CN114634577A - Fusion protein and application thereof in preparing medicine for treating tumor and virus infection - Google Patents

Abstract

The invention relates to a fusion protein and its application for preparing a medicament for treating tumor and virus infection, the fusion protein comprises an antibody or an antigen binding fragment thereof specifically binding to human OX40 and human interferon IFN alpha-2 a or a mutant thereof; wherein the human interferon IFN α -2a or mutant thereof is linked directly or through a peptide linker to the carboxy-terminus or amino-terminus of the light or heavy chain of the antibody or antigen-binding fragment thereof that specifically binds to human OX 40. The fusion protein of the invention has the advantages of unique drug effect and compliance in the aspects of anti-tumor and anti-virus treatment.

Description

Fusion protein and application thereof in preparing medicine for treating tumor and virus infection

Technical Field

The invention belongs to the field of biological medicine. In particular, the invention relates to a fusion protein and uses thereof. More specifically, the invention relates to fusion proteins of anti-OX 40 antibodies and human interferons and their use in the preparation of medicaments for the treatment of tumors and/or viral infections.

Background

Human OX40 is mainly expressed on activated T cells, including CD4, CD8, Th and Treg cells, among others. In that

Expression of OX40 on T cells was low, but its expression levels were up-regulated after antigen-induced stimulation and peaked within 12 hours to 5-6 days. Similarly, expression of OX40L is also affected by the activation state of the cells. APC cells can detect expression of OX40L 1-3 days after antigen stimulation. Interestingly, in addition to immune cells, muscle cells also express OX40L under stimulation by inflammatory factors, suggesting that the OX40L-OX40 signaling pathway may be broadly active in the body's inflammatory response.

Activation of the antigen-dependent OX40L/OX40 co-stimulatory molecule was coupled to multiple signaling pathways within T cells. Crystal structure studies have shown that binding of OX40L to OX40 induces trimerization of the OX40-OX40L complex, thereby forming binding sites for receptor-associated factors (TRAFs) within the cell. The latter (TRAF2, 5) can further activate NF-kB signal channel and inhibit T cell apoptosis. It has been found that activation of OX40 can lead to high expression of Bcl-2 and Bcl-xL, suggesting that OX40 can realize the function of inhibiting T cell apoptosis by inducing expression of anti-apoptotic proteins through NF-kB signaling pathway.

PKB/PI3K is another important signal pathway downstream of OX 40. Studies have found that on the one hand, co-stimulatory signals of OX40 on T cells are essential for maintaining PKB activation, and on the other hand, constitutively activated PKB can antagonize down-regulation of anti-apoptotic proteins in T cells caused by defects in OX 40. OX40 costimulatory signals can maintain Survivin protein expression through the PKB/PI3K signaling pathway.

Finally, activation of TCR and OX40 on T cells can also synergistically cause activation of calcium flux and NFAT signaling pathways, modulating the expression of cytokines including IL-2, IL-4, IL-5, and IFN- γ.

In conclusion, the above studies indicate that the activation of OX40 can regulate the proliferation, apoptosis and cytokine secretion activity of T cells through NF-kB signal pathway, PKB/PI3K signal pathway and NFAT signal pathway, thereby achieving the effect of enhancing the activity of immune system.

Based on the above findings, OX40 has become an important target for immunotherapy, and numerous preclinical and clinical studies suggest that OX40 can be an important target for tumor immunotherapy. Interestingly, recent studies have also found a role for the OX40 signaling pathway in inhibiting hepatitis b virus infection, suggesting that OX40 agonists may be potential as a therapeutic approach for combating viral infections, such as hepatitis b patients.

Interferons are a highly active, multifunctional glycoprotein. In one aspect. The interferon can play a strong anti-tumor role by regulating and controlling the proliferation of tumor cells, inhibiting tumor metastasis and angiogenesis and activating anti-tumor immune response; on the other hand, interferon has important clinical application value in the aspect of antivirus by regulating the immune system of human body, for example, interferon becomes one of important means for clinically treating hepatitis B virus infection. Human interferons IFN alpha-2 a and IFN alpha-2 b are two types of human interferons. The 23 rd amino acid of human interferon IFN alpha-2 a is lysine (Lys), and unlike IFN alpha-2 a, the 23 rd amino acid of IFN alpha-2 b is arginine (Arg). Further, recombinant human INF α -2b exists as a non-covalent dimer crystal with zinc ion mediated dimer surface interactions. Of all INF α isoforms, only two INF α -2b, INF α -14c are glycosylated, INF α -2b being the only INF α isoform with a threonine residue at position 106 and also the only O-glycosylated human INF α protein. The structural difference may affect the immunogenicity and immunotoxicity of different IFN alpha-2 subtypes, and the prior art shows that IFN alpha-2 a and INF alpha-2 b have certain immunogenicity difference but no immunotoxicity difference.

OX40 agonist and interferon have important application values or potentials in the aspects of tumor resistance and virus resistance, but the existing evidence indicates that the OX40 agonist and the interferon have defects in the aspects of patient response rate and drug effect. Thus, there is a current need for more therapeutically effective OX40 agonists and interferons.

Disclosure of Invention

The present invention aims to provide a fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to human OX40 and human interferon IFN α -2 a. The invention also provides the use of the fusion protein for treating tumors and/or viral infections.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a fusion protein comprising:

a) an antibody or antigen-binding fragment thereof that specifically binds human OX 40; and

b) human interferon IFN alpha-2 a or mutants thereof;

wherein the human interferon IFN α -2a or mutant thereof is linked directly or through a peptide linker to the carboxy terminus or amino terminus of the light or heavy chain of the antibody or antigen-binding fragment thereof that specifically binds to human OX 40.

The fusion protein according to the invention, wherein the antibody or antigen-binding fragment thereof that specifically binds to human OX40 comprises:

a heavy chain variable region comprising 3 complementarity determining regions (HCDR1, HCDR2 and HCDR3) in a heavy chain variable region (VH) as set forth in SEQ ID NO:1, wherein each HCDR is defined as set forth in Table 1 of the following descriptive section of the present patent application; and

a light chain variable region comprising 3 complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the heavy chain variable region (VL) set forth in SEQ ID NO:2, wherein each LCDR is defined as set forth in Table 1 of the following description of the present application.

Preferably, the heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 1 and the light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 2.

The fusion protein according to the invention, wherein the antibody or antigen binding fragment thereof that specifically binds to human OX40 is a camelized single domain antibody, scFv dimer, BsFv, dsFv2, dsFv-dsFv ', Fv fragment, Fab ', F (ab ')2, ds diabody, nanobody, domain antibody, or diabody.

The fusion protein according to the invention, wherein the antibody further comprises an immunoglobulin constant region, such as the constant region of human IgG1, IgG2, or IgG 4.

The fusion protein of the invention, wherein the amino acid sequence of the human interferon IFN alpha-2 a is shown in SEQ ID NO. 3;

further preferably, the amino acid sequence of the human interferon IFN alpha-2 a mutant is mutated in the amino acid sequence shown in SEQ ID NO. 3 by one or more selected from the following:

T106A, R149A, a145G, a145D, R120A, or L117A;

still further preferably, the amino acid sequence of said mutant of human interferon IFN alpha-2 a comprises one or more double mutations selected from the group consisting of:

T106A/A145D, T106A/R149A, T106A/A145G, T106A/R120A or T106A/L117A.

The fusion protein according to the invention, wherein the peptide linker is selected from the group consisting of: (G)_n、KESGSVSSEQLAQFRSLD、EGKSSGSGSESKST、GSAGSAAGSGEF、(GGGGS)_nor (GGSGG)_n(ii) a Preferably, the peptide linker is (GGGGS)_nWherein n is an integer between 0 and 5; preferably, n is an integer between 1 and 3.

The fusion protein according to the invention, wherein the fusion protein is selected from the group consisting of:

UM06-L9, consisting of an antibody specifically binding to human OX40 comprising a heavy chain as set forth in SEQ ID NO. 5 and a light chain as set forth in SEQ ID NO.6, a peptide linker and human interferon IFN α -2a as set forth in SEQ ID NO. 3, wherein said human interferon IFN α 2a is linked to the carboxy terminus of the light chain of said antibody specifically binding to human OX40 by a peptide linker GGGGS.

UM06-L9.1 consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:7, a peptide linker and human interferon IFN alpha-2 a as shown in SEQ ID NO:3, wherein said human interferon IFN alpha 2a is linked via a peptide linker (GGGGS)₂Linking the carboxy terminus of the light chain of the antibody that specifically binds human OX 40.

UM06-L21 consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:9, a peptide linker and a human interferon IFN alpha-2 a mutant as shown in SEQ ID NO:8 (T106A/L117A), wherein the human interferon IFN alpha-2 a mutant is linked via a peptide linker (GGGGS)₂Is linked to the carboxy terminus of the light chain of the antibody that specifically binds human OX 40.

In another aspect, the invention provides an isolated polynucleotide encoding the fusion protein.

In yet another aspect, the invention provides a vector comprising the isolated polynucleotide.

In yet another aspect, the invention provides a host cell comprising the vector.

The invention also provides a method of expressing the fusion protein comprising culturing the host cell under conditions capable of expressing the isolated polynucleotide.

The invention also provides a kit comprising the fusion protein.

The invention also provides a pharmaceutical composition, which comprises the fusion protein and a pharmaceutically acceptable carrier.

The invention also provides the use of the fusion protein in the manufacture of a medicament for the treatment of a condition which can benefit from an enhanced immune response and/or from exposure to interferon.

Preferably, the condition is cancer or a viral infection, such as hepatitis B virus infection.

The invention also provides a method of treating a condition that can benefit from an enhanced immune response and/or from exposure to interferon, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of the fusion protein; preferably, the condition is cancer or a viral infection, such as hepatitis B virus infection.

The invention also provides a fusion protein for the treatment of a disorder that can benefit from an enhanced immune response and/or from exposure to interferon; preferably, the condition is cancer or a viral infection, such as hepatitis B virus infection.

The invention provides a fusion protein of OX40 agonist antibody and interferon, which combines different action mechanisms of the OX40 agonist antibody and interferon, and forms synergistic enhancement effect: on the one hand, when the interferon moiety in the fusion protein binds to the surface of a cell that highly expresses an interferon receptor (e.g., a tumor or virus-infected cell), it is possible to enhance the activity of OX 40-activating antibodies in a receptor-mediated manner, thereby better enhancing the immune system activity; on the other hand, the half-life of the fusion protein is greatly prolonged compared with that of interferon molecules, so that the fusion protein can be used for clinical administration with lower frequency, and has great advantages compared with the interferon which needs to be injected daily clinically at present.

In conclusion, the fusion protein of the invention has unique drug effect and compliance advantages in the aspects of anti-tumor and anti-virus treatment.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows a schematic structural diagram of a fusion protein according to the present invention.

FIG. 2 shows SDS-PAGE and HPLC detection profiles of the fusion proteins according to the present invention.

FIG. 3 shows the results of ELISA binding experiments of fusion proteins according to the invention with human OX40 protein.

FIG. 4 shows the proliferation inhibitory effect of the fusion protein according to the present invention on Daudi cells.

FIG. 5 shows that the fusion protein according to the present invention activates the NF-kB signaling pathway activity of Jurkat cells.

Fig. 6 shows a plasma concentration time profile of the fusion protein according to the present invention in mice.

Detailed Description

The following description of the present application is intended to be illustrative of various embodiments of the present application. Therefore, the specific embodiments discussed herein should not be construed as limiting the scope of the application. Numerous equivalents, changes, and modifications will readily occur to those skilled in the art without departing from the scope of the present application, and it is intended that such equivalents be included within the scope of the present invention. All documents, including publications, patents, and patent applications, cited in this application are incorporated by reference in their entirety.

Definition of

The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds a particular antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains as λ or κ. The antibody is "Y" shaped, with the neck of the Y structure consisting of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure includes the variable and first constant regions of one of the heavy chains, which are associated with the variable and constant regions of one of the light chains. The variable regions of the light and heavy chains determine the binding of the antigen. The variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs). The CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3, and the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR 3.

A "CDR" is a region of an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any one or combination of a number of well known antibody CDR assignment systems, including, for example: chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et Al, Nature 342:877-883 (1989); Al-Lazikani et Al, "Standard transformations for the structural of immunoglobulins", Journal of Molecular Biology,273,927-948 (1997)); kabat based on antibody sequence variability (Kabat et al, Sequences of Proteins of Immunological Interest, 4 th edition, u.s.department of Health and Human Services, National Institutes of Health (1987), abm (university of bath), contact (university College London), international ImmunoGeneTics database (IMGT) (on world wide web: circles. fr.)); and North CDR definition based on affinity propagation clustering with a large number of crystal structures.

For example, according to different CDR determination schemes, the residues of each CDR of an antibody in a fusion protein according to the present invention are defined as follows (table 1).

TABLE 1 antibody CDR definitions

Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses a combination of CDR sequences determined in any of the ways described above.

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the region of minimum overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be appreciated by those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure and protein folding of the antibody. Thus, the present invention also contemplates variants of any of the CDRs given herein. For example, in a variant of one CDR, the amino acid residue of the smallest binding unit may remain unchanged, while the remaining CDR residues according to Kabat or Chothia definition may be replaced by conserved amino acid residues.

The three CDRs are separated by flanking continuous portions called Framework Regions (FRs), which are more highly conserved than the CDRs and form a scaffold-supported hypervariable loop. The constant regions of the heavy and light chains are not involved in antigen binding, but have multiple effector functions. Antibodies can be classified into several classes depending on the amino acid sequence of the heavy chain constant region. Depending on whether it contains alpha, delta, epsilon, gamma and mu heavy chains, antibodies can be classified into five major classes or isoforms, respectively: IgA, IgD, IgE, IgG and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4(γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain), among others.

The term "antigen-binding fragment" as used herein refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have an intact antibody structure. Examples of antigen binding fragments include, but are not limited to, such as bifunctional antibodies (diabodies), Fab ', F (ab ')2, Fv fragments, disulfide stabilized Fv fragments dsFv, (dsFv)2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized bifunctional antibodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), bivalent single chain antibodies (BsFv), multispecific antibodies, camelized single domain antibodies (camelized single domain antibodies), nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding fragment may bind to the same antigen as the maternal antibody. In certain embodiments, an antigen-binding fragment can comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

An "Fab" fragment of an antibody refers to the portion of the antibody molecule that is disulfide bonded to one light chain (which includes both the variable and constant regions) and to the variable and partial constant regions of one heavy chain.

By "Fab'" fragment is meant a Fab fragment comprising part of the hinge region.

"F (ab') 2" refers to a dimer of Fab.

The Fc portion of antibodies is responsible for a variety of different effector functions, such as ADCC and CDC, but is not involved in antigen binding.

The "Fv" segment of an antibody refers to the smallest antibody fragment that contains the entire antigen-binding site. The Fv fragment consists of the variable region of one light chain and the variable region of one heavy chain.

"fusion protein" refers to a recombinant protein in which a cDNA encoding a protein of interest is ligated to a cDNA encoding an antibody or antibody fragment at the gene level and expressed in a eukaryotic or prokaryotic expression system.

The "linker" refers to a peptide chain consisting of 1 to 50 amino acids forming a peptide bond or a derivative thereof, and the N-terminus and the C-terminus of the peptide chain form a covalent bond with either the anti-OX 40 antibody or the interferon, respectively, to bind the anti-OX 40 antibody to the interferon. The anti-OX 40 antibody and interferon may be integrated by binding to the N-terminus or C-terminus of interferon, respectively, on the C-terminal side or N-terminal side of the heavy chain or light chain of the anti-OX 40 antibody via a linker sequence or directly using a peptide bond. As preferred embodiments of the fusion protein of interferon with an anti-OX 40 antibody, mention may be made of: a fusion protein obtained by binding the C-terminus of the heavy chain or light chain of an anti-OX 40 antibody to the N-terminus of interferon via a linker sequence; alternatively, a fusion protein obtained by binding the C-terminus of interferon to the N-terminus of the heavy or light chain of an anti-OX 40 antibody via a linker sequence.

"Single chain Fv antibody" or "scFv" refers to an engineered antibody having a light chain variable region directly linked to a heavy chain variable region or linked via a peptide chain (Huston JS et al, Proc Natl Acad Sci USA,85:5879 (1988)).

"Single chain antibody Fv-Fc" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to an Fc portion of an antibody.

"Camelidized single domain antibodies", "Heavy chain antibodies" or "HCAb (Heavy-chain-only antibodies, HCAb)" all refer to antibodies that contain two VH domains but no light chain (Riechmann L. and Muydermans S., J Immunol Methods,231(1-2):25-38 (1999); Muydermans S., J Biotechnol,74(4):277-302 (2001); WO 94/04678; WO 94/25591; U.S. patent No.6,005,079). Heavy chain antibodies were originally derived from camelidae (camels, dromedary and llamas). Despite the absence of the light chain, camelized antibodies (camelized antibodies) have a confirmed full function of antigen binding (polymers Casterman C. et al, Nature363(6428):446-8 (1993); Nguyen VK. et al, Heavy-chain antibodies in camelids: a case of evolution innovation, immunogenetics.54(1):39-47 (2002); Nguyen VK. et al, immunology.109(1):93-101 (2003)). The variable region (VH domain) of heavy chain antibodies is the smallest known antigen-binding unit produced by acquired immunity (Koch-Nolte F. et al, FASEB J.21(13):3490-8.Epub (2007)).

"Nanobody" refers to an antibody fragment consisting of one VH domain from a heavy chain antibody and two constant regions CH2 and CH 3.

"bifunctional antibodies" (diabodies) include small antibody fragments with two antigen-binding sites, where the fragment contains a VH domain and a VL domain linked on the same polypeptide chain (see Holliger P. et al, Proc Natl Acad Sci USA.90(14):6444-8 (1993); EP 404097; WO 93/11161). The linker between the two domains is so short that the two domains on the same chain do not pair with each other, thereby forcing the two domains to pair with the complementary domains of the other two chains to form two antibody binding sites. The two antibody binding sites may be targeted to bind to the same or different antigens (or epitopes).

"Domain antibody" refers to an antibody fragment containing only one heavy chain variable region or one light chain variable region. In some cases, two or more VH domains are covalently bound by one polypeptide linker and form a bivalent domain antibody. The two VH domains of a bivalent domain antibody may be targeted to the same or different antigens.

In certain embodiments, "(dsFv) 2" comprises three peptide chains: two VH groups are linked by a polypeptide linker and are bound to two VL groups by disulfide bonds.

In certain embodiments, a "bispecific ds bifunctional antibody" comprises VL1-VH2 (linked by two polypeptide linkers) and VH1-VL2 (also linked by two polypeptide linkers), which are bound by a disulfide bond between VH1 and VLl.

A "bispecific dsFv" or "dsFv-dsFv" comprises three polypeptide chains: VH1-VH2 groups in which the heavy chains of both are linked by polypeptide linkers (e.g., long flexible linkers) and are bound by disulfide bonds to VL1 and VL2 groups, respectively, each pair of disulfide-paired heavy and light chains having different antigen specificity.

In certain embodiments, an "scFv dimer" is a bivalent diabody or bivalent single chain antibody (BsFv) comprising two VH-VL (joined by a polypeptide linker) groups that dimerize, wherein the VH of two groups cooperates with the VL of another group to form two binding sites that can be targeted to bind to the same antigen (or epitope) or to different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific diabody comprising interconnected V_L1-V_H2(ligated by polypeptide linker) and V_H1-V_L2(ligated by polypeptide linkers) wherein V_H1And V_L1Collaboration, V_H2And V_L2In cooperation, and each cooperative pair has a different antigen specificity.

The term "fully human", when used in this application, means that the antibody or antigen-binding fragment has or consists of an amino acid sequence corresponding to that of an antibody produced by a human or human immune cell or derived from a non-human source, e.g., a transgenic non-human animal utilizing a human antibody repertoire, or other sequence encoding a human antibody. In certain embodiments, a fully human antibody does not comprise amino acid residues (particularly antigen binding residues) derived from a non-human antibody.

The term "humanized" as used herein, when applied to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that includes CDRs derived from a non-human animal, FR regions derived from a human, and constant regions derived from a human, where applicable. Because the humanized antibody or antigen binding fragment has reduced immunogenicity, it may be used in certain embodiments as a therapeutic agent in humans. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen-binding fragment consists essentially entirely of human-derived sequences, except for CDR sequences that are non-human. In some embodiments, the human-derived FR region may include the same amino acid sequence as the human-derived antibody from which it is derived, or it may include some amino acid changes, e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid change. In some embodiments, the amino acid change can be present in only the heavy chain FR region, only the light chain FR region, or both chains. In some preferred embodiments, the humanized antibody comprises human FRl-3 and human JH and JK.

The term "chimeric" as used herein refers to an antibody or antigen-binding fragment having a portion of a heavy and/or light chain derived from one species, and the remainder of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody can include constant regions derived from a human and variable regions derived from a non-human animal, such as a mouse.

The term "OX 40" refers to a receptor that binds to OX 40L. It is a type I membrane protein belonging to the TNF receptor family, otherwise designated ACT-4, OX40L receptor, CD134 antigen, ACT35 antigen, TNFRSF 4. It has a molecular weight of 50kDa and is stored in SwissProt under accession number P43489.

The human interferon used in the application refers to a high-activity multifunctional secretory glucoprotein with antiviral, immunoregulation and antitumor functions. Based on the gene sequence, receptor specificity, and the like, these IFN genes can be classified into type I, type II, and type III. Human type I interferons include 13 subtypes of IFN alpha, IFN beta, IFN epsilon, IFN kappa and IFN omega. Type I IFN has a common cell surface receptor IFNAR, composed of IFNAR1 and IFNAR2 two subunits. Type II interferons are only 1, IFN γ. The cell surface receptor for IFN γ is IFNGR, consisting of two subunits, IFNGR1 and IFNGR 2. Type III IFN including IFN lambda 1, IFN lambda 2, IFN lambda 3 and IFN lambda 4. The interferon in the fusion protein of the present application also includes interferon variants known in the art.

"specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, an antibody or antigen-binding fragment thereof of the present application specifically binds to human and/or monkey OX40, and its binding affinity (K)_D)≤10^-6And M. K in the present application_DIs the ratio (k) of dissociation rate to binding rate_off/k_on) It can be determined by means of surface plasmon resonance, for example using an instrument such as Biacore.

A fusion protein UM06-L9 as described in the present application, consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:6, a peptide linker and human interferon IFN α 2a as shown in SEQ ID NO:3, wherein said human interferon IFN α 2a is linked to the carboxy-terminus of the light chain of said antibody specifically binding to human OX40 via the peptide linker GGGGS.

The fusion protein UM06-L9.1 described in this application, consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:7, a peptide linker and human interferon IFN alpha 2a as shown in SEQ ID NO:3, wherein the human interferon IFN alpha 2a is linked via a peptide linker (GGGGS)₂Linking the carboxy terminus of the light chain of the antibody that specifically binds human OX 40.

The fusion protein UM06-L18 described in the present application, consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:11, a peptide linker and a human interferon IFN alpha 2b mutant as shown in SEQ ID NO:10 (T106A/L117A), wherein the human interferon IFN alpha 2b mutant is linked via a peptide linker (GGGGS)₂Linking the carboxy terminus of the light chain of the antibody that specifically binds human OX 40. Wherein the amino acid sequence of the human interferon IFN alpha 2b mutant is shown in SEQ ID NO. 4. The fusion protein UM06-L20 described in the present application consists of a specificity comprising a heavy chain as shown in SEQ ID NO. 5 and a light chain as shown in SEQ ID NO. 13An antibody that binds to human OX40, a peptide linker, and a human interferon IFN α 2b mutant (L117A) as shown in SEQ ID NO:12, wherein the human interferon IFN α 2b mutant is formed by a peptide linker (GGGGS)₂Linking the carboxy terminus of the light chain of the antibody that specifically binds human OX 40.

The fusion protein UM06-L21 described in the present application, consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:9, a peptide linker and a human interferon FN α 2a mutant as shown in SEQ ID NO:8 (T106A/L117A), wherein the human interferon IFN α 2a mutant is linked via a peptide linker (GGGGS)₂Linking the carboxy terminus of the light chain of the antibody that specifically binds human OX 40.

In the present application, "conservative substitution" when used in reference to an amino acid sequence means the substitution of one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions may be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, VaL, Leu, and Ile), between neutral hydrophilic side chain amino acid residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain amino acid residues (e.g., Asp, Glu), between basic side chain amino acid residues (e.g., His, Lys, and Arg), or between aromatic side chain amino acid residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions do not generally result in significant changes in the conformational structure of a protein, and therefore the biological activity of the protein can be retained.

"percent sequence identity," when used with respect to an amino acid sequence (or nucleic acid sequence), refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to a reference sequence to amino acid (or nucleic acid) residues in the candidate sequence, after alignment of the sequences and, if necessary, introduction of a spacer to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of the amino acid residues may or may not be considered identical residues. Sequences can be aligned by means disclosed in the art to determine the percent sequence identity of amino acid (or nucleic acid) sequences. One skilled in the art can use default parameters for the tool or adjust the parameters appropriately as needed for the alignment, for example by choosing an appropriate algorithm.

As used herein, "T cell" includes CD4+ T cells, CD8+ T cells, T helper type 1T cells, T helper type 2T cells, T helper type 17T cells, and suppressor T cells.

"effector function" as used herein refers to the biological activity of the Fc region of an antibody to bind its effectors such as the C1 complex and Fc receptor. Exemplary effector functions include Complement Dependent Cytotoxicity (CDC) induced by interaction of the antibody with C1q on the C1 complex, antibody dependent cell mediated cytotoxicity (ADCC) induced by binding of the Fc region of the antibody to Fc receptors on effector cells, and phagocytosis.

As used herein, "cancer" or "cancerous condition" refers to any medical condition that is mediated by the growth, proliferation or metastasis of neoplastic or malignant cells and that causes solid and non-solid tumors, such as leukemia. The term "tumor" as used herein refers to a solid substance of a tumor and/or malignant cells.

"viral infection" as used herein refers to a pathogenic process in which the virus enters the human body through various routes and proliferates in human cells, causing damage to the body, including chronic viral infections such as hepatitis b virus, hepatitis c virus, herpes virus, EB (Epstein-Barr) virus, hiv, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, viral infections of adenovirus, kaposi's sarcoma-associated herpes virus epidemics, thin-ring virus (Torquetenovirus), JC virus, or BK virus infections, and the like.

"treating" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof. For cancer, "treatment" or "therapy" may refer to inhibiting or slowing the growth, reproduction, or metastasis of a tumor or malignant cell, or some combination of the above. In the context of a tumor, "treating" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

The "separated" material has been artificially altered from its natural state. If a "separated" substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not isolated, but is considered to be "isolated" if the polynucleotide or polypeptide is sufficiently isolated from substances with which it coexists in its natural state and is present in a sufficiently pure state. In certain embodiments, the antibodies and antigen-binding fragments are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure, as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC).

The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein is operably inserted and which allows expression of the protein. The vector may be used to transform, transduce or transfect a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses, among others. Animal virus species used as vectors are retroviruses (including lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papova papilloma viruses (e.g., SV 40)). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin. The vector may also include components that facilitate its entry into the cell, including, but not limited to, viral particles, liposomes, or protein coats.

The term "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or vector is introduced.

As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount or concentration of a drug effective to treat a disease. For example, for use of the antibodies or antigen-binding fragments thereof disclosed herein, a therapeutically effective amount means that at such a dose or concentration, the antibody or antigen-conjugate can eliminate all or a portion of the tumor, inhibit or slow tumor growth, inhibit tumor cell metastasis, reduce any symptoms or markers associated with the tumor or cancer condition, prevent or delay the development of the tumor or cancer condition, inhibit or eliminate virus or virus-infected cells, or some combination thereof.

By "pharmaceutically acceptable" is meant that the carrier, vehicle, diluent, adjuvant and/or salt in general is chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

Fusion protein of the present invention

In certain embodiments, the present application provides exemplary fusion proteins UM06-L9, UM06-L9.1, and UM 06-L21.

It will be appreciated by those skilled in the art that the foregoing CDR sequences may be modified to include substitutions of one or more amino acids, thereby resulting in improved biological activity, such as improved binding affinity to human OX 40. For example, libraries of antibody variants (e.g., Fab or FcFv variants) can be produced and expressed using phage display technology, and subsequently screened for antibodies having affinity for human OX 40. In another example, computer software can be used to simulate the binding of the antibody to human OX40 and to identify amino acid residues on the antibody that form a binding interface. Substitutions of these residues can be avoided to prevent a decrease in binding affinity, or can be targeted for substitution to form stronger binding. In certain embodiments, at least one (or all) substitution in a CDR sequence is a conservative substitution.

In certain embodiments, the fusion proteins and antigen-binding fragments comprise one or more CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the sequences listed for the exemplary fusion proteins UM06-L9, UM06-L9.1, and UM06-L21 provided herein, while retaining similar or even higher binding affinity to human OX40 than its parent antibody, which has substantially the same sequence but whose corresponding CDR sequences have 100% sequence identity to the listed sequences.

In some embodiments, the fusion proteins described herein can be at ≦ 10^-7Binding affinity of M (K)_D) Binds specifically to human OX40, as measured by surface plasmon resonance. The binding affinity value may be K_DThe value is expressed as the ratio of the off-rate to the on-rate (k) at which the binding of antigen and antigen binding molecule reaches equilibrium_off/k_on) And (4) calculating. The antigen binding affinity (e.g. K)_D) May be suitably determined by suitable methods known in the art, including, for example, plasmon resonance binding using an instrument such as Biacore.

In certain embodiments, a fusion protein described herein has an EC with human OX40 of 10ng/mL to 10 μ g/mL₅₀(i.e., half the binding concentration) binding. Binding of the antibody or fusion protein to human OX40 can be determined by methods known in the art, such as sandwich methods, e.g., ELISA, Western blot, FACS, or other binding assays. In an illustrative example, a test antibody (i.e., a primary antibody) is bound to immobilized human OX40 or cells expressing human OX40, followed by washing away unbound antibody, and introducing a labeled secondary antibody that is capable of binding to the primary antibody and thus detecting the bound secondary antibody. The detection may be performed on microplate readers when using immobilized OX40, or may be performed using FACS analysis when using cells expressing human OX 40.

In certain embodiments, the fusion proteins described herein have an EC of 0.1 μ g/mL to 10 μ g/mL (as determined using FACS analysis)₅₀(i.e., an effective concentration of 50%) binds to human OX 40.

In certain embodiments, the fusion proteins described herein can activate the human OX40 signaling pathway by FcR-mediated or interferon receptor-mediated means, and thereby provide biological activities including, for example, inducing cytokine production by activated T cells (e.g., CD4+ T cells and CD8+ T cells), inducing proliferation of activated T cells (e.g., CD4+ T cells and CD8+ T cells), and reversing the suppressive function of regulatory tregs.

The fusion protein is specific for human OX 40. In certain embodiments, the fusion protein does not bind to murine OX40, but binds to monkey OX40 with similar binding affinity to human OX 40. For example, the binding of the monoclonal antibody MT01-L1 having the same CDR sequences as the fusion protein of the invention to murine OX40 could not be detected by conventional binding assays such as FACS analysis, whereas FACS detected binding of MT01-L1 to both monkey OX40 and human OX 40.

In some embodiments, the fusion protein has a constant region of the IgG2 isotype with reduced or eliminated effector function. Equivalent functions such as ADCC and CDC can result in cytotoxicity to OX 40-expressing cells. Some normal cells are capable of expressing OX 40. To avoid potential undesirable toxicity to these normal cells, certain embodiments of the antibodies of the invention have reduced or even eliminated effector function. Numerous assays are known for assessing ADCC or CDC activity, such as Fc receptor binding assays, complement Clq binding assays and cell lysis methods, which can be readily selected by one skilled in the art. Without wishing to be bound by theory, it is believed that antibodies with reduced or eliminated effector functions such as ADCC and CDC do not cause or minimize cytotoxicity to OX 40-expressing cells (e.g., those normal cells), thus avoiding undesirable side effects.

In some embodiments, the fusion proteins described herein have an extended duration of action in an organism compared to interferon molecules. This is due to the longer half-life and drug retention time of the fusion protein in the animal. This property is beneficial to reduce the times of administration for patients and improve the drug effect of the drug.

In some embodiments, the fusion proteins described herein have reduced side effects. For example, the anti-OX 40 antibodies and antigen-binding fragments thereof can have fully human IgG sequences and are therefore less immunogenic than humanized antibodies. As another example, the fusion proteins and antigen-binding fragments thereof may have an IgG2 or IgG4 format to eliminate ADCC and CDC.

In some embodiments, the fusion protein described herein is advantageous in that it can be used in combination with immunogenic substances, such as tumor cells, purified tumor antigens and cells transfected with an encoding immunostimulatory factor, tumor vaccines. Furthermore, the fusion proteins can be included in combination therapies, including combination therapies with standard chemotherapy and radiotherapy, target-based small molecule therapies, other emerging immune checkpoint modulator therapies. In some embodiments, the antibodies and antigen-binding fragments thereof can be used as the base molecule in the form of antibody-drug conjugates, bispecific or multivalent antibodies.

In some embodiments, the fusion proteins and antigen-binding fragments thereof described herein are camelized single domain antibodies (camelized single chain domain antibodies), diabodies (diabodies), scfvs, scFv dimers, BsFv, dsFv, (dsFv)2, dsFv-dsFv ', Fv fragments, Fab ', F (ab ')2, ds diabodies (ds diabodies), nanobodies, domain antibodies, or diabodies.

In some embodiments, the fusion protein described herein comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region and/or a light chain constant region. The heavy chain constant region includes the CH1, CH1-CH2, or CH1-CH3 regions. In some embodiments, the immunoglobulin constant region may further comprise one or more modifications to achieve a desired property. For example, the constant region may be modified to reduce or eliminate one or more effector functions, to enhance FcRn receptor binding or to introduce one or more cysteine residues.

In certain embodiments, the antibodies and antigen-binding fragments thereof further comprise a conjugate. It is contemplated that the antibodies or antigen-binding fragments thereof of the invention may be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", constraints to Microbiology and Immunology, j.m.cruse and r.e.lewis.jr. (eds.), Carger Press, New York (1989)). These conjugates may be attached to the antibody or antigen conjugate by covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or addition, among other means. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites other than the epitope-binding portion that can be used to bind to one or more conjugates. For example, such sites may comprise one or more reactive amino acid residues, such as cysteine and histidine residues, for facilitating covalent attachment to the conjugate. In certain embodiments, the antibody may be linked indirectly to the conjugate, or via another conjugate. For example, the antibody or antigen-binding fragment thereof can bind to biotin and then indirectly bind to a second conjugate, which is linked to avidin. The conjugate can be a detectable label, a pharmacokinetic modifying moiety, a purifying moiety, or a cytotoxic moiety. Examples of detectable labels may include fluorescent labels (e.g., fluorescein, rhodamine, dansyl chloride, phycoerythrin or texas red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase or β -D-galactoxinase), stable isotopes or radioisotopes, chromophore moieties, digoxin, biotin/avidin, DNA molecules or gold for detection. In certain embodiments, the conjugate may be a pharmacokinetic modifying moiety such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A "cytotoxic moiety" may be any agent that is harmful to or may damage or kill a cell. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine glance sideways at Line, cytarabine, 5-fluorouracil dacarbazide), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (DDP), Cisplatin, anthracyclines (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and Amitomycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

Polynucleotides and recombinant methods

The amino acid sequence in the fusion protein of the present application can be converted into the corresponding DNA coding sequence using genetic engineering techniques well known in the art. Due to the degeneracy of the genetic code, the resulting DNA sequences can be completely identical while the encoded protein sequence remains unchanged.

Vectors comprising polynucleotides encoding the fusion proteins can be introduced into host cells for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art. In another embodiment, the fusion protein can be made by methods of homologous recombination as are well known in the art. Various carriers can be selected. Carrier components typically include, but are not limited to, two or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter (e.g., SV40, CMV, EF-1a) and a transcription termination sequence.

In some embodiments, the vector system comprises a mammalian, bacterial, yeast system, etc., and will include plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2, and other laboratory-available or commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

A vector comprising a polynucleotide encoding the fusion protein can be introduced into a host cell for cloning or gene expression. The host cells suitable for cloning or expressing the DNA in the vector of the invention are prokaryotic cells, yeast or higher eukaryotic cells. Prokaryotic cells suitable for use in the present invention include eubacteria, such as gram-negative or gram-positive bacteria, for example, Enterobacteriaceae such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescens, and Shigella, and bacilli such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Peucella and Streptomyces.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast may also be used as host cells for cloning or expressing vectors encoding fusion proteins. Saccharomyces cerevisiae or Saccharomyces cerevisiae are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species and strains are more commonly used and are suitable for use in the present invention, such as Schizosaccharomyces pombe; kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC12, 424), Kluyveromyces bulgaricus (ATCC16, 045), Kluyveromyces williamsii (ATCC24, 178), Kluyveromyces lactis (ATCC56, 500), Kluyveromyces drosophilus (ATCC36, 906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; yarrowia lipolytica (EP402, 226); pichia pastoris (EP183, 070); candida species; trichoderma reesei (EP244, 234); performing Neurospora; schwann western yeast, such as schwann western yeast; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus such as Aspergillus nidulans and Aspergillus niger.

Host cells provided herein that are suitable for expression of glycosylated antibodies or antigen-binding fragments thereof can be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Various baculovirus strains (bacterial strains) and variants thereof, and corresponding permissive insect host cells (permissive insect host cells), have been found to be derived from hosts such as: spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as Autographa californica nuclear polyhedrosis virus and Bm-5 variants of Bombyx mori nuclear polyhedrosis virus, all of which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

However, the most interesting are the vertebrate cells, and the culture of the vertebrate cells (tissue culture) has become a routine practice. Examples of mammalian host cells that may be used are SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cell subclones in suspension culture, Graham et al, Gen Virol.36:59 (1977)); baby hamster kidney cells (B blood, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse testicular support cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC5 cells; FS4 cells; and a human liver cancer cell line (HepG 2). In certain preferred embodiments, the host cell is a 293F cell.

Host cells are transformed with the above-described expression or cloning vectors that produce the fusion proteins and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformed cells, or amplifying genes encoding the sequences of interest.

The host cells of the invention used to produce the fusion proteins can be cultured in a variety of media well known in the art. The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH and the like, which are previously used to select the host cell for expression, are well known to the skilled person.

When using recombinant techniques, the antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the particulate debris of the host cells or lysed fragments is first removed, for example, by centrifugation or sonication. Carter et al, Bio/Technology 10:163-167(1992) describe methods for isolating antibodies secreted into the membrane space of E.coli walls. Briefly, the cell paste (cell paste) was lysed in the presence of uranium acetate (pH 3.5), EDTA, and phenylmethanesulfonic fluoride (PMSF) for about 30 minutes or more. Cell debris was removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is typically first concentrated using a commercially available protein concentration filter, such as the lAmicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be added in any of the foregoing steps to inhibit proteolytic degradation, as well as antibiotics to prevent the growth of adventitious contaminants.

The antibody produced from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique. The class of the antibody and the presence of the Fc domain of any immunoglobulin in the antibody determines whether protein a is suitable as an affinity ligand. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J.Immunol. meth.62:13 (1983)). Protein G is applicable to all murine isoforms and human gamma 3(Guss et al, EMBO J.5:1567-1575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, it can be purified using Bakerbond ABX. TM. resin (J.T.Baker, Phillipsburg, N.J.). Other techniques for protein purification may also be determined depending on the antibody to be obtained, such as fractionation in ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (e.g.polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification step, the mixture containing the antibody of interest and impurities can be treated by low pH hydrophobic interaction chromatography, using an elution buffer at a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., from about 0 to 0.25M salt concentration).

Reagent kit

Kits comprising the fusion proteins are provided. In some embodiments, the kit is used to detect the presence or level of OX40 in a biological sample. The biological sample may comprise a cell or tissue.

In some embodiments, the kit comprises a fusion protein conjugated to a detectable label. In some embodiments, the kit comprises an unlabeled fusion protein and further comprises a secondary antibody that is capable of binding to the unlabeled fusion protein. The kit may further include instructions for use and packaging separating each component in the kit.

In some embodiments, the fusion protein is linked to a substrate or instrument for use in a sandwich assay such as an ELISA or immunochromatographic assay. Suitable substrates or instruments may be, for example, microplates and test strips.

Pharmaceutical compositions and methods of treatment

The present application further provides pharmaceutical compositions comprising the fusion proteins and one or more pharmaceutically acceptable carriers.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending/dispersing agents, sequestering agents, diluents, adjuvants or nontoxic auxiliary substances, other components well known in the art, or various combinations thereof.

Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents, or stabilizing agents such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and/or propyl gallate. The inclusion of one or more antioxidants, such as methionine, in a composition containing a fusion protein as disclosed herein will reduce oxidation of the fusion protein. The reduction in oxidation prevents or reduces the reduction in binding affinity, thereby improving antibody stability and extending shelf life.

Further, the pharmaceutically acceptable carrier may include, for example, an aqueous medium such as sodium chloride injection, ringer's solution injection, isotonic glucose injection, sterile water injection, or dextrose and lactated ringer's injection, a non-aqueous medium such as a fixed oil of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, an antibacterial substance at a bacteria-inhibiting or fungistatic concentration, an isotonic agent such as sodium chloride or dextrose, a buffer such as phosphate or citrate buffer, an antioxidant such as sodium bisulfate, a local anesthetic such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, an emulsifying agent such as polysorbate 80 (Tween-80), an integration agent such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethyleneglycol bis (2-aminoethylether) tetraacetic acid), Ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agent may be added to the pharmaceutical composition in a multi-dose container and includes phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzalkonium chloride. Suitable excipients may include, for example, water, salt, glucose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions may be prepared in any conventional form, for example, liquid solvents, suspending agents, emulsifying agents, or solid forms suitable for the production of liquid solvents, suspending agents or emulsifying agents. Injectable preparations may include ready-to-use sterile and/or pyrogen-free solutions, sterile dried solubles combined with solvent prior to use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dried insoluble products combined with vehicle prior to use, and sterile and/or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

In certain embodiments, a unit dose of an injectable formulation is packaged in an ampoule, a manifold, or a syringe with a needle. It is well known in the art that all formulations for injection administration should be sterile pyrogen free.

In certain embodiments, sterile lyophilized powders can be prepared by dissolving the fusion proteins disclosed herein in an appropriate solvent. The solvent may contain a compound that enhances the stability of the powder or reconstituted solution prepared from the powder, or improves the pharmacological properties of the powder or reconstituted solution. Suitable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, brown sugar, or other suitable materials. The solvent may contain a buffer, such as a citric acid buffer, a sodium or potassium phosphate buffer, or other buffers known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. Subsequent sterile filtration of the solution is carried out under standard conditions well known in the art and then lyophilized to produce the desired formulation. In one embodiment, the resulting solvent is dispensed into vials for lyophilization. Each tubule may contain a single dose or multiple doses of the fusion protein. The loading per vial may be slightly higher than that required for each dose or for multiple doses (e.g., 10% excess), thereby ensuring accurate sampling and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as in the range of about 4 ℃ to room temperature.

And re-dissolving the freeze-dried powder with water for injection to obtain the preparation for injection administration. In one embodiment, the lyophilized powder can be reconstituted by addition to sterile pyrogen-free water or other suitable liquid carrier. The precise amount is determined by the selected therapy and can be determined empirically.

Also provided are methods of treatment comprising administering a therapeutically effective amount of a fusion protein described herein to a subject in need thereof.

The therapeutically effective dose of the fusion protein provided herein depends on a variety of factors well known in the art, such as body weight, age, past medical history, current therapy, the health status and potential for cross-infection of the subject, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of tumor development. One skilled in the art (e.g., a physician or veterinarian) can proportionately lower or raise the dosage based on these or other conditions or requirements.

In certain embodiments, the fusion proteins provided herein can be administered at a therapeutically effective dose of between about 0.0lmg/kg to about 100 mg/kg. In certain embodiments, the fusion protein is administered at a dose of about 50mg/kg or less, and in certain embodiments, 10mg/kg or less, 5mg/kg or less, 1mg/kg or less, 0.5mg/kg or less, or 0.1mg/kg or less. A particular dose may be administered at multiple intervals, such as once daily, twice or more monthly, once weekly, once every two weeks, once every three weeks, once monthly, or once every two or more months. In certain embodiments, the dosage administered may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the dosage administered is adjusted during the course of treatment according to the response of the subject to whom it is administered.

The dosage regimen may be adjusted to achieve an optimal response (e.g., therapeutic response). For example, administration may be carried out as a single dose or in multiple divided doses over a period of time.

The fusion protein disclosed in the present invention can be administered by a known administration means in the art, such as injection (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection including intravenous drip, intramuscular injection or intradermal injection) or non-injection (e.g., oral, nasal, sublingual, rectal or topical administration).

In certain embodiments, the fusion proteins are useful for treating disorders associated with their molecular mechanisms, including tumors and cancers, such as non-small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, granulomatous grass (mycoses fungoides), merkel cell carcinoma and other hematologic malignancies, such as Classical Hodgkin Lymphoma (CHL), primary mediastinal large B cell lymphoma, B-rich lymphoma of T cells/histiocytes, EBV positive and negative PTLD, and EBV-related Diffuse Large B Cell Lymphoma (DLBCL), plasmacytic lymphoma, extranodal NK/T cell lymphoma, and other malignant hematologic disorders, Nasopharyngeal carcinoma and HHV 8-associated primary effusion lymphoma, hodgkin's lymphoma, Central Nervous System (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brain stem glioma. In certain embodiments, the disorders treated by the fusion protein include chronic viral infections, such as viral infections of hepatitis b virus, hepatitis c virus, herpes virus, Epstein-Barr virus, aids virus, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type 2, human papilloma virus, adenovirus, kaposi's sarcoma-associated herpes virus epidemics, thin-ring virus (Torquetenovirus), JC virus, or BK virus infections, and the like.

Application method

The application further provides methods of using the fusion proteins.

In some embodiments, the present application provides a method of treating a disease or disorder associated with the mechanism of the fusion protein in an individual comprising administering a therapeutically effective amount of a fusion protein described herein.

The fusion proteins disclosed herein can be administered alone or in combination with one or more other therapeutic means or substances. For example, the fusion proteins disclosed herein can be used in combination with chemotherapy, radiation therapy, cancer treatment surgery (e.g., tumor resection), antiviral drugs, one or more anti-emetic drugs or other therapies for complications resulting from chemotherapy, or any other therapeutic substance for cancer or viruses. In certain such embodiments, the fusion proteins disclosed herein, when used in combination with one or more therapeutic agents, may be administered simultaneously with the one or more therapeutic agents, and in certain such embodiments, the fusion proteins may be administered simultaneously as part of the same pharmaceutical composition. However, a fusion protein that is "in combination" with another therapeutic agent need not be administered simultaneously or in the same composition as the therapeutic agent. The meaning of "in combination" in the present invention also includes that a fusion protein administered before or after another therapeutic substance is also considered to be "in combination" with the therapeutic substance, even if the fusion protein and the second substance are administered by different administration means. Where possible, other therapeutic substances to be used in combination with the fusion proteins disclosed herein may be administered by methods according to the product specifications for the other therapeutic substance, or by Reference to surgeon's docket No. 2003(Physicians' Desk Reference,57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (11 months 2002)), or by other methods known in the art.

In certain embodiments, the therapeutic substance is capable of inducing or enhancing an immune response against the cancer. For example, tumor vaccines can be used to induce an immune response to certain tumors or cancers. Cytokine therapy can be used to enhance the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, but are not limited to, interferons such as interferon alpha, beta and gamma, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF, interleukins such as IL-1, IL-1a, IL-2, IL 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-ll and IL-12, tumor necrosis factors such as TNF-alpha and TNF-beta. Agents that inactivate immunosuppressive targets, such as PD-L1/PD-1 antibodies, TGF- β inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, may also be used. Another group of agents include those that activate an immune response against a tumor or cancer cell, for example, those that increase T cell activation (e.g., T cell costimulatory molecule agonists such as CTLA-4, ICOS), as well as those that increase dendritic cell function and antigen presentation.

The following examples are intended to better illustrate the invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. These specific compositions, materials and methods are not intended to limit the invention, but are merely illustrative of specific embodiments within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without adding inventive step and without departing from the scope of the present invention. It will be appreciated that various modifications to the method of the invention are still included within the scope of the invention. The inventors intend such variations to be included within the scope of the present invention.

Example 1: preparation of the fusion protein of the invention

This example illustrates the design and expression of several anti-OX 40 antibody-human interferon fusion proteins. "MT 01-C1" refers to a monoclonal antibody having VH (SEQ ID NO: 1) and VL (SEQ ID NO: 2) sequences identical to UM06-L9, UM06-L9.1, UM06-L18, UM06-L20 and UM06-L21, and heavy and light chain constant regions are human IgG4(S228P) and kappa chain, respectively.

The interferon IFN alpha 2 sequence is taken from human interferon IFN alpha 2a (P01563), and the amino acid sequence is shown in SEQ ID NO:3 or human interferon IFN alpha 2b, and the amino acid sequence is shown in SEQ ID NO. 4.

An exemplary fusion protein design is shown in FIG. 1. Wherein the content of the first and second substances,

"UM 06-L9" refers to a polypeptide consisting of a sequence as set forth in SEQ ID NO:5 and the heavy chain as set forth in SEQ ID NO:6, a peptide linker and human interferon IFN α 2a as shown in SEQ ID No. 3, wherein the human interferon IFN α 2a is linked at the carboxy-terminus of the light chain of OX40 antibody via the peptide linker GGGGS.

"UM 06-L9.1" refers to a polypeptide consisting of a sequence as set forth in SEQ ID NO:5 and the heavy chain as set forth in SEQ ID NO:7, a peptide linker and a human interferon IFN α 2a as shown in SEQ ID NO:3, wherein the carboxyl terminus of the light chain of OX40 antibody is linked through the peptide linker (GGGGS)₂Connecting human interferon IFN alpha 2 a.

"UM 06-L21" refers to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:5 and the heavy chain as set forth in SEQ ID NO:9, a peptide linker and a human interferon IFN alpha 2a mutant (T106A/L117A) as shown in SEQ ID NO:8, wherein the carboxyl terminal of the light chain of the OX40 antibody is connected through the peptide linker (GGGGS)₂Connecting human interferon IFN alpha 2a mutant.

"UM 06-L18" refers to a polypeptide consisting of a sequence as set forth in SEQ ID NO:5 and the heavy chain as set forth in SEQ ID NO:11, a peptide linker and a human interferon IFN alpha 2b mutant (T106A/L117A) as shown in SEQ ID NO:10, wherein the carboxyl end of the light chain of the OX40 antibody is connected through the peptide linker (GGGGS)₂Connecting human interferon IFN alpha 2b mutant.

"UM 06-L20" refers to a polypeptide consisting of a sequence as set forth in SEQ ID NO:5 and the heavy chain as set forth in SEQ ID NO:13, a peptide linker and a human interferon IFN α 2b mutant (L117A) as shown in SEQ ID NO:12, wherein the carboxyl end of the light chain of OX40 antibody is flanked by a peptide linker (GGGGS)₂Connecting human interferon IFN alpha 2b mutant.

The cDNA sequences encoding the antibody heavy and light chains of the fusion protein were cloned into the mammalian cell expression vector pcDNA3.4, respectively. The heavy chain expression plasmid and the light chain expression plasmid were transfected into HEK293 cells using Lipofectamine 2000 transfection reagent (Invitrogen) at a molar ratio of 2:1 and cultured at 37 ℃ under 5% carbon dioxide for 7 days. The culture supernatant was collected, and the antibody in the supernatant was purified by Protein A affinity chromatography. The purified antibody was dialyzed against PBS solution, freeze-dried, concentrated, and stored at-20 ℃. The SDS-PAGE and HPLC profiles of the purified proteins are shown in FIG. 2.

Example 2: ELISA binding assay

A96-well high affinity plate was coated with 1. mu.g/mL human OX40 protein solution at 100. mu.L/well and shaken overnight at 4 ℃. The next day, the cells were washed 3 times with 300. mu.L of PBST (Tween 20: 0.5 ‰), then blocked with 100. mu.L/well of 5% BSA/PBS for 2 hours, and shaken at room temperature. 300 u L PBST washing 3 times. Gradient dilutions of the fusion protein samples were made with PBS. Add to 96-well plate at 100. mu.L/well and shake for 1 hour at room temperature. 300 u L PBST washing 3 times. A mouse anti-human IgG HRP (Kingsler Biotech, cat # A01854) solution was prepared, added to a 96-well plate at 100. mu.L/well, and shaken at room temperature for 1 hour. 300 u L PBST washing 4 times. Add 100. mu.L/well TMB and develop for 20 min. Adding 100 mu L/well of 0.6N H₂SO₄The color development was terminated, and OD450nm was detected.

The results are shown in FIG. 3, and the fusion proteins UM06-L9, UM06-L9.1, UM06-L18, UM06-L20 and UM06-21 are combined with EC by ELISA₅₀EC of 1.880, 1.836, 1.347, 1.198 and 1.382nM, respectively, which are all in agreement with the OX40 antibody MT01-C1₅₀(1.214nM) was essentially equivalent (FIG. 3).

Example 3: daudi cell proliferation inhibition assay

Since IFN alpha-2 is a physiologically active cytokine, when it is fused with an OX40 antibody, it is considered to appropriately reduce the interferon activity of the fusion protein. The clinical dose of therapeutic OX40 antibody and long-acting interferon (PEG interferon) suggests that it is preferable that the interferon activity in the fusion protein be hundreds of times lower than that of long-acting interferon. Human interferon activity can be characterized using the Daudi cell proliferation assay. The Daudi cells (ATCC) highly express interferon receptors, and thus interferon has biological activity thereto. The Daudi cells are paved on a 96-well plate by 20000 cells/90 mu L/hole, a sample to be detected is prepared into 10 multiplied working solution with concentration gradient dilution, the 10 mu L/hole is respectively added into the 96-well plate, the 96-well plate is placed in an incubator at 37 ℃, CCK8 is added after 72 hours to detect OD450, and the proliferation inhibition rate of the cells of each hole is calculated. This inhibition rate reflects the activity of interferon in the sample. The experimental results showed that the proliferation inhibitory activity of PEG-IFN alpha 2 (Pagabin, Xiamen Biotech Co., Ltd., batch No. 201811JS19), fusion proteins UM06-L9, UM06-L9.1, UM06-L18, UM06-L20 and UM06-L21 on Daudi cells (IC)₅₀) 10.64, 85.87, 92.06, 7886, 8556 and 2964pM respectively (fig. 4).

Example 4: OX40 Signal pathway activation assay

The inventors of the present application constructed a cell assay system for detecting OX40 activators. Specifically, the inventors of the present invention constructed a "HEK 293-OX 40-NF-. kappa.B-luciferase reporter gene (Luc)" stable transgenic cell line, and when OX40 activating antibody was mixed with the stable transgenic cell line and Raji cells expressing FcR, expression of NF-. kappa.B-luciferase reporter gene was activated.

PBS is used for preparing fusion protein concentration gradient solution, 2 Xworking solution with final concentration is prepared, and the operation is carried out on ice. The "HEK 293-OX40-NF kappa B-Luc" cells and Raji cells were collected, centrifuged, resuspended in culture medium, and plated into 384-well plates. Adding the fusion protein working solution and a proper amount of cell suspension into a 384-well plate, incubating for 5 hours, adding an One-glo (Promega) detection reagent, mixing uniformly, and detecting a fluorescent signal by TECAN SPARK 20M.

As shown in FIG. 5, the mean value of the signal from the wells to which no OX40 agonist was added was set as 0% activation, the concentration group having the largest mean activation signal among the wells to which different concentration gradients of MT01-C1 mab was added was set as 100% activation, and the values of the detected fluorescence signals of the other samples were normalized based on this. As can be seen from the test results, PEG interferon alpha 2 (Pagemin, Xiamen Biotechnology GmbH, batch No. 201811JS19) has no activation effect on HEK293-OX40-NF kappa B-Luc, which indicates that the interferon alpha 2 activity in the fusion protein molecule has no interference on the OX40 activity test system. OX40 antibody MT01-C1, fusion proteins UM06-L9, UM06-L9.1, UM06-L18, UM06-L20 and UM06-L21 Activity to activate NF kappa B-luciferase reporter Gene in the above described assay System₅₀It was judged that the difference between the molecules was not large. However, UM06-L18 and UM06-L21 are significantly larger than the other molecules in view of the maximal fold activation of the OX40 reporter. In combination, UM06-L21 has the greatest efficacy (efficacy) and potency (potency).

Example 5: balb/c mouse pharmacokinetic experiment

6 female Balb/c mice, 6-8 weeks old, were given UM06-L21 by intravenous injection. The dose administered was 5 mg/kg. Peripheral venous blood was collected from the animals before and 1, 2, 6, 24, 48, 72, 96, 168, 240, 336 hours after the administration. Serum was collected from 3 animals at each time point and blood was collected from 6 animals at different time points in alternation.

A solution of human OX40 protein at a concentration of 1. mu.g/mL was coated in 100. mu.L/well in 96-well high affinity plates and shaken overnight at 4 ℃. The next day, the cells were washed 3 times with 300. mu.L of PBST (Tween 20: 0.5 ‰), then blocked with 100. mu.L/well of 5% BSA/PBS for 1 hour, and shaken at room temperature. 300 u L PBST washing 4 times. PBS is used for preparing 100-fold diluted solutions of the serum sample to be detected and the serum solution of the control substance with different concentrations. Add to 96 well plate at 100. mu.L/well and shake for 1.5 hours at room temperature. 300 u L PBST washing 4 times. A solution of 0.5. mu.g/mL of rabbit anti-human IFN (Abcam, cat # ab222552) was prepared, added to a 96-well plate at 100. mu.L/well, and shaken at room temperature for 1.5 hours. 300 u L PBST washing 4 times. A solution of goat anti-rabbit IgG HRP (King Ray Biotech, cat # A00098) was prepared, added to a 96-well plate at 100. mu.L/well, and shaken at room temperature for 1 hour. 300 u L PBST washing 4 times. Add 100. mu.L/well TMB and develop for 20 min. Adding 100 mu L/well of 0.6N H₂SO₄The color development was terminated, and OD450nm was detected. And performing Logistic four-parameter fitting on the detection values of the reference substance solutions with different concentrations and the reference substance concentration to obtain a standard curve regression equation. Substituting the detection value of the sample to be detected into an equation for calculation to obtain the serum drug concentration at different time points.

As shown in FIG. 6, mice were injected intravenously with 5mg/kg of UM06-L18 and UM06-L21 in the experiment. After administration of UM06-L21 for 14 days, the serum drug concentration was still above 10. mu.g/mL, but UM06-L18 could not be detected, indicating that the fusion protein UM06-L21 has better pharmacokinetic properties in mouse models than UM 06-L18. Further calculating the area under the curve (AUC) of the two, the AUC of UM06-L21 and UM06-L18 are 14469+/-3220 and 11062+/-1282 h.mu.g/mL respectively. The AUC of UM06-L21 was significantly greater than that of UM06-L18(P ═ 0.025) by statistical analysis of t-test.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any way. Those skilled in the art can make various changes, substitutions and alterations on the technical solutions and contents disclosed in the present invention without departing from the technical scope of the present invention, and the technical solutions and contents do not depart from the contents of the technical solutions of the present invention.

Sequence listing

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Ala Asn Tyr Tyr Gly Ser Lys Phe Ala Met Asp Tyr Trp Gly Gln Gly

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Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

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Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu

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Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

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Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

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Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

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Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

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Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

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Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

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Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

355 360 365

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

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Leu Arg Ser Lys Glu

385

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Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

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Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys

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Glu Asp Ser Ile Ala Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

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Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

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Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

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Leu Arg Ser Lys Glu

165

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

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20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gly Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ile Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

225 230 235 240

Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp

245 250 255

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

260 265 270

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

275 280 285

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

290 295 300

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

305 310 315 320

Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys

325 330 335

Glu Asp Ser Ile Ala Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

340 345 350

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

355 360 365

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

370 375 380

Leu Arg Ser Lys Glu

385

<210> 10

<211> 165

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys

100 105 110

Glu Asp Ser Ile Ala Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

Leu Arg Ser Lys Glu

165

<210> 11

<211> 389

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gly Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ile Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

225 230 235 240

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

245 250 255

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

260 265 270

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

275 280 285

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

290 295 300

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

305 310 315 320

Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys

325 330 335

Glu Asp Ser Ile Ala Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

340 345 350

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

355 360 365

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

370 375 380

Leu Arg Ser Lys Glu

385

<210> 12

<211> 165

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

100 105 110

Glu Asp Ser Ile Ala Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

Leu Arg Ser Lys Glu

165

<210> 13

<211> 389

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gly Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ile Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

225 230 235 240

Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

245 250 255

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

260 265 270

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

275 280 285

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

290 295 300

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

305 310 315 320

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

325 330 335

Glu Asp Ser Ile Ala Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

340 345 350

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

355 360 365

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

370 375 380

Leu Arg Ser Lys Glu

385

Claims (14)

1. A fusion protein comprising:

a) an antibody or antigen-binding fragment thereof that specifically binds human OX 40; and

b) human interferon IFN alpha-2 a or mutants thereof;

wherein the human interferon IFN α -2a or mutant thereof is linked directly or through a peptide linker to the carboxy terminus or amino terminus of the light or heavy chain of the antibody or antigen-binding fragment thereof that specifically binds to human OX 40.

2. The fusion protein of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds human OX40 comprises:

a heavy chain variable region comprising 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region set forth in SEQ ID NO: 1; and

a light chain variable region comprising 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region set forth in SEQ ID NO: 2;

wherein the HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 are defined as shown in the following table:

preferably, the heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 1 and the light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 2.

3. The fusion protein of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof that specifically binds human OX40 is a camelized single domain antibody, scFv dimer, BsFv, dsFv2, dsFv-dsFv ', Fv fragment, Fab ', F (ab ')2, ds diabody, nanobody, domain antibody, or diabody.

4. The fusion protein of any one of claims 1 to 3, wherein the antibody further comprises a constant region of an immunoglobulin; preferably, the constant region is that of human IgG1, IgG2 or IgG 4.

5. The fusion protein of any one of claims 1 to 4, wherein the amino acid sequence of human interferon IFN alpha-2 a is as set forth in SEQ ID NO 3;

preferably, the amino acid sequence of the human interferon IFN alpha-2 a mutant is the amino acid sequence shown in SEQ ID NO. 3, and the mutant has one or more mutations selected from the following:

T106A, R149A, a145G, a145D, R120A, or L117A;

more preferably, the amino acid sequence of the human interferon IFN alpha-2 a mutant has one or more double mutations selected from the following amino acid sequences shown in SEQ ID NO. 3:

T106A/A145D, T106A/R149A, T106A/A145G, T106A/R120A or T106A/L117A.

6. The fusion protein of any one of claims 1 to 5, wherein the peptide linker is selected from the group consisting of: (G)_n、KESGSVSSEQLAQFRSLD、EGKSSGSGSESKST、GSAGSAAGSGEF、(GGGGS)_nor (GGSGG)_n(ii) a Preferably, the peptide linker is (GGGGS)_nWherein n is an integer between 0 and 5; preferably, n is an integer between 1 and 3.

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein is selected from one or more of the following:

a fusion protein consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO. 5 and a light chain as shown in SEQ ID NO.6, a peptide linker and human interferon IFN α -2a as shown in SEQ ID NO. 3, wherein said human interferon IFN α 2a is linked to the carboxy-terminus of the light chain of said antibody specifically binding to human OX40 by a peptide linker GGGGS;

fusion protein consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO. 5 and a light chain as shown in SEQ ID NO. 7, a peptide linker and human interferon IFN alpha-2 a as shown in SEQ ID NO. 3, wherein said human interferon IFN alpha 2a is linked via a peptide linker (GGGGS)₂Ligating the carboxy terminus of the light chain of said antibody that specifically binds human OX 40;

fusion protein consisting of an antibody specifically binding to human OX40 comprising a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:9, a peptide linker and a human interferon IFN alpha-2 a mutant as shown in SEQ ID NO:8, wherein the human interferon IFN alpha-2 a mutant is linked via a peptide linker (GGGGS)₂Is linked to the carboxy terminus of the light chain of the antibody that specifically binds human OX 40.

8. An isolated polynucleotide encoding the fusion protein of any one of claims 1 to 7.

9. A vector comprising the isolated polynucleotide of claim 8.

10. A host cell comprising the vector of claim 9.

11. A method of expressing the fusion protein of any one of claims 1 to 7, comprising culturing the host cell of claim 10 under conditions capable of expressing the isolated polynucleotide of claim 8.

12. A kit comprising the fusion protein of any one of claims 1 to 7.

13. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

14. Use of a fusion protein according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment of a condition that can benefit from an enhanced immune response and/or from exposure to interferon; preferably, the condition is cancer or a viral infection; more preferably, the viral infection is a hepatitis B virus infection.

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