several positions in , E16, D53, E44, E50 and E36 were substituted by positively charged amino acids.

In some specific embodiments, the amino acid substitution to increase the isoelectric point of the dimer or fusion protein formed by the pairing domain comprises: D22, E48 and Position D53 is substituted with a positively charged amino acid, and the engineered β2 microglobulin is substituted with a positively charged amino acid at position E69 of the sequence of SEQ ID NO:2. In some embodiments, the positively charged amino acid is K or R.

In a more specific embodiment, the amino acid substitution to increase the isoelectric point of the dimer or fusion protein formed by the pairing domain comprises: the engineered HLA-I α3 comprises amino acid substitutions D22R, E48K in the sequence of SEQ ID NO:1 and D53K, and the engineered β2 microglobulin comprises the amino acid substitution E69R in the sequence of SEQ ID NO:2.

As a specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:3, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:5. As a specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:3, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:6. As a specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:4, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:5. As a specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:4, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:6.

In another specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:7, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:9. In another specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:7, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:10. In another specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:8, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:9. In another specific example, the engineered HLA-I α3 includes the amino acid sequence shown in SEQ ID NO:8, and the engineered β2 microglobulin includes the amino acid sequence shown in SEQ ID NO:10.

GKETLQRADPPKTHVTHHPXSRHEATLRCWALGFYPAEITLTWQRDGKDQTQKTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPS (SEQ ID NO: 3, X=I or V);

GKETLQRADPPKTHVTHHPISRHEATLRCWALGFYPAEITLTWQRDGKDQTQKTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPS (SEQ ID NO: 4);

IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDRGP (SEQ ID NO: 5);

IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDR (SEQ ID NO: 6)

GKETLQRADPPKTHVTHHPXSRHEATLRCWALGFYPAEITLTWQRDGKDQTQKTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPS (SEQ ID NO: 7, X=I or V)

GKETLQRADPPKTHVTHHPVSRHEATLRCWALGFYPAEITLTWQRDGKDQTQKTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPS (SEQ ID NO: 8)

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDRGP (SEQ ID NO: 9)

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDR (SEQ ID NO: 10)

In some embodiments, one of the first bioactive peptide and the second bioactive peptide is IL-15 with a propeptide linked to its N-terminus, and the other is an IL-15Rα sushi domain. In some specific embodiments, the first bioactive peptide is IL-15 with a propeptide linked to its N-terminus, and the second bioactive peptide is IL-15Rα sushi domain. In some other specific embodiments, the first biologically active peptide is the sushi domain of IL-15Rα, and the second biologically active peptide is IL-15 with a propeptide linked to its N-terminus.

In some embodiments, the IL-15 with a propeptide at the N-terminus is mammalian IL-15 with a propeptide at its N-terminus, preferably primate IL-15 with a propeptide at its N-terminus, More preferred is human IL-15 with a propeptide linked to its N-terminus. In some embodiments, the N-terminal propeptide-linked IL-15 comprises at least 85%, 86%, 87%, 88%, 89%, 90%, Amino acid sequences that are 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. In some embodiments, the N-terminal propeptide-linked IL-15 comprises the amino acid sequence shown in SEQ ID NO:11. In some specific embodiments, the amino acid sequence of IL-15 with a propeptide linked to its N-terminus is shown in SEQ ID NO:11.

In some embodiments, the IL-15Rα sushi domain is a mammalian IL-15Rα sushi domain, preferably a primate IL-15Rα sushi domain, more preferably a human IL-15Rα sushi domain. In some embodiments, the IL-15Rα sushi domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% of the amino acid sequence shown in SEQ ID NO: 13 Amino acid sequences that are %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the IL-15Rα sushi domain comprises the amino acid sequence shown in SEQ ID NO:13. In some specific embodiments, the amino acid sequence of the IL-15Rα sushi domain is shown in SEQ ID NO:13.

In some embodiments, the N-terminal propeptide-linked IL-15 comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, said IL-15Rα sushi domain comprising the same as SEQ ID NO: 13 The amino acid sequence shown has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Amino acid sequences with % or 100% identity. In some embodiments, the IL-15 with a propeptide linked to its N-terminal comprises the amino acid sequence shown in SEQ ID NO:11, and the IL-15Rα sushi domain comprises the amino acid sequence shown in SEQ ID NO:13. In some specific embodiments, the amino acid sequence of IL-15 with a propeptide attached to its N-terminal is shown in SEQ ID NO: 11, and the amino acid sequence of the IL-15Rα sushi domain is shown in SEQ ID NO: 13 Show.

In some embodiments, one of the first bioactive peptide and the second bioactive peptide is IL-15 and the other is an IL-15Rα sushi domain. In some specific embodiments, the first bioactive peptide is IL-15, and the second bioactive peptide is IL-15Rα sushi domain. In other specific embodiments, the first bioactive peptide is IL-15Rα sushi domain, and the second bioactive peptide is IL-15.

In some embodiments, the IL-15 is mammalian IL-15, preferably primate IL-15, more preferably human IL-15. In some embodiments, the IL-15 comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the amino acid sequence shown in SEQ ID NO: 12 Amino acid sequences that are %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the IL-15 comprises the amino acid sequence shown in SEQ ID NO:12. In some specific embodiments, the amino acid sequence of IL-15 is shown in SEQ ID NO:12.

In some embodiments, the IL-15 comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the amino acid sequence shown in SEQ ID NO: 12 %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, the IL-15Rα sushi domain comprises an amino acid sequence shown in SEQ ID NO: 13 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence. In some embodiments, the IL-15 comprises the amino acid sequence shown in SEQ ID NO:12, and the IL-15Rα sushi domain comprises the amino acid sequence shown in SEQ ID NO:13. In some specific embodiments, the amino acid sequence of the IL-15 is shown in SEQ ID NO:12, and the amino acid sequence of the IL-15Rα sushi domain is shown in SEQ ID NO:13.

GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 11);

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 12);

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 13).

In some embodiments, the Fc polypeptide is an immunoglobulin G (IgG) Fc polypeptide comprising a CH2 domain and a CH3 domain of an IgG heavy chain constant region. In some embodiments, the Fc polypeptide is an IgG1 Fc polypeptide. In some embodiments, the Fc polypeptide is an IgG4 Fc polypeptide. In some embodiments, the Fc polypeptide is a human IgG Fc polypeptide. In some embodiments, the Fc polypeptide is a human IgG1 Fc polypeptide. In some embodiments, the Fc polypeptide is a human IgG4 Fc polypeptide.

In some embodiments, the Fc domain is an IgG Fc domain. The IgG Fc domain is a dimer, each Fc polypeptide of which comprises the CH2 domain and the CH3 domain of the IgG heavy chain constant region. The two Fc polypeptides of the Fc domain are capable of stably associating with each other. For example, two Fc polypeptides are stably associated through one or more of linkers, disulfide bonds, hydrogen bonds, electrostatic interactions, salt bridges, and hydrophobic-hydrophilic interactions. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is an IgG4 Fc domain. In some embodiments, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228, particularly amino acid substitution S228P, which enhances the stability of the fusion protein.

In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fc domain is a human IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG4 Fc domain comprising an amino acid substitution at position S228, particularly amino acid substitution S228P, which enhances the stability of the fusion protein.

In some embodiments, the Fc domain comprises modifications, such as amino acid substitutions. The modification can be, for example, a modification to increase its binding to FcRn, a modification to reduce or eliminate its binding to FcγI, FcγIIA, FcγIIB, FcγIIIA and/or FcγIIIB, etc. The modification can, for example, prolong the in vivo half-life of the fusion protein, or/and reduce or eliminate the ADCC effect of the fusion protein.

In some embodiments, the Fc domain comprises modifications that increase its binding to FcRn. In some embodiments, the IgG Fc domain comprises amino acid substitutions at positions 252, 254, and 256, in particular, replacing positions 252, 254, and 256 with Tyr, Thr, and Glu, respectively. In some specific embodiments, said IgG Fc domain comprises amino acid substitutions M252Y, S254T and T256E (YTE mutation). In a more specific example, the IgG Fc domain is a human IgG Fc domain, especially a human IgG1 Fc domain. In a specific embodiment, said human IgG Fc domain comprises amino acid substitutions M252Y, S254T and T256E (YTE mutation). The amino acid substitution increases the binding of the Fc domain to FcRn and prolongs the in vivo half-life of the fusion protein.

In some embodiments, the Fc domain comprises a modification that reduces or eliminates its binding to FcyI, FcyIIA, FcyIIB, FcyIIIA, and/or FcyIIIB. In some embodiments, the IgG Fc domain comprises an amino acid substitution at positions 234 and 235, in particular, replacing both positions 234 and 235 with Ala. In some embodiments, the IgG Fc domain comprises an amino acid substitution of Ala at both positions 234 and 235. In a more specific example, the IgG Fc domain is a human IgG Fc domain, especially a human IgG1 Fc domain. In a specific embodiment, said human IgGl Fc domain comprises amino acid substitutions L234A and L235A (LALA mutation). The amino acid substitution reduces or eliminates the binding of the Fc domain to FcγI, FcγIIA, FcγIIB, FcγIIIA and/or FcγIIIB, and reduces or eliminates the ADCC effect of the fusion protein.

In some specific embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14, and the second polypeptide comprises SEQ ID NO: 14 Amino acid sequence shown in ID NO:15. In some specific embodiments, the fusion protein comprises two first polypeptides and two second polypeptides, wherein the first polypeptides comprise the amino acid sequence shown in SEQ ID NO: 14, and the second The polypeptide comprises the amino acid sequence shown in SEQ ID NO:15. In some specific embodiments, the fusion protein consists of two first polypeptides and two second polypeptides, wherein the first polypeptides comprise the amino acid sequence shown in SEQ ID NO: 14, the second The two polypeptides comprise the amino acid sequence shown in SEQ ID NO:15. In a specific embodiment, the fusion protein is composed of two first polypeptides and two second polypeptides, wherein the amino acid sequence of the first polypeptides is shown in SEQ ID NO: 14, the second The amino acid sequence of the two polypeptides is shown in SEQ ID NO:15. In a specific embodiment, the fusion protein is composed of two first polypeptides and two second polypeptides, wherein the amino acid sequence of the first polypeptides is shown in SEQ ID NO: 14, the second The amino acid sequence of the two polypeptides is shown in SEQ ID NO: 15, and the structure of the fusion protein is shown in FIG. 3 .

In some specific embodiments, the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 18, and the second polypeptide comprises SEQ ID NO: Amino acid sequence shown in ID NO:15. In some specific embodiments, the fusion protein comprises two first polypeptides and two second polypeptides, wherein the first polypeptides comprise the amino acid sequence shown in SEQ ID NO: 18, and the second The polypeptide comprises the amino acid sequence shown in SEQ ID NO:15. In some specific embodiments, the fusion protein consists of two first polypeptides and two second polypeptides, wherein the first polypeptides comprise the amino acid sequence shown in SEQ ID NO: 18, the second The two polypeptides comprise the amino acid sequence shown in SEQ ID NO:15. In a specific embodiment, the fusion protein is composed of two first polypeptides and two second polypeptides, wherein the amino acid sequence of the first polypeptides is shown in SEQ ID NO: 18, and the first polypeptide The amino acid sequence of the two polypeptides is shown in SEQ ID NO:15.

GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGKETLQRADPPKTHVTHHPVSRHEATLRCWALGFYPAEITLTWQRDGKDQTQ KTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSEPKSSDKTHTCPPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 14);

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLECVLNKATNVAHWTTPSLKCIRIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDRGP (SEQ ID NO: 15).

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGKETLQRADPPKTHVTHHPVSRHEATLRCWALGFYPAEITLTWQRDGKDQTQKTELVETRPAGDRTFQKWAAV VVPSGEEQRYTCHVQHEGLPKPLTLRWEPSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18)

pharmaceutical composition

The present invention provides a pharmaceutical composition, which comprises the fusion protein described in the present invention and one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers or other agents.

isolated nucleic acid

The invention provides isolated nucleic acids encoding the fusion proteins described herein. The nucleic acid sequences of the polypeptide chains of some fusion proteins are exemplarily listed in the sequence listing.

carrier

The invention provides vectors comprising the isolated nucleic acids described herein. In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector, a specific example, the expression vector is pcDNA3.1. The expression vector can optionally be any expression vector capable of expressing the fusion protein described herein.

host cell

The invention provides a host cell comprising an isolated nucleic acid or vector of the invention. Host cells are suitable host cells for cloning or encoding fusion proteins. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for making fusion proteins. Mammalian cells are, for example, Chinese Hamster Ovary (CHO) cells, CHO-S cells.

Preparation of fusion protein

The present invention provides a method for preparing the fusion protein of the present invention, which comprises culturing the host cell under the condition that the fusion protein is expressed, and isolating and purifying the expressed fusion protein.

In order to produce the fusion protein, the nucleic acid encoding the fusion protein is isolated and inserted into one or more vectors for further cloning or/and expression in host cells. The nucleic acid can be obtained by various methods well known in the art, such as gene splicing and chemical synthesis.

As a specific example, the nucleic acid sequences encoding the first polypeptide and the second polypeptide of the fusion protein of the present invention are respectively shown in SEQ ID NO: 16 and 17, and the encoded amino acid sequences are shown in SEQ ID NO: 14 and 15 respectively Show. In certain embodiments, when the fusion protein is expressed in a host cell, the propeptide sequence of the first polypeptide (i.e., amino acid residues 1 to 19 of the amino acid sequence shown in SEQ ID NO: 14) After being cleaved by an enzyme, the amino acid sequence of the first polypeptide in the expression product is shown in SEQ ID NO:18.

use

The present invention provides the use of the fusion protein described in the present invention in the preparation of medicines for treating diseases of subjects.

The present invention also provides the use of the pharmaceutical composition described in the present invention in the preparation of medicines for treating diseases of subjects.

The present invention also provides a method for treating a disease in a subject in need, comprising administering a therapeutically effective amount of the fusion protein or pharmaceutical composition of the present invention to the subject.

In some embodiments, the disease is selected from autoproliferative diseases, neoplastic diseases, bacterial diseases, viral diseases, immune diseases. In some embodiments, the disease is an IL-15-related disease. In some embodiments, the IL-15-related disease is selected from smallpox virus infection, HIV infection, bacterial infection, fungal infection, HBV infection, melanoma, colorectal cancer, skin cancer, lymphoma, renal cell carcinoma, thyroid cancer , brain cancer, liver cancer, lung cancer, stomach cancer, breast cancer, bladder cancer, prostate cancer, multiple myeloma, esophagus cancer, pancreatic cancer, testicular cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, leukemia, Anemia, myelodysplastic syndrome, multiple sclerosis, psoriasis, rheumatoid arthritis, gastritis, and mucositis.

The present invention also provides the following specific embodiments, but protection scope of the present invention is not limited thereto:

Embodiment 1. A fusion protein comprising a first polypeptide and a second polypeptide, wherein

The first polypeptide comprises a first biologically active peptide, a first pairing domain operably linked to the first biologically active peptide, and a first pairing domain operably linked to the first biologically active peptide from the N-terminus to the C-terminus. Fc polypeptide, and

The second polypeptide comprises a second biologically active peptide and a second pairing domain operably linked to the second biologically active peptide from the N-terminus to the C-terminus,

Wherein, one of the first pairing domain and the second pairing domain includes the amino acid sequence of engineered HLA-Iα3, and the other pairing domain includes the amino acid sequence of engineered β2 microglobulin;

The first pairing domain and the second pairing domain are capable of forming a dimer, and the first bioactive peptide and the second bioactive peptide are capable of forming a dimer;

The amino acid sequence of the engineered HLA-I α3 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, the amino acid sequence of the engineered β2 microglobulin has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

Embodiment 2. The fusion protein of embodiment 1, wherein the fusion protein comprises two of the first polypeptides and two of the second polypeptides, the Fc polypeptides of the two first polypeptides are associated to form Fc domain.

Embodiment 3. The fusion protein according to embodiment 1 or 2, wherein the first pairing domain comprises the amino acid sequence of engineered HLA-Iα3 and the second pairing domain comprises engineered β2 microglobulin amino acid sequence.

Embodiment 4. The fusion protein of embodiment 1 or 2, wherein the first pairing domain comprises the amino acid sequence of engineered β2 microglobulin and the second pairing domain comprises engineered HLA-1 Amino acid sequence of α3.

Embodiment 5. The fusion protein according to any one of embodiments 1-4, wherein at least one non-native interchain bond is capable of forming between the first pairing domain and the second pairing domain and the non-native Interchain linkages are able to stabilize the dimer.

Embodiment 6. The fusion protein of embodiment 5, wherein the amino acid residue forming the non-native interchain bond is at the contact interface of the first pairing domain and the second pairing domain.

Embodiment 7. The fusion protein according to embodiment 5 or 6, wherein the number of non-native interchain bonds is 1-3, preferably 1.

Embodiment 8. The fusion protein according to any one of embodiments 5-7, wherein the non-native interchain linkage is a disulfide linkage.

Embodiment 9. The fusion protein according to any one of embodiments 1-8, wherein said engineered HLA-Iα3 comprises an amino acid sequence having amino acid substitutions in SEQ ID NO: 1, said engineered β2 micron The globulin comprises an amino acid sequence having amino acid substitutions in SEQ ID NO:2.

Embodiment 10. The fusion protein according to embodiment 9, wherein the amino acid substitutions in the engineered HLA-I α3 and the engineered β2 microglobulin both comprise the interface between the two and are capable of forming Cysteine residue substitution for disulfide bonds.

Embodiment 11. The fusion protein of embodiment 10, wherein the cysteine residue substitutions are selected from one to at most pairs of the group consisting of:

(1) R60C in SEQ ID NO:1 and Y26C in SEQ ID NO:2;

(2) A62C in SEQ ID NO:1 and R12C in SEQ ID NO:2; and

(3) G63C in SEQ ID NO:1 and Y67C in SEQ ID NO:2.

Embodiment 12. The fusion protein according to any one of embodiments 9-11, wherein the amino acid substitutions in the engineered HLA-I α3 or/and engineered β2 microglobulin comprise improved pairing domain formation Amino acid substitution of the isoelectric point of the dimer or fusion protein.

Embodiment 13. The fusion protein according to embodiment 12, wherein the isoelectric point of the dimer or fusion protein formed by the pairing domain is increased to 6.5-9.0.

Embodiment 14. The fusion protein according to embodiment 12 or 13, wherein the amino acid substitutions that increase the dimer formed by the pairing domain or the isoelectric point of the fusion protein comprise: engineered HLA-I α3 in SEQ ID NO: One or several positions in E3, D22, E48, D53, E90 and E101 of the 1 sequence were replaced by positively charged amino acids.

Embodiment 15. The fusion protein according to any one of embodiments 12-14, wherein the amino acid substitution to increase the dimer formed by the pairing domain or the isoelectric point of the fusion protein comprises: in engineered β2 microglobulin One or several positions in E74, E47, E69, D34, E16, D53, E44, E50 and E36 of the sequence of SEQ ID NO: 2 are substituted with positively charged amino acids.

Embodiment 16. The fusion protein according to embodiment 15, wherein the amino acid substitution to increase the dimer formed by the pairing domain or the isoelectric point of the fusion protein comprises: said engineered HLA-I α3 in SEQ ID NO: Positions D22, E48 and D53 of the 1 sequence are substituted with positively charged amino acids, and the engineered β2 microglobulin is substituted with positively charged amino acids at position E69 of the sequence of SEQ ID NO:2.

Embodiment 17. The fusion protein according to any one of embodiments 14-16, wherein the positively charged amino acid is K or R.

Embodiment 18. The fusion protein according to embodiment 12, wherein the amino acid substitution to increase the dimer formed by the pairing domain or the isoelectric point of the fusion protein comprises: said engineered HLA-I α3 in SEQ ID NO: 1 sequence comprises amino acid substitutions D22R, E48K and D53K, and said engineered β2 microglobulin comprises amino acid substitution E69R in the sequence of SEQ ID NO:2.

Embodiment 19. The fusion protein according to any one of embodiments 1-4, wherein the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO: 3, and the engineered β2 microsphere The protein comprises the amino acid sequence shown in SEQ ID NO:5 or 6.

Embodiment 20. The fusion protein according to any one of embodiments 1-4, wherein the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO: 4, and the engineered β2 microsphere The protein comprises the amino acid sequence shown in SEQ ID NO:5 or 6.

Embodiment 21. The fusion protein according to any one of embodiments 1-4, wherein the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO:7, and the engineered β2 microsphere The protein comprises the amino acid sequence shown in SEQ ID NO:9 or 10.

Embodiment 22. The fusion protein according to any one of embodiments 1-4, wherein the engineered HLA-I α3 comprises the amino acid sequence shown in SEQ ID NO:8, and the engineered β2 microsphere The protein comprises the amino acid sequence shown in SEQ ID NO:9 or 10.

Embodiment 23. The fusion protein according to any one of embodiments 1-22, wherein

One of the first biologically active peptide and the second biologically active peptide is IL-15 with a propeptide attached to the N-terminal, and the other is IL-15Rα sushi domain; or the first biologically active peptide and the second biologically active peptide One of the active peptides is IL-15, and the other is IL-15Rα sushi domain.

Embodiment 24. The fusion protein of embodiment 23, wherein

The first bioactive peptide is IL-15 with a propeptide attached to the N-terminal, and the second bioactive peptide is IL-15Rα sushi domain; or the first bioactive peptide is IL-15, and the second bioactive peptide It is IL-15Rα sushi domain.

Embodiment 25. The fusion protein of embodiment 23, wherein

The first bioactive peptide is the IL-15Rα sushi domain, and the second bioactive peptide is IL-15 with a propeptide attached to the N-terminal; or the first bioactive peptide is the IL-15Rα sushi domain, and the second The bioactive peptide is IL-15.

Embodiment 26. The fusion protein according to any one of embodiments 23-25, wherein the N-terminal propeptide-linked IL-15 comprises at least 85% of the amino acid sequence shown in SEQ ID NO: 11, Amino acid sequences that are 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

Embodiment 27. The fusion protein according to any one of embodiments 23-25, wherein said IL-15 comprises at least 85%, 86%, 87%, 88% of the amino acid sequence shown in SEQ ID NO: 12 Amino acid sequences that are %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

Embodiment 28. The fusion protein according to any one of embodiments 23-27, wherein the IL-15Rα sushi domain comprises at least 85%, 86%, 87% of the amino acid sequence shown in SEQ ID NO: 13 Amino acid sequences that are 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

Embodiment 29. The fusion protein according to any one of embodiments 23-25, wherein the IL-15 with a propeptide attached to its N-terminus comprises the amino acid sequence shown in SEQ ID NO: 11, and the IL-15Rα The sushi domain comprises the amino acid sequence shown in SEQ ID NO:13.

Embodiment 30. The fusion protein according to any one of embodiments 23-25, wherein the IL-15 comprises the amino acid sequence shown in SEQ ID NO: 12, and the IL-15Rα sushi domain comprises SEQ ID NO : the amino acid sequence shown in 13.

Embodiment 31. The fusion protein according to any one of embodiments 2-30, wherein said Fc domain is an IgG Fc domain, preferably a human IgG Fc domain.

Embodiment 32. The fusion protein of embodiment 31, wherein the Fc domain is a human IgGl Fc domain or a human IgG4 Fc domain.

Embodiment 33. The fusion protein according to any one of embodiments 2-32, wherein the two Fc polypeptides are separated by a linker, disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, hydrophobic-hydrophilic interaction One or several stable associations.

Embodiment 34. The fusion protein of any one of embodiments 31-33, wherein the Fc domain comprises a modification that increases its binding to FcRn.

Embodiment 35. The fusion protein according to any one of embodiments 31-34, wherein said IgG Fc domain comprises amino acid substitutions M252Y, S254T and T256E according to the EU numbering system.

Embodiment 36. The fusion protein according to any one of embodiments 31-35, wherein the Fc domain comprises a modification that reduces or eliminates its binding to FcyI, FcyIIA, FcyIIB, FcyIIIA and/or FcyIIIB.

Embodiment 37. The fusion protein according to any one of embodiments 31-36, wherein the IgG Fc domain comprises an amino acid substitution of Ala at both positions 234 and 235 according to the EU numbering system.

Embodiment 38. The fusion protein of embodiment 1 or 2, wherein

The first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 14, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 15; or

The first polypeptide comprises the amino acid sequence shown in SEQ ID NO:18, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:15.

Embodiment 39. An isolated nucleic acid encoding the fusion protein of any one of embodiments 1-38.

Embodiment 40. A vector comprising the isolated nucleic acid of embodiment 39.

Embodiment 41. A host cell comprising the isolated nucleic acid of embodiment 39, or the vector of embodiment 40.

Embodiment 42. A method for preparing the fusion protein according to any one of embodiments 1-38, which comprises culturing the host cell according to embodiment 41 under the condition that the fusion protein is expressed, and isolating and purifying Expressed fusion protein.

Embodiment 43. A pharmaceutical composition comprising the fusion protein of any one of embodiments 1-38 and a pharmaceutically acceptable carrier.

Embodiment 44. Use of the fusion protein of any one of embodiments 1-38 in the manufacture of a medicament for treating a disease in a subject.

Embodiment 45. Use of the pharmaceutical composition of embodiment 43 for the preparation of a medicament for treating a disease in a subject.

Embodiment 46. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion protein of any one of embodiments 1-38 or the fusion protein of embodiment 43. pharmaceutical composition.

Embodiment 47. Use of the fusion protein of any one of embodiments 23-38 in the manufacture of a medicament for the treatment of IL-15-related diseases.

Embodiment 48. The use according to embodiment 47, wherein the IL-15-related disease is selected from smallpox virus infection, HIV infection, bacterial infection, fungal infection, HBV infection, melanoma, colorectal cancer, skin Cancer, Lymphoma, Renal Cell Carcinoma, Thyroid Cancer, Brain Cancer, Liver Cancer, Lung Cancer, Stomach Cancer, Breast Cancer, Bladder Cancer, Prostate Cancer, Multiple Myeloma, Esophageal Cancer, Pancreatic Cancer, Testicular Cancer, Ovarian Cancer, Cervical Cancer , endometrial cancer, head and neck cancer, leukemia, anemia, myelodysplastic syndrome, multiple sclerosis, psoriasis, rheumatoid arthritis, gastritis, and mucositis.

The present invention will be further described below in conjunction with specific examples, however, these examples in the present invention are only for illustration and do not limit the scope of the present invention. Likewise, the invention is not limited to any specific preferred embodiments described herein. Those skilled in the art should understand that equivalent replacements or corresponding improvements to the technical features of the present invention still fall within the protection scope of the present invention. Unless otherwise specified, the reagents used in the following examples are all commercially available products, and the preparation of the solution can adopt conventional techniques in the art.

Unless otherwise specified, the practice of the present invention will use conventional techniques of biochemistry, protein purification, etc. within the skill of the art, or in accordance with product instructions, and the techniques are fully explained in the literature. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. In this example, the fusion protein ALT-803 (structure shown in Figure 2 ) was used as a reference substance, and its amino acid sequence was shown in SEQ ID NO: 2 and 5 in the patent application US20190022187A1; hIgG1 was used as an isotype control.

Embodiment 1: Design of MHC-I element sequence

According to IPD-IMGT/HLA (https://www.ebi.ac.uk/ipd/imgt/hla/stats.html) database (version 3.42) statistics (Table 1), relative to HLA-II, HLA-I has More allele numbers and wider distribution, so HLA-I molecules are selected to design the structural elements of the fusion protein to reduce the risk of immunogenicity.

According to IPD-IMGT/HLA database (version 3.42) statistics (table 2), in HLA-I molecule, the quantity of HLA-I-B allele is maximum, therefore selects the structural element of HLA-I-B molecular design fusion protein, to further reduce Immunogenicity Risk.

Table 1. Number of HLA alleles

allele number HLA-I 20,597 HLA-II 7,723 HLA 28,320

Table 2. HLA-I alleles and proteins

type A B C E. f G allele 6,291 7,562 6,223 256 45 82 protein 3,896 4,803 3,618 110 6 twenty two Invalid (nulls) 325 253 272 7 0 4

1.1 Determination of the initial sequence of MHC-I elements

(表4)。In the Uniprot (https://www.uniprot.org/) database, obtain a series of natural HLA-IB molecular α chain full-length sequences (Table 3); in the Uniprot database, obtain the HLA-I molecule β chain (also known as β-microglobulin) full-length sequence (Uniprot ID: P61769). Through the cross-reference system of the Uniprot database page, the protein crystal data information containing the α-chain of the HLA-IB molecule was further obtained. In order to ensure the accuracy of the crystal data, a high-resolution crystal structure was selected.

(Table 4).

Through sequence analysis, combined with the information of these crystal structures, the relative starting position of the constant region of the α chain of the natural HLA-I-B molecule (defined here as MHCICα, constant region alpha of MHC I, or Cα for short) was determined. There is a certain sequence diversity in the constant region of the α chain of HLA-I-B molecules. By using the online comparison tool ClustalW2 (https://www.ebi.ac.uk/Tools/phylogeny/simple_phylogeny/), the sequences in Table 3 were multiplexed. Sequence alignment, and then analyze and display the alignment results through webLogo (http://weblogo.berkeley.edu/), and obtain the consistency of the constant region of the α chain of the HLA-I-B molecule according to the frequency of amino acids at each position sequence (Figure 1), and the consensus sequence of the constant region of the HLA-I-B molecule α chain was used as the initial sequence for subsequent operations and transformation. Since human β2 microglobulin has no sequence diversity, by analyzing the sequence (Uniprot ID: P61769), determine the pairing region of natural β2 microglobulin and MHCICα (defined here as MHCICβ, constant region beta of MHC I, or Cβ for short )the sequence of.

The initial sequence of the MHC-I element is as follows:

>MHC ICαori (SEQ ID NO:1):

GKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPS

>MHC ICβori (SEQ ID NO:2):

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR

Table 3. The sequence information of the natural HLA-I-B molecular α chain contained in the Uniprot database

serial number Uniprot accession number 1 A0A140T9S9 2 A0A140T9G0 3 A0A140T9H3 4 A0A140T9A9 5 A0A140T951 6 A0A1W2PPR8 7 D9J307 8 S6AU73 9 Q2L6G2 10 D5H3J5 11 Q5SS57 12 P30685 13 P01889

Table 4. Crystal structures of proteins containing the α-chain of HLA-I-B molecules

1.2 Optimization of the initial sequence of MHC-I elements

a) Introduction of interchain disulfide bonds to enhance pairing stability of MHC-I elements

The above-mentioned MHCICαori and MHCICβori amino acid sequences were simultaneously loaded into the MOE (Molecular Operating Environment) software, and heterodimer modeling was carried out through the Homology modeling module of MOE to obtain the simulated structure of the MHC-I element. In the natural state, there are no disulfide bonds between the constant regions of MHC molecules. In order to improve the pairing stability of the MHC-I constant region and optimize its developability, on the domain contact interface of MHCICα and MHCICβ, for MHCICαori, cysteine (Cys) can be introduced at the R60, A62 or G63 position; for MHCICβori, Cys at Y26, R12 or Y67 position. Through the Disulfide scan module of MOE, the deltaStability after the introduction of the disulfide bond was simulated and calculated. The A62C mutation was introduced into the MHCICαori, and the R12C mutation was introduced into the MHCICβori. The most stable disulfide bonds can be formed at the interface of Cα and Cβ (Table 5).

Table 5. Simulation results of interfacial disulfide bond introduction of MHCICαori and MHCICβori

MHC ICαori MHC ICβori delta Stability(kcal/mol) R60C Y26C 1.96 A62C R12C -1.62 G63C Y67C 1.86

b) Isoelectric point optimization

The theoretical isoelectric point (pI, isoelectric point) of the MHC-I element after introducing disulfide bonds (A62C and R12C in Table 5) into the initial sequence is 5.80, which is not conducive to the later process development. Therefore, we carried out relevant charge modification on MHC-I components to improve their druggability. The MHC-I element was analyzed by the Protein-properties module of the MOE software for solvent accessible surface (SAS, solvent accessible surface) and charged amino acid residues. In the MHC-I element, amino acid residues with a solvent exposure degree of >40% and exhibiting negative charge/acidity are shown in Table 6. At the same time, in combination with the structural environment, chemical environment and application purpose of the relevant residues, one or more of these acidic amino acid residues are replaced with basic amino acid residues (arginine (R) or lysine (K) ), the theoretical isoelectric point of the MHC-I element is increased to 6.5-9.0. In a specific scheme, in MHCICα, amino acid substitutions D22R, E48K, and D53K are introduced; in MHCICβ, amino acid substitutions E69R are introduced to increase the theoretical isoelectric point of MHC-I elements to 7.8.

The MHC-I element sequence optimized by a) and b) is as follows:

>MHC ICα1 (SEQ ID NO:4):

GKETLQRADPPKTHVTHHPISRHEATLRCWALGFYPAEITLTWQRDGKDQTQKTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPS

>MHCICβ1 (SEQ ID NO:6):

IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDR

Table 6. MHC-I element SAS and residue charge properties molecules

c) Other optimizations

Considering the 4 amino acids at the end of the Cβ region (-KWDR), their side chains are large, which may cause potential steric hindrance. If a flexible G4S linking peptide is used, the stability of the protein conformation will be reduced. The structural characteristics of glycine (G) and proline (P) determine that these two amino acids are mainly located at random coil or corner positions, and secondly, glycine has no side chain group, which can well alleviate the steric hindrance effect, while proline The side chain of the acid is a five-membered ring structure, which has a certain rigidity and can improve conformational stability. Therefore, GP is finally added to the carboxyl terminus of the Cβ region to balance potential steric hindrance effects and conformational stability.

The sequence of the terminally modified MHCICβ element is as follows:

>MHCICβ1_GP (SEQ ID NO:5)

IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDRGP

d) Another optimization scheme

Based on the initial sequence of the MHC-I element in 1.1, refer to the optimization scheme of 1.2b) and c), and introduce the amino acid substitution I20V in MHCICα, another optimized MHC-I element is obtained, and its sequence is as follows:

>MHC ICα2 (SEQ ID NO:8):

GKETLQRADPPKTHVTHHPVSRHEATLRCWALGFYPAEITLTWQRDGKDQTQKTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPS

>MHC ICβ2 (SEQ ID NO: 10):

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDR

>MHCICβ2_GP (SEQ ID NO:9)

IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTRFTPTEKDEYACRVNHVTLSQPKIVKWDRGP

Example 2. Construction of pro15MHCin fusion protein and protein expression and purification

The fusion protein pro15MHCin consists of two first polypeptides and two second polypeptides. The genes encoding the first polypeptide and the second polypeptide of the fusion protein (the nucleic acid sequences are shown in SEQ ID NO: 16 and 17 respectively, and the amino acid sequences encoded by it are shown in SEQ ID NO: 14 and 15 respectively) are artificially degenerated Synthesize, respectively insert vectors (such as the pcDNA3.1 vector disclosed in CN107001463A, the pCHO1.0 vector disclosed in CN109422811A, etc.), and construct the expression vectors expressing the corresponding polypeptide chains. The expression vectors encoding the first polypeptide and the second polypeptide were co-transfected into ExpiCHO-S cells (manufacturer: Shanghai Institute of Pharmaceutical Industry, article number: 127200005) at a mass ratio of 1:1, and the transfected expression volume was 0.1- 1L, the cell density is 6E+06cells/mL.

After transfection, the cells were expressed and cultured at 32°C, 5% CO ₂ , and 130rpm. On the 10th day, the cell supernatant was collected, and the target protein was captured using the AKTAPure 25L system and MabSelect SuRe LX filler from GE. In this experiment, 0.1M NaOH solution was used to clean the filler, and then equilibrated with AC-A (50mM Tris-HAc, 150mMNaCl, pH 7.4) for 3 column volumes (CV). After loading and equilibrating, use AC-B (50mM NaAc-HAc, 1M NaCl, pH5.5) rinse 3CV, equilibrate with phase A: AC-C1 (50mM NaAc-HAc, pH 5.0), phase B: AC-C2 (50mM NaAc-HAc, pH 3.0 ), 0-100% B, and an elution volume of 20CV for gradient elution. After elution, NanoDrop Lite (Thermo Fisher Scientific) was used to measure the protein concentration of the eluted harvest, and SEC-HPLC was used to detect the purity of the eluted protein. Use 3CV AC-D (1M HAc) to regenerate the column, fill the regenerated column with 20% ethanol and store it in a refrigerator at 2-8°C.

Example 3. Detection of the proliferation effect of fusion protein on PBMC by CTL luminescence method

Luminescent Cell Viability Assay(厂家：Promega，货号：G7572)检测试剂震荡孵育2min，室温孵育10min后酶标仪(厂家：Thermo，型号：Variosken Flash)检测。Draw a corresponding volume of protein sample according to the concentration of the fusion protein, and prepare a 200nM 60μL protein solution with RPMI 1640 (manufacturer: Gibco, article number: 22400-089) + 10% FBS (manufacturer: Gibco, article number: 10099-141C). The protein solution was serially diluted 3 times. Resuscitate human peripheral blood mononuclear cells (PBMC), add 10mL preheated complete medium to resuscitate the cells, centrifuge at 1330rpm for 10min; add RPMI 1640+10% FBS to resuspend the cells for counting, take 9E+06 cells and adjust the cell density to 5E+05cells/mL, add 90 μL of cell suspension to each well of the 96-well plate and detach the cells immediately, culture at 37°C for 2 hours, then add 10 μL of the above-mentioned gradiently diluted protein solution sample to each well according to the layout of the well plate, and continue to store at 37°C Cultivate for 3 days. After incubation, add 100 μL to each well

Luminescent Cell Viability Assay (manufacturer: Promega, product number: G7572) detection reagent was incubated with shaking for 2 minutes, incubated at room temperature for 10 minutes, and then detected by a microplate reader (manufacturer: Thermo, model: Variosken Flash).

The results of the PBMC cell proliferation experiment ( FIG. 4 ) showed that when the concentration of the control substance ALT-803 was ≥1.0 nM, it would significantly inhibit the proliferation of PBMC. However, pro15MHCin has a pro-proliferative effect on PBMC, and does not show any proliferation-inhibiting phenomenon. Compared with ALT-803, pro15MHCin has better safety.

Example 4. Using CTL luminescence method to detect the proliferation effect of fusion protein on NK cells

Luminescent Cell Viability Assay(厂家：Promega，货号：G7572)检测试剂震荡孵育2min，室温孵育10min后酶标仪(厂家：Thermo，型号：Variosken Flash)检测。The preparation of protein samples refers to the method in Example 3. Resuscitate NK cells (manufacturer: Schbio, article number: PB56005C), wash twice with RPMI 1640 (manufacturer: Gibco, article number: 22400-089)+20% FBS (manufacturer: Gibco, article number: 10099-141C). Add RPMI 1640+10% FBS to resuspend the cells, adjust the cell density to 5E+05cells/mL, add 90 μL of cell suspension to each well of the 96-well plate and spin off the cells, culture at 37°C for 2-4 hours and press the well plate 10 μL of protein samples were added to each well, and cultured at 37°C for 3 days. After incubation, add 100 μL to each well

Luminescent Cell Viability Assay (manufacturer: Promega, product number: G7572) detection reagent was incubated with shaking for 2 minutes, incubated at room temperature for 10 minutes, and then detected by a microplate reader (manufacturer: Thermo, model: Variosken Flash).

The results of the NK cell proliferation experiment (Figure 5) showed that when the concentration of the fusion protein was ≥0.1nM, compared with ALT-803, pro15MHCin had a better effect on promoting the proliferation of NK cells.

Example 5. Detection of the proliferative effect of the fusion protein on CD8+ T cells by CTL luminescence method

CD8+ T cells were sorted according to the instructions of EasySep ^TM Human CD8+T Cell Isolation Kit (manufacturer: Stemcell, product number: 17953). Resuscitate 2 human PBMCs, add 10 mL medium (RPMI 1640 (manufacturer: Gibco, product number: 22400-089) + 20% FBS (manufacturer: Gibco, product number: 10099-141C)), centrifuge at 1330 rpm for 10 min, discard the supernatant; add 900 μL PBS + 100 μL DNaseI (manufacturer: Stemcell, product number: 07900) for 15 min at room temperature; use 1×EasySep buffer, filter the cells with a 40 μm filter membrane, and perform cell counting. Take an appropriate amount of cell suspension, add 1×EasySep buffer according to 1mLEasySep buffer/5E+07cells and resuspend in the flow tube. Add Enrichment Cocktail according to 50μL Enrichment Cocktail/mL cell suspension, incubate at room temperature for 5min. Vortex Magnetic Particles thoroughly, add Magnetic Particles according to 50 μL Magnetic Particles/mL cell suspension, resuspend cells, and mix well. Make up the volume of 1×EasySep buffer to 2.5mL, put the flow tube on the magnetic stand, and let it stand at room temperature for 2.5min. Pour the supernatant into a new centrifuge tube, add medium to 10mL, centrifuge, resuspend with 1mL medium, and count the cells.

Luminescent Cell Viability Assay检测试剂震荡孵育2min，室温孵育10min后酶标仪(厂家：Thermo，型号：Variosken Flash)检测。The preparation of protein samples refers to the method in Example 3. Adjust the density of the sorted CD8+ T cells to 5E+05 cells/mL, add 90 μL of cell suspension to each well of a 96-well plate and centrifuge the cells briefly, culture at 37°C for 2-4 hours, and arrange each well according to the well plate Add 10 μL protein sample and continue to culture at 37°C for 3 days. After incubation, add 100 μL to each well

The Luminescent Cell Viability Assay detection reagent was incubated with shaking for 2 minutes, incubated at room temperature for 10 minutes, and then detected by a microplate reader (manufacturer: Thermo, model: Variosken Flash).

The results of the CD8+ T cell proliferation experiment (Figure 6) showed that when the concentration of the fusion protein was ≥1.0 nM, compared with ALT-803, pro15MHCin had a comparable proliferation-promoting effect on CD8+ T cells.

Claims (10)

1. A fusion protein comprising a first polypeptide and a second polypeptide, wherein

The first polypeptide comprises, from N-terminus to C-terminus, a first bioactive peptide, a first pairing domain operably linked to the first bioactive peptide, and an Fc polypeptide operably linked to the first pairing domain, and

the second polypeptide comprises, from N-terminus to C-terminus, a second bioactive peptide and a second pairing domain operably linked to the second bioactive peptide,

wherein one of the first and second mating domains comprises an amino acid sequence of an engineered HLA-iα3 and the other mating domain comprises an amino acid sequence of an engineered β2 microglobulin;

The first and second mating domains are capable of forming a dimer, and the first and second bioactive peptides are capable of forming a dimer;

the amino acid sequence of the engineered HLA-I.alpha.3 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 1, and the amino acid sequence of the engineered beta.2 microglobulin has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 2; alternatively, the process may be carried out in a single-stage,

the fusion protein comprises two of the first polypeptides and two of the second polypeptides, the Fc polypeptides of the two first polypeptides being associated to form an Fc domain.

2. The fusion protein of claim 1, wherein the engineered HLA-iα3 comprises an amino acid sequence having an amino acid substitution in SEQ ID No. 1 and the engineered β2 microglobulin comprises an amino acid sequence having an amino acid substitution in SEQ ID No. 2.

3. The fusion protein of claim 2, wherein the amino acid substitutions in the engineered HLA-iα3 or/and engineered β2 microglobulin comprise amino acid substitutions that increase the isoelectric point of the dimer or fusion protein formed by the mating domain; alternatively, the isoelectric point of the dimer or fusion protein formed by the paired domains is increased to 6.5-9.0.

4. A fusion protein according to claim 3 wherein the amino acid substitution that increases the isoelectric point of the dimer or fusion protein formed by the partner domain comprises: the engineered HLA-I alpha 3 is substituted by positively charged amino acids at one or more positions E3, D22, E48, D53, E90 and E101 of the sequence of SEQ ID NO. 1, or/and

an engineered β2 microglobulin having one or more positions E74, E47, E69, D34, E16, D53, E44, E50 and E36 of the sequence SEQ ID NO. 2 replaced with a positively charged amino acid; preferably, the method comprises the steps of,

amino acid substitutions that increase the isoelectric point of the dimer or fusion protein formed by the paired domains comprise: the engineered HLA-I.alpha.3 is substituted with positively charged amino acids at positions D22, E48 and D53 of the sequence of SEQ ID NO. 1, and the engineered beta.2 microglobulin is substituted with positively charged amino acids at position E69 of the sequence of SEQ ID NO. 2; optionally, the positively charged amino acid is K or R; more preferably, the amino acid substitution that increases the isoelectric point of the dimer or fusion protein formed by the paired domains comprises: the engineered HLA-I.alpha.3 comprises the amino acid substitutions D22R, E K and D53K in the SEQ ID NO. 1 sequence and the engineered beta.2 microglobulin comprises the amino acid substitution E69R in the SEQ ID NO. 2 sequence.

5. The fusion protein of claim 1, wherein:

the engineering HLA-I alpha 3 comprises an amino acid sequence shown as SEQ ID NO. 3, and the engineering beta 2 microglobulin comprises an amino acid sequence shown as SEQ ID NO. 5 or 6;

the engineering HLA-I alpha 3 comprises an amino acid sequence shown as SEQ ID NO. 4, and the engineering beta 2 microglobulin comprises an amino acid sequence shown as SEQ ID NO. 5 or 6;

the engineering HLA-I alpha 3 comprises an amino acid sequence shown as SEQ ID NO. 7, and the engineering beta 2 microglobulin comprises an amino acid sequence shown as SEQ ID NO. 9 or 10; or (b)

The engineering HLA-I alpha 3 comprises an amino acid sequence shown as SEQ ID NO. 8, and the engineering beta 2 microglobulin comprises an amino acid sequence shown as SEQ ID NO. 9 or 10.

6. The fusion protein of any one of claims 1-5, wherein

One of the first bioactive peptide and the second bioactive peptide is IL-15 with a propeptide connected with an N end, and the other is IL-15 Ralpha sushi structural domain; or (b)

One of the first bioactive peptide and the second bioactive peptide is IL-15, and the other is IL-15Rα sushi domain; preferably, the first bioactive peptide is IL-15 with a propeptide attached to the N-terminus, and the second bioactive peptide is IL-15Rα sushi domain;

The first bioactive peptide is IL-15, and the second bioactive peptide is IL-15Rα sushi domain;

the first bioactive peptide is an IL-15 Ralpha sushi domain, and the second bioactive peptide is IL-15 with a propeptide connected with the N end; or (b)

The first bioactive peptide is IL-15Rα sushi domain, and the second bioactive peptide is IL-15.

7. The fusion protein of claim 6, wherein the N-terminally propeptide-linked IL-15 comprises an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 11; or/and (or)

Wherein the IL-15 comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO 12; or/and (or)

The IL-15Rα sushi domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 13.

8. The fusion protein of claim 1, wherein

The first polypeptide comprises an amino acid sequence shown in SEQ ID NO. 14, and the second polypeptide comprises an amino acid sequence shown in SEQ ID NO. 15; or (b)

The first polypeptide comprises an amino acid sequence shown in SEQ ID NO. 18, and the second polypeptide comprises an amino acid sequence shown in SEQ ID NO. 15.

9. Use of the fusion protein of any one of claims 1-8 in the manufacture of a medicament for treating a disease in a subject.

10. Use of the fusion protein of any one of claims 6-8 in the manufacture of a medicament for the treatment of an IL-15 related disorder; preferably, the IL-15 related disease is selected from the group consisting of smallpox infection, HIV infection, bacterial infection, fungal infection, HBV infection, melanoma, colorectal cancer, skin cancer, lymphoma, renal cell carcinoma, thyroid cancer, brain cancer, liver cancer, lung cancer, stomach cancer, breast cancer, bladder cancer, prostate cancer, multiple myeloma, esophageal cancer, pancreatic cancer, testicular cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, leukemia, anemia, myelodysplastic syndrome, multiple sclerosis, psoriasis, rheumatoid arthritis, gastritis, and mucositis.

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