CN116135885A

CN116135885A - Fusion proteins targeting CD73 protein

Info

Publication number: CN116135885A
Application number: CN202211458337.8A
Authority: CN
Inventors: 张振清; 贾云莉; 李志远
Original assignee: Biotheus Inc
Current assignee: Biotheus Inc
Priority date: 2021-11-18
Filing date: 2022-11-17
Publication date: 2023-05-19

Abstract

The present application relates to a fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to CD73 protein. The fusion proteins described herein may further comprise a TGF-beta binding moiety. The application also provides multispecific molecules, immunoconjugates, chimeric antigen receptors, immune effector cells, pharmaceutical compositions comprising the fusion proteins; nucleic acid molecules encoding said fusion proteins, expression vectors and host cells for the preparation of said fusion proteins, and uses thereof.

Description

Fusion proteins targeting CD73 protein

Technical Field

The present application relates to the field of biological medicine, in particular to a fusion protein comprising an antibody or antigen binding fragment thereof capable of specifically binding to CD73 protein.

Background

Extracellular-5' -nucleotidase (encoded by the NT5E gene, also known as CD 73), full length 522aa of the mature protein, approximately 70KD in molecular weight, has a high degree of glycosylation, and the mature protein forms homodimers by being anchored to the cell surface by Glucosyl Phosphatidylinositol (GPI), while also having a cutter and circulating in soluble form (double CD73, sCD 73). CD39 upstream of the adenosine pathway hydrolyzes ATP to produce AMP, CD73 hydrolyzes AMP to produce adenosine, acts on the adenosine receptor (A2 AR) of immune cells, activates downstream Protein Kinase A (PKA) and CSK kinase, inhibits LCK, MAPK, PKC and the like a series of signal pathways related to immune activation, and thus exerts an immunosuppressive effect.

CD73 has low tissue specificity and is highly expressed in various normal tissues such as lung, pancreas, stomach, small intestine, ovary, cerebellum, etc. CD73 is up-regulated in various tumor species such as thyroid cancer, colorectal cancer, liver cancer, glioma, renal cancer, and the like, and is closely related to poor prognosis. In addition, immune cells, DC cells, treg (regulatory T cells), NK (natural killer cells), MDSC (myeloid suppressor cells), TAM (tumor-associated macrophages) and other cell surfaces in the tumor microenvironment are expressed. CD73 expression is regulated by multiple factors, mainly hypoxia inducible factor-1 (HIF-1) in the tumor microenvironment. Hypoxia conditions of the tumor microenvironment induce up-regulation of HIF-1 expression, thereby inducing extensive expression of CD73 in TME. The radiotherapy and chemotherapy treatment causes tumor killing release ATP to pass through CD39-CD 73-adenosine pathway, which is favorable for proliferation and function of various cancer cells, but is unfavorable for inhibiting cancer cells. Based on the above mechanism, several researchers have explored the anti-tumor effect of CD73, and preclinical mouse model results show that CD 73-targeting antibodies or gene knockout CD73 can effectively block tumor growth and metastasis.

TGF- β mediated signaling in the tumor microenvironment can promote tumor invasion, migration, and metastasis through a variety of mechanisms including epithelial-mesenchymal transition (EMT). TGF-beta is expressed in cancers and in mesenchymal cells including cancer-associated fibroblasts (CAFs). TGF-beta can maintain tumor progression by activating CAFs, stimulating immunosuppression, and promoting vascular factors. The CD 73-adenosine pathway and the TGF-beta pathway have independent and complementary immunosuppressive functions. Therefore, if the medicine can simultaneously block two immune signal paths of TGF-beta and CD73, the anti-tumor activity and the curative effect can be further improved.

Disclosure of Invention

The present application provides a fusion protein comprising an antibody or antigen-binding fragment thereof capable of specifically binding CD 73. In the present application, the fusion protein may further comprise a TGF-beta binding moiety. The fusion proteins described herein have one or more of the following properties: 1) Capable of specifically binding to human and/or monkey CD73 protein; 2) Capable of binding TGF-beta; 3) Has inhibiting effect on CD73 enzyme activity on tumor cell surface; 4) Has inhibiting effect on soluble CD73 enzyme activity; 5) Capable of blocking the TGF- β/SMAD signaling pathway; 6) Can relieve the inhibition of adenosine to T cells; and 7) can inhibit the growth and/or proliferation of tumor cells.

In one aspect, the present application provides a fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to CD73 protein, said antibody or antigen-binding fragment thereof comprising at least one CDR in the heavy chain variable region amino acid sequence as depicted in SEQ ID No. 41.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence of SEQ ID No. 3.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence of SEQ ID No. 2.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence of SEQ ID No. 1.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence of SEQ ID No. 1, the HCDR2 comprising the amino acid sequence of SEQ ID No. 2 and the HCDR3 comprising the amino acid sequence of SEQ ID No. 3.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises H-FR1, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of said HCDR1, and said H-FR1 comprises the amino acid sequence shown in SEQ ID NO. 7, SEQ ID NO. 12 or SEQ ID NO. 27.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 8 or SEQ ID NO. 13.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO 9 or SEQ ID NO 14.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises H-FR4, the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 10 or SEQ ID NO. 15.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises an antibody heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 41.

In certain embodiments, the VH in the fusion protein comprises the amino acid sequence shown as SEQ ID NO. 11, SEQ ID NO. 16 or SEQ ID NO. 28.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises at least one CDR of the light chain variable region amino acid sequence as shown in SEQ ID NO. 42.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID No. 6.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID No. 5.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID No. 4.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence of SEQ ID No. 4, the LCDR2 comprising the amino acid sequence of SEQ ID No. 5 and the LCDR3 comprising the amino acid sequence of SEQ ID No. 6.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises L-FR1, the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of said LCDR1, and said L-FR1 comprises the amino acid sequence shown in SEQ ID NO. 17, SEQ ID NO. 22 or SEQ ID NO. 29.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises L-FR2, said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises the amino acid sequence set forth in SEQ ID NO. 18 or SEQ ID NO. 23.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises L-FR3, said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises the amino acid sequence shown in SEQ ID NO. 19 or SEQ ID NO. 24.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO. 20 or SEQ ID NO. 25.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises an antibody light chain variable region, VL, comprising the amino acid sequence set forth in SEQ ID NO. 42.

In certain embodiments, the VL in the fusion protein comprises the amino acid sequence set forth in SEQ ID NO. 21, SEQ ID NO. 26 or SEQ ID NO. 30.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises a VH and a VL comprising an amino acid sequence selected from any one of the following groups:

1) The VH comprises an amino acid sequence shown in SEQ ID NO. 11, and the VL comprises an amino acid sequence shown in SEQ ID NO. 21;

2) The VH comprises an amino acid sequence shown in SEQ ID NO. 16, and the VL comprises an amino acid sequence shown in SEQ ID NO. 26; and

3) The VH comprises the amino acid sequence shown in SEQ ID NO. 28, and the VL comprises the amino acid sequence shown in SEQ ID NO. 30.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises an antibody heavy chain constant region.

In certain embodiments, the heavy chain constant region of the antibody or antigen binding fragment thereof in the fusion protein is derived from an IgG constant region.

In certain embodiments, the heavy chain constant region of the antibody or antigen binding fragment thereof in the fusion protein is derived from a human IgG constant region.

In certain embodiments, the heavy chain constant region is derived from a heavy chain constant region selected from the group consisting of: igG1, igG2, igG3 and IgG4.

In certain embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 33 or SEQ ID NO. 34.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 31.

In certain embodiments, the antibody or antigen binding fragment thereof in the fusion protein comprises an antibody light chain constant region.

In certain embodiments, the light chain constant region is derived from igkappa.

In certain embodiments, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 35.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO. 32.

In certain embodiments, the antibody or antigen-binding fragment thereof in the fusion protein comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 31 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 32.

In certain embodiments, the fusion protein further comprises a TGF-beta binding moiety.

In certain embodiments, the TGF-beta binding-moiety in the fusion protein specifically binds to human TGF-beta.

In certain embodiments, the TGF-beta binding-portion of the fusion protein comprises an extracellular domain of a human TGF-beta receptor.

In certain embodiments, the human TGF-beta receptor comprises human TGF-beta RII.

In certain embodiments, the TGF-beta binding-portion of the fusion protein comprises the amino acid sequence shown in SEQ ID NO. 37.

In certain embodiments, the TGF- β binding moiety in the fusion protein is linked to the heavy chain variable region of the antibody or antigen binding fragment thereof that specifically binds CD73 protein.

In certain embodiments, the TGF- β binding moiety in the fusion protein is directly or indirectly linked to the C-terminus of the heavy chain constant region of the antibody or antigen binding fragment thereof that specifically binds the CD73 protein.

In certain embodiments, the N-terminus of the TGF- β binding moiety in the fusion protein is directly or indirectly linked to the C-terminus of the heavy chain constant region of the antibody or antigen binding fragment thereof that specifically binds the CD73 protein.

In certain embodiments, the TGF- β binding moiety in the fusion protein is linked to the heavy chain variable region of the antibody or antigen binding fragment thereof that specifically binds CD73 protein through a peptide linker.

In certain embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO. 36.

In certain embodiments, the fusion protein comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 39 and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 32.

In certain embodiments, the fusion protein comprises two polypeptides each comprising the amino acid sequence set forth in SEQ ID NO. 39 and two polypeptides each comprising the amino acid sequence set forth in SEQ ID NO. 32.

In certain embodiments, the fusion protein has one or more of the following properties:

1) Capable of specifically binding to human and/or monkey CD73 protein;

2) Has inhibiting effect on CD73 enzyme activity on tumor cell surface;

3) Has inhibiting effect on soluble CD73 enzyme activity;

4) Capable of blocking the TGF- β/SMAD signaling pathway; and

5) Can relieve the inhibition of adenosine to T cells.

In another aspect, the present application provides isolated nucleic acid molecules encoding the fusion proteins described herein.

In another aspect, the present application provides a vector comprising a nucleic acid molecule described herein.

In certain embodiments, the vector comprises a plasmid vector or a viral vector.

In another aspect, the present application provides a host cell comprising a nucleic acid molecule as described herein or a vector as described herein.

In another aspect, the present application provides immunoconjugates comprising the fusion protein.

In another aspect, the present application provides a multispecific molecule comprising a fusion protein described herein.

In certain embodiments, the multispecific molecule additionally specifically binds one or more other targets.

In another aspect, the present application also provides chimeric antigen receptors comprising the fusion proteins described herein.

In another aspect, the present application also provides an immune effector cell comprising the chimeric antigen receptor.

In another aspect, the present application also provides a method of preparing the fusion protein, the method comprising culturing the host cell under conditions such that the fusion protein is expressed.

In another aspect, the present application also provides a pharmaceutical composition comprising the fusion protein, the nucleic acid molecule, the vector, the host cell, the immunoconjugate, the multispecific molecule, the chimeric antigen receptor and/or the immune effector cell, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition further comprises other pharmaceutically active ingredients.

In another aspect, the present application also provides a kit comprising the fusion protein, the nucleic acid molecule, the vector, the host cell, the immunoconjugate, the multispecific molecule, the chimeric antigen receptor, the immune effector cell, and/or the pharmaceutical composition.

In another aspect, the application also provides the use of the fusion protein, the nucleic acid molecule, the vector, the host cell, the immunoconjugate, the multispecific molecule, the chimeric antigen receptor, the immune effector cell, the pharmaceutical composition and/or the kit for the preparation of a medicament for the prevention and/or treatment of a disease and/or disorder.

In certain embodiments, the disease and/or condition comprises a tumor.

In certain embodiments, the disease and/or disorder comprises a solid tumor and/or a hematological tumor.

In certain embodiments, the tumor comprises a CD73 positive tumor.

In certain embodiments, the tumor is selected from one or more of the following: colorectal cancer, renal cell carcinoma, non-small cell carcinoma, and pancreatic cancer.

In another aspect, the present application provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of the fusion protein or the pharmaceutical composition.

In another aspect, the present application provides a method of detecting the presence or amount of CD73 and/or TGF- β in a sample, the method comprising administering the fusion protein.

In another aspect, the application provides the use of the fusion protein in the preparation of a detection reagent for determining the presence or amount of CD73 and/or TGF- β in a sample.

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the invention as described herein. Accordingly, the drawings and descriptions herein are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention that are related to the present application will be better understood by reference to the exemplary embodiments and the drawings that are described in detail below. The drawings are briefly described as follows:

FIG. 1 shows the binding curve of murine antibody 13D12 to tumor surface CD 73.

FIG. 2 shows a flow chart of the binding of murine antibody 13D12 to monkey CD3+CD8+T cells.

FIG. 3 shows the binding of humanized antibody 7002-01 to soluble recombinant CD 73.

FIGS. 4A-4B show the inhibition of CD73 enzyme activity by humanized antibody 7002-01 in serum of liver cancer patient (A) and melanoma patient (B).

FIG. 5 shows the alleviating effect of humanized antibody 7002-01 on AMP-mediated inhibition of CD4+ T cells.

FIG. 6 shows the recovery effect of humanized antibody 7002-01 on killing of tumor cells by PBMC.

FIG. 7 shows a schematic structural diagram of a fusion protein.

FIG. 8 shows the detection of affinity of fusion proteins for human CD73 overexpressing cells.

FIG. 9 shows the detection of the affinity of fusion proteins for monkey CD73 overexpressing cells.

FIG. 10 shows the detection of the enzymatic inhibition of CD73 by the fusion protein on human breast cancer cells.

FIG. 11 shows the detection of the enzymatic inhibition of human melanoma cell CD73 by the fusion protein.

FIG. 12 shows the enzyme activity inhibition assay of the soluble CD73 antigen by the fusion protein.

FIG. 13 shows that the fusion proteins block TGF-beta/SMAD signaling pathway detection.

FIG. 14 shows the experimental results of the inhibition of the inverted T cells by the fusion proteins.

Detailed Description

Further advantages and effects of the invention of the present application will be readily apparent to those skilled in the art from the disclosure of the present application by describing embodiments of the invention with specific examples.

Definition of terms

In the present application, the term "cluster of differentiation 73" or "CD73" is also referred to as extracellular-5 '-nucleotidase, which is capable of converting extracellular 5' nucleoside monophosphates to nucleosides, i.e., adenosine Monophosphate (AMP) to adenosine. The term CD73 includes membrane-bound forms (also referred to as membrane-bound CD 73) or soluble forms (also referred to as soluble or non-membrane-bound CD 73). CD73 may be isolated from cells or tissues that naturally express them, or produced recombinantly using techniques well known in the art. The sequence of CD73 is well known in the art and can be found in NCBI database accession No. nm_002526.

The term "fusion protein" generally refers to a protein/polypeptide having at least two moieties covalently linked together. Wherein each moiety may be a protein/polypeptide having different properties. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties may be linked by a single peptide bond or by a peptide linker.

In this application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibodies", for the purposes of the present invention the term "antibody" also includes antibody fragments, such as Fab, F (ab') ₂ Fv, scFv, fd, dAb and other antibody fragments that retain antigen binding function (e.g., specifically bind CD73 protein). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetramer units with another polypeptide called the J chain and contain 10 antigen binding sites, whereas IgA antibodies comprise 2-5 basic 4-chain units that can polymerize in conjunction with the J chain to form multivalent combinations. For IgG, the 4-chain unit is typically about 15 0,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, followed by four CH domains for the mu and epsilon isoforms. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The VH and VL pairs together form a single antigen binding site. For the structure and properties of antibodies of different classes, see e.g. Basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw (eds), appleton&Lange, norwalk, conn, 1994, pages 71 and chapter 6. L chains from any vertebrate species can be divided into one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of the constant domain of the heavy Chain (CH). Currently there are five classes of immunoglobulins: igA, igD, igE, igG and IgM, have heavy chains named α, δ, ε, γ and μ, respectively.

In this application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (e.g., CD 73). In the present application, the antigen binding fragment may include Fab, fab', F (ab) ₂ Fv fragment, F (ab') ₂ scFv, di-scFv and/or dAb.

In this application, the term "Fab" generally refers to antigen binding fragments of antibodies. As described above, papain can be used to digest intact antibodies. The antibodies, after digestion with papain, produce two identical antigen-binding fragments, a "Fab" fragment, and a residual "Fc" fragment (i.e., fc region, supra). Fab fragments can consist of a complete L chain with a heavy chain variable region and the first constant region (CH 1) of the H chain (VH).

In this application, the term "Fab' fragment" generally refers to a monovalent antigen binding fragment of a human monoclonal antibody that is slightly larger than the Fab fragment. For example, a Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of a heavy chain. For example, a Fab' fragment can also include part or all of the 220-330 amino acid residues of the heavy chain.

In the present application, the term "F (ab') 2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F (ab') 2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. F (ab') 2 fragments have divalent antigen binding activity and are capable of cross-linking antigens.

In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, fv fragments comprise all or part of the amino terminal variable region of about 110 amino acids of the heavy and light chains.

In the present application, the term "scFv" generally refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g. via a synthetic linker such as a short flexible polypeptide linker) and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically stated otherwise, as used herein, an scFv may have the VL and VH variable regions described in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

In this application, the term "dAb" generally refers to an antigen binding fragment having a VH domain, a VL domain or having a VH domain or a VL domain, reference is made, for example, to Ward et al (Nature, 1989Oct 12;341 (6242): 544-6), reference is made to Holt et al, trends Biotechnol.,2003,21 (11): 484-490; and other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd. The term "dAb" generally includes sdabs. The term "sdAb" generally refers to a single domain antibody. Single domain antibodies generally refer to antibody fragments consisting of only the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL).

In this application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" refers to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies as used herein may be prepared in hybridoma cells or may be prepared by recombinant DNA methods.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody") and the constant region is derived from a human antibody such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent antibody.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody are replaced with the corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible, provided that they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. A "humanized" form of a non-human antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) having the desired properties, affinity and/or ability, such as alpaca, mouse, rat, rabbit or non-human primate. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.

The term "fully human antibody" generally refers to an antibody comprising only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell, the fully human antibody may contain a murine sugar chain. Similarly, "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans by phage display or other molecular biological methods in transgenic animals with human immunoglobulin germline sequences. Exemplary techniques that can be used to make antibodies are in U.S. patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as the use of libraries, are known in the art.

In this application, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, dev. Comparat. Immunol.27:55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

In this application, the CDRs contained in the antibody or antigen binding fragment thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the present application can be determined by Kabat, chothia or IMGT numbering system. In certain embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the present application can be determined by the Kabat numbering system.

In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain. The variable domains of the heavy and light chains, respectively, may be referred to as "V _H "and" V _L "(or" VH "and" VL ", respectively). These domains are typically the most variable portions of an antibody (relative to other antibodies of the same type) and comprise antigen binding sites.

In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ greatly in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. It is typically concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody (see Kabat et al, sequences of Immunological Interest, fifth Edition, national Institute of Health, bethesda, md. (1991)).

In this application, the term "FR" generally refers to the more highly conserved portion of the antibody variable domain, which is referred to as the Framework Region (Framework Region). Typically, the variable domains of the natural heavy and light chains each comprise four FR regions, namely four in VH (H-FR 1, H-FR2, H-FR3 and H-FR 4), and four in VL (L-FR 1, L-FR2, L-FR3 and L-FR 4).

In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds a target (which may be an epitope) may be an antibody that binds the target with greater affinity, avidity, more readily, and/or for a longer duration than it binds other targets. In certain embodiments, the antibodies specifically bind to epitopes on proteins that are conserved among proteins of different species. In certain embodiments, specific binding may include, but is not required to be, exclusively binding. The term also applies, for example, to antigen binding proteins that are specific for a particular epitope that cross-reacts with multiple antigens, wherein specific antibodies bind to multiple antigens that carry the cross-reactive epitope. The binding sites of such antigen binding proteins and/or antigen binding proteins having specific binding cross-reactive epitopes are also referred to as multi-specific or cross-specific binding site antigen binding proteins, respectively. For example, an antigen binding protein may have multiple specific binding sites that specifically bind to epitopes of multiple different antigens cross-reactive.

In this application, the terms "transforming growth factor beta" and "TGF-beta" generally refer to any TGF-beta family protein encoded by the TGFB1, TGFB2, and/or TGFB3 genes in humans. As used herein, the term "human TGF- β1" generally refers to a tgfβ1 protein encoded by a human TGFB1 gene (e.g., a wild-type human TGFB1 gene). An exemplary wild-type human TGF-beta 1 protein is provided by GenBank accession number NP-000651.3. As used herein, the term "human tgfβ2" generally refers to a tgfβ2 protein encoded by a human TGFB2 gene (e.g., a wild-type human TGFB2 gene). Exemplary wild-type human TGF-beta 2 proteins are provided by GenBank accession numbers NP-001129071.1 and NP-003229.1. As used herein, the term "human tgfβ3" refers to a tgfβ3 protein encoded by a human TGFB3 gene (e.g., a wild-type human TGFB3 gene). Exemplary wild-type human TGF-beta 3 proteins are provided by GenBank accession numbers NP-003230.1, NP-001316868.1, and NP-001316867.1.

In this application, the terms "transforming growth factor beta receptor" and "TGF-beta receptor" refer to any TGF-beta receptor family protein encoded by the TGFBR1, TGFBR2 and TGFBR3 genes in humans. As used herein, the term "human tgfβr1" refers to a TGF- βri protein encoded by a human TGFBR1 gene (e.g., a wild-type human TGFBR1 gene). Exemplary wild-type human TGF- βRII proteins are provided by GenBank accession numbers NP-004603.1, NP-001124388.1, and NP-001293139.1. In this application, the term "TGF- βRII" may comprise wild-type human TGF- βRII or a functionally active fragment thereof. For example, in the present application, the TGF-. Beta.RII may comprise the amino acid sequence shown as SEQ ID NO. 37.

In this application, the term "binding moiety" generally refers to a moiety that has the ability to specifically bind to a target molecule or complex. The binding moiety may comprise a small molecule, a peptide, a modified peptide (e.g., a peptide having an unnatural amino acid residue and/or a stapled peptide), a polypeptide, a protein, an antibody fragment, an scFv, an Fc-containing polypeptide, a fusion antibody, a ligand, an aptamer, a nucleic acid, a variant thereof, or any combination thereof. In certain embodiments, the binding moiety comprises a domain of a native protein or variant thereof, wherein the native protein binds to the target molecule or complex via the domain. For example, the term "TGF-beta binding-moiety" generally refers to a moiety that specifically binds to one or more proteins of the TGF-beta family (e.g., TGF-beta 1, TGF-beta 2, or TGF-beta 3), and in certain embodiments may comprise an extracellular domain of a TGF-beta receptor (e.g., TGF-beta RII, or TGF-beta RIII).

In this application, the term "linked" generally refers to covalent or non-covalent bonding between two molecules or moieties. The connection may comprise a direct connection or an indirect connection. For example, the indirect connection may be via an intermediate molecule or moiety.

In this application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance.

In this application, the term "nucleic acid molecule" generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to allow expression of the genetic material elements carried thereby within the host cell. For example, the carrier may comprise: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors may include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these.

In this application, the term "host cell" generally refers to a single cell, cell line or cell culture that may or may not be the recipient of a subject plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention. Cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. Cells may include cells transfected in vitro with the vectors described herein. The cells may be bacterial cells (e.g., E.coli), yeast cells, or other eukaryotic cells, such as COS cells, chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells.

In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (e.g., covalent attachment via a linker molecule) of the other agent (e.g., a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent) to the fusion protein, which conjugate can specifically bind to an antigen on a target cell through the antibody or antigen-binding fragment thereof, delivering the other agent to the target cell.

In the present application, the term "pharmaceutical composition" generally refers to a composition for the prevention/treatment of a disease or disorder. The pharmaceutical composition may comprise a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein and/or a cell described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counter ions, such as sodium, and/or nonionic surfactants.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

In the present application, the term "prevention and/or treatment" generally refers to the therapeutic or prophylactic measures described herein. For example, a "treatment" method may employ administration of a fusion protein to a subject suffering from a disease or susceptible to such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurrent disease or disorder, or in order to extend the survival of the subject beyond that expected without such treatment.

In this application, the term "effective amount" in the context of administering a therapy to a subject generally refers to the amount of therapy that achieves the desired prophylactic or therapeutic effect.

In the present application, reference to proteins, polypeptides and/or amino acid sequences is also to be understood as comprising at least the following ranges: variants or homologues having the same or similar function as the protein or polypeptide.

In the present application, the variant may be, for example, a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or antigen binding fragment thereof that specifically binds CD 73). For example, the functional variant may comprise a protein or polypeptide that has been altered in amino acids by at least 1, such as 1-30, 1-20, or 1-10, and yet another such as 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%,70%,80%,90%, or 100% of the biological activity (e.g., antigen binding capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution.

In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or antigen binding fragment thereof that specifically binds CD 73).

In this application, homology generally refers to similarity, similarity or association between two or more sequences. "percent sequence homology" can be calculated by: the two sequences to be aligned are compared in a comparison window, the number of positions in the two sequences where the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) is present is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to produce the percent sequence homology. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence. The homology can also be determined by the following method: FASTA and BLAST. For a description of FASTA algorithm, see w.r.pearson and d.j.lipman, "improved tools for biological sequence comparison", proc.Natl. Acad.Sci., U.S. Proc., 85:2444-2448, 1988; "quick sensitive protein similarity search" by d.j.lipman and w.r.pearson, science,227:1435-1441, 1989. For a description of the BLAST algorithm, see "a basic local contrast (alignment) search tool", journal of molecular biology, 215:403-410, 1990.

In this application, the term "comprising" generally refers to the meaning of including, generalizing, containing or comprising. In some cases, the meaning of "as", "consisting of … …" is also indicated.

In this application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

Detailed Description

The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be contacted with an antigen or epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, abM, a combination of Kabat/Chothia et al. These coding systems are known in the art and can be found, for example, in http:// www.bioinf.org.uk/abs/index. The CDR regions can be determined by one skilled in the art using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, CDR regions may differ. In this application, the CDRs encompass CDR sequences partitioned according to any CDR partitioning scheme; variants thereof are also contemplated, including amino acid substitutions, deletions and/or additions to the amino acid sequence of the CDRs. Such as 1-30, 1-20 or 1-10, and further such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; homologues thereof are also contemplated, which may be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequences of the CDRs. In the present application, the isolated antigen binding protein may be defined by Kabat.

In one aspect, the present application provides a fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to CD73 protein. In certain embodiments, the CD73 antibody or antigen-binding fragment thereof comprises at least one CDR in the heavy chain variable region (VH) amino acid sequence as set forth in SEQ ID NO. 41. For example, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO. 11, SEQ ID NO. 16 and SEQ ID NO. 28.

In the present application, the CDRs of the CD73 antibody or antigen-binding fragment thereof may be divided in any form, so long as the VH is identical to the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:16 and SEQ ID NO:28, and the HCDR obtained by division in any form may fall within the scope of the present application.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID No. 3.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID No. 2.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID No. 1.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence of SEQ ID No. 1, the HCDR2 may comprise the amino acid sequence of SEQ ID No. 2, and the HCDR3 may comprise the amino acid sequence of SEQ ID No. 3.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise H-FR1, the C-terminus of said H-FR1 is directly or indirectly linked to the N-terminus of said HCDR1, and said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 7, SEQ ID NO. 12 or SEQ ID NO. 27.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise H-FR2, said H-FR2 being located between said HCDR1 and said HCDR2, and said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 13.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO 9 or SEQ ID NO 14.

In this application, the CD73 antibody or antigen binding fragment thereof may comprise H-FR4, the N-terminus of H-FR4 is linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 10 or SEQ ID NO. 15.

In this application, the CD73 antibody or antigen binding fragment thereof may comprise H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 7, SEQ ID NO. 12 or SEQ ID NO. 27, said H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 13, said H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 14, and said H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 10 or SEQ ID NO. 15.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise a VH comprising the amino acid sequence shown in SEQ ID NO. 41. For example, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO. 11, SEQ ID NO. 16 and SEQ ID NO. 28.

In the present application, the CD73 antibody or antigen-binding fragment thereof may include an antibody heavy chain constant region. For example, the antibody heavy chain constant region may be derived from the heavy chain constant region of any immunoglobulin, including IgM, igD, igG, igA and IgE. For example, the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In the present application, the heavy chain constant region of the immunoglobulin may comprise a mutant thereof. In the present application, the antibody heavy chain constant region may be derived from the heavy chain constant region of any of human IgG 1-4. For example, the CD73 antibody or antigen binding fragment thereof may comprise a constant region derived from a human IgG1 heavy chain. For example, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO. 33. For example, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO. 34.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise at least one CDR in the antibody light chain variable region VL, which may comprise the amino acid sequence shown in SEQ ID NO. 42. For example, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NO. 21, SEQ ID NO. 26 and SEQ ID NO. 30. In the present application, the LCDR of the CD73 antibody or antigen-binding fragment thereof may be divided in any form, so long as VL is identical to the amino acid sequence shown in any one of SEQ ID NOS: 42, and the LCDR divided in any form falls within the scope of the present application.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 6.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 5.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 4.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID No. 4, the LCDR2 may comprise the amino acid sequence shown in SEQ ID No. 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID No. 6.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise L-FR1, the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of said LCDR1, and said L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:17, SEQ ID NO:22 and SEQ ID NO: 29.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise L-FR2, said L-FR2 being located between said LCDR1 and said LCDR2, and said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 18 or SEQ ID NO. 23.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise L-FR3, said L-FR3 being located between said LCDR2 and said LCDR3, and said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 19 or SEQ ID NO. 24.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise L-FR4, the N-terminus of L-FR4 is linked to the C-terminus of LCDR3, and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO. 20 or SEQ ID NO. 25.

In this application, the CD73 antibody or antigen binding fragment thereof may comprise L-FR1, L-FR2, L-FR3 and L-FR4, said L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:17, SEQ ID NO:22 and SEQ ID NO:29, said L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:23, said L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:19 or SEQ ID NO:24, and said L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO: 25.

In this application, the CD73 antibody or antigen-binding fragment thereof may comprise a VL comprising the amino acid sequence shown as SEQ ID NO. 42. For example, the VL may comprise an amino acid sequence set forth in any one of SEQ ID NO. 21, SEQ ID NO. 26 and SEQ ID NO. 30.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise an antibody light chain constant region. For example, the light chain constant region may be derived from a human antibody light chain constant region. For example, the light chain constant region may be derived from a human igκ constant region. For example, the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO. 35.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. For example, the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 4, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 6.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise VH and VL. For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 41, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 42.

For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 11, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 21.

For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 16, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 26.

For example, the VH may comprise the amino acid sequence shown in SEQ ID NO. 28, and the VL may comprise the amino acid sequence shown in SEQ ID NO. 30.

In the present application, the CD73 antibody may comprise a heavy chain, for example, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 31.

In the present application, the CD73 antibody may comprise a light chain, for example, the light chain may comprise the amino acid sequence shown in SEQ ID NO. 32.

In the present application, the CD73 antibody or antigen-binding fragment thereof may comprise an antibody or antigen-binding fragment thereof. In the present application, the antigen binding fragments may include Fab, fab ', fv fragments, F (ab') ₂ ，F(ab) ₂ scFv, di-scFv and/or dAb. In the present application, the antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies, and/or fully human antibodies. For example, the VH of the humanized CD73 antibody may comprise the amino acid sequence shown in SEQ ID NO. 28. For example, the VL of the humanized CD73 antibody may comprise the amino acid sequence shown in SEQ ID NO. 30. For example, the VH of the humanized CD73 antibody may comprise the amino acid sequence shown in SEQ ID NO. 16. For example, the VL of the humanized CD73 antibody may comprise the amino acid sequence set forth in SEQ ID NO. 26.

Furthermore, it is contemplated that the CD73 antibodies or antigen binding fragments thereof described herein may comprise heavy and/or light chain sequences modified with one or more conserved sequence modifications present therein. By "conservative sequence modifications" is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR regions of a CD73 antibody or antigen binding fragment thereof described herein may be replaced with other amino acid residues of the same side chain set. Those skilled in the art know that some conservative sequence modifications do not result in the disappearance of antigen binding, see, for example, brummell et al, (1993) Biochem 32:1180-8; de Wildt et al, (1997) Prot.Eng.10:835-41; komissarov et al, (1997) J.biol. Chem.272:26864-26870; hall et al, (1992) J.Immunol.149:1605-12; kelley and O' Connell (1993) biochem.32:6862-35; adib-Conquy et al, (1998) int. Immunol.10:341-6and beer et al, (2000) Clin. Can. Res.6:2835-43.

In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) may be introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG 1) and/or CH3 domain (residues 341-447 of human IgG 1) and/or hinge region (numbered according to the EU numbering system) of an antibody described herein to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell.

In certain embodiments, one, two, or more amino acid substitutions may be introduced into the Fc region of an IgG constant domain to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332 and 396 (numbered according to the EU numbering system) may be substituted with a different amino acid residue such that the antibody has an altered affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand that alters affinity thereto may be, for example, an Fc receptor or a C1 component of complement. This method is described in more detail in U.S. Pat. nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In certain embodiments, deletion or inactivation (by point mutation or otherwise) of the constant region domains may reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. For a description of mutations that delete or deactivate constant domains, thereby increasing tumor localization, see, e.g., U.S. patent nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., shields RL et al, (2001) J Biol Chem 276:6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations in the constant regions of the antibodies described herein can be made: a N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; L235A substitution; c236 is deleted; P238A substitution; S239D substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L is substituted; a327Q substitution; P329A substitution; a332L substitution; I332E substitution; or a P396L substitution (numbered according to the EU numbering system). In certain embodiments, in the constant region of the antibody, the Fc region of the IgG may comprise L234A and L235A mutations; optionally, the Fc fragment of the IgG further comprises a G237A mutation.

In the present application, the fusion protein may comprise a moiety that specifically binds to CD73 protein and a TGF- β binding moiety. For example, the moiety that specifically binds to CD73 protein may comprise a CD73 antibody as described herein.

In the present application, the TGF-beta binding-moiety may be linked to any part of the immunoglobulin chain of the CD73 antibody or antigen binding fragment thereof, e.g., comprising an N-terminal or C-terminal amino acid residue. For example, a TGF-beta binding-moiety may be linked (covalently or non-covalently) to an immunoglobulin chain directly or via a linker (e.g., a peptide linker). Covalent attachment may be chemical or genetic (i.e., to form a fusion protein). In certain embodiments, the TGF-beta binding-moiety may be linked (e.g., covalently) to a C-terminal amino acid residue of an immunoglobulin chain. In certain embodiments, the TGF- β binding moiety may be linked (e.g., via a peptide bond) to a C-terminal amino acid residue of an immunoglobulin chain without a linker. In certain embodiments, the TGF- β binding moiety may be linked (e.g., covalently) to a C-terminal amino acid residue of an immunoglobulin chain via a linker. In certain embodiments, the linker may be a peptide linker. In certain embodiments, the peptide structure has the general formula (G ₄ S) n, where n is an integer. In certain embodiments, the peptide linker comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 36.

In certain embodiments, the TGF- β binding moiety may be linked (e.g., covalently) to the heavy chain constant region of a CD73 antibody. The TGF-beta binding-moiety may be linked (e.g., covalently) to any portion of the heavy chain constant region of a CD73 antibody. In certain embodiments, the TGF-beta binding-moiety may be linked to a C-terminal amino acid residue of the heavy chain constant region of a CD73 antibody. For example, the linkage may be via a peptide linker. For example, the peptide linker may comprise the amino acid sequence shown in SEQ ID NO. 36.

In the present application, the TGF-beta binding-moiety may be any moiety that specifically binds to one or more members of the family or isoforms of TGF-beta. In certain embodiments, the TGF-beta binding-moiety specifically binds TGF-beta 1 (e.g., human TGF-beta 1). In certain embodiments, the TGF-beta binding-moiety specifically binds TGF-beta 2 (e.g., human TGF-beta 2). In certain embodiments, the TGF-beta binding-moiety specifically binds TGF-beta 3 (e.g., human TGF-beta 3). In certain embodiments, the TGF-beta binding-moiety specifically binds to at least two of TGF-beta 1 (e.g., human TGF-beta 1), TGF-beta 2 (e.g., human TGF-beta 2), and TGF-beta 3 (e.g., human TGF-beta 3). In certain embodiments, the TGF-beta binding-moiety specifically binds to TGF-beta 1 (e.g., human TGF-beta 1) and TGF-beta 3 (e.g., human TGF-beta 3). In certain embodiments, the TGF-beta binding-moiety specifically binds TGF-beta 1 (e.g., human TGF-beta 1), TGF-beta 2 (e.g., human TGF-beta 2), and TGF-beta 3 (e.g., human TGF-beta 3). One skilled in the art will appreciate that a TGF-beta binding-moiety that specifically binds one family member or isoform of TGF-beta may bind one or more other family members or isoforms of TGF-beta with similar or higher affinity. Exemplary TGF-beta binding moieties are disclosed in De Crescendzo et al (2008) Transforming Growth Factor-beta in Cancer Therapy, volume II, cancer Drug Discovery and Development, humana Press; zwaagstra et al (2012) Mol Cancer Ther.11 (7): 1477-87; ravi et al (2018) nat. Commun.9:741; EP0975771B1, US7786261B2, US8993524B2 and US20150225483A1, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the TGF-beta binding-portion comprises a domain of a protein that binds TGF-beta 1 (e.g., human TGF-beta 1), TGF-beta 2 (e.g., human TGF-beta 2), and/or TGF-beta 3 (e.g., human TGF-beta 3), or a variant thereof having similar or improved TGF-beta binding affinity. In certain embodiments, the domain is an extracellular domain of a TGF- β receptor (e.g., a human TGF- β receptor). In certain embodiments, the domain is a tgfβ binding domain of a TGF- β receptor (e.g., a human TGF- β receptor). In certain embodiments, the TGF- β receptor is selected from the group consisting of: TGF-. Beta.RII (e.g., human TGF-. Beta.RII), and TGF-. Beta.RII (e.g., human TGF-. Beta.RII). In certain embodiments, the TGF-beta binding-portion comprises an extracellular domain of TGF-beta RII. In certain embodiments, the TGF-beta binding-portion comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO. 37. In certain embodiments, the TGF-beta binding-portion comprises the amino acid sequence shown in SEQ ID NO. 37. In certain embodiments, the TGF-beta binding-portion consists of an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO. 37.

In the present application, the fusion protein may comprise: 1) A CD73 antibody or antigen-binding fragment thereof; 2) TGF-beta binding moiety, and 3) peptide linker.

In this application, the fusion protein may comprise a first polypeptide and a second polypeptide, wherein the first polypeptide may comprise the amino acid sequence shown in SEQ ID NO. 39 and the second polypeptide may comprise the amino acid sequence shown in SEQ ID NO. 32. Wherein the first polypeptide may comprise, in order from the N-terminus to the C-terminus: heavy chain of CD73 antibody, peptide linker, TGF- βrii extracellular domain, wherein the second polypeptide may comprise a light chain of CD73 antibody. For example, the first polypeptide may comprise the amino acid sequence shown in SEQ ID NO. 39. For example, the second polypeptide may comprise the amino acid sequence shown in SEQ ID NO. 32.

In the present application, the fusion protein may comprise two copies of the first polypeptide and two copies of the second polypeptide, wherein the first polypeptide may comprise, in order from the N-terminus to the C-terminus: heavy chain of CD73 antibody, peptide linker, TGF- βrii extracellular domain, wherein the second polypeptide may comprise a light chain of CD73 antibody. For example, the first polypeptide may comprise the amino acid sequence shown in SEQ ID NO. 39. For example, the second polypeptide may comprise the amino acid sequence shown in SEQ ID NO. 32.

In the present application, the desired properties of the fusion protein include: (1) Specifically binds to CD73 (e.g., membrane binds to human CD73 or soluble human CD 73); (2) Inhibit or reduce the enzymatic activity of CD73 (e.g., membrane bound human CD73 or soluble human CD 73); (3) Increasing proliferation of anti-CD 3/anti-CD 28 stimulated T cells (e.g., cd4+ T cells) in the presence of Adenosine Monophosphate (AMP); (4) mediates CD73 internalization; (5) decreasing adenosine levels in CD73 expressing tumor cells; (6) Stimulating an immune response (e.g., an immune response against a tumor or immunogen); (7) Preventing and/or treating tumors (e.g., CD 73-expressing tumors). The fusion proteins of the present application have one or more of the above-described desirable properties.

Nucleic acid molecules, vectors and host cells

In another aspect, the present application provides isolated nucleic acid molecules that can encode the fusion proteins described herein. For example, it may be produced or synthesized by: (i) Amplified in vitro, for example by Polymerase Chain Reaction (PCR) amplification; (ii) produced by clonal recombination; (iii) Purified, e.g., fractionated by cleavage and gel electrophoresis; or (iv) synthesized, for example by chemical synthesis.

In another aspect, the present application provides a vector that may comprise a nucleic acid molecule as described herein. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not exclusively.

In another aspect, the present application provides a host cell, which may comprise a nucleic acid molecule as described herein or a vector as described herein. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a 293T cell, a COS-1 cell, a SP2/0 cell, a NS0 cell, or a myeloma cell. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.

Immunoconjugates, multispecific molecules, chimeric antigen receptors, immune effector cells, pharmaceutical compositions, and methods of making

In another aspect, the present application also provides immunoconjugates that may comprise the fusion proteins described herein.

In certain embodiments, the fusion proteins described herein, or fragments thereof, may be linked to another therapeutic agent. The linkage may be through one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of linkers (which may be known in the art) may be used to form the immunoconjugate. The immunoconjugate may further comprise, for example, an antibody-drug conjugate (ADC).

In certain embodiments, the therapeutic agent may be coupled to the fusion proteins of the present application using linker techniques known in the art. Examples of the types of linkers that have been used to couple therapeutic agents to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. A linker may be selected that is easily cleaved, e.g., by low pH within the lysosomal compartment or by proteases (e.g., proteases preferentially expressed in tumor tissue, such as cathepsins, such as cathepsin B, C, D).

In another aspect, the present application provides multispecific molecules that may comprise a fusion protein described herein, and additional specificity binding antigen moieties.

For example, the fusion proteins of the present application may be linked to other antibodies or antigen-binding fragments thereof capable of specifically binding any protein that may be used as a potential target for combination therapy. To produce the multispecific molecules, a fusion protein of the invention may be linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

Thus, in another aspect, the present application provides a multispecific molecule comprising a fusion protein of the present invention.

In certain embodiments, the multispecific molecule specifically binds CD73 (e.g., membrane binds human CD73 and/or soluble human CD 73), and TGF- β, and additionally specifically binds one or more other targets.

In another aspect, the present application also provides a chimeric antigen receptor comprising a fusion protein described herein.

In another aspect, the present application also provides an immune effector cell comprising the chimeric antigen receptor described herein. In certain embodiments, the immune effector cell comprises a lymphocyte.

In another aspect, the present application also provides a pharmaceutical composition comprising a fusion protein described herein, the nucleic acid molecule, the vector, the host cell, the immunoconjugate, the multispecific molecule, the chimeric antigen receptor and/or the immune effector cell, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, typically those active compounds having complementary activity that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents compatible with drug administration, generally safe, non-toxic.

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, endoluminal, intra-arterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient, as described in WO 2015/036583.

In the present application, the pharmaceutical composition may further comprise other pharmaceutically active ingredients. For example, the pharmaceutically active ingredient may comprise a drug having anti-tumor activity. For example, the pharmaceutically active ingredient may comprise an immune checkpoint inhibitor.

In another aspect, the present application also provides a method of preparing the fusion protein, comprising culturing a host cell described herein under conditions such that the fusion protein is expressed.

Method and use

In another aspect, the present application provides a method of preventing and/or treating a disease and/or disorder, the method comprising administering to a subject in need thereof the fusion protein, the nucleic acid molecule, the vector, the host cell, the immunoconjugate, the multispecific molecule, the chimeric antigen receptor, the immune effector cell, and/or the pharmaceutical composition described herein.

In another aspect, the present application also provides said fusion protein, said nucleic acid molecule, said vector, said host cell, said immunoconjugate, said multispecific molecule, said chimeric antigen receptor, said immune effector cell and/or said pharmaceutical composition for preventing and/or treating a disease and/or disorder.

In another aspect, the present application also provides the use of the fusion protein, the nucleic acid molecule, the vector, the host cell, the immunoconjugate, the multispecific molecule, the chimeric antigen receptor, the immune effector cell and/or the pharmaceutical composition in the preparation of a medicament for preventing and/or treating a disease and/or disorder.

In certain embodiments, the disease and/or condition comprises a tumor.

In certain embodiments, the tumor expresses CD73. In certain embodiments, the CD73 may be membrane bound human CD73 and/or soluble human CD73.

In certain embodiments, the tumor involves tumor cells that express CD73. In certain embodiments, the CD73 is expressed on the surface of the tumor cells.

In certain embodiments, the tumor is a CD73 positive tumor.

In this application, the fusion proteins may also be administered in combination with other therapeutic agents. For example, the therapeutic agent may include an immune checkpoint inhibitor. In this application, the fusion protein may also be administered with other therapies, for example, the therapies may be any therapies known for tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy or palliative therapy.

In another aspect, the present application provides a method for stimulating an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a fusion protein described herein, the nucleic acid molecule, the vector, the host cell, the immunoconjugate, the multispecific molecule, the chimeric antigen receptor, the immune effector cell, and/or the pharmaceutical composition. In another aspect, there is provided the use of said fusion protein, said nucleic acid molecule, said vector, said host cell, said immunoconjugate, said multispecific molecule, said chimeric antigen receptor, said immune effector cell and/or said pharmaceutical composition for stimulating an immune response in a subject, or for the manufacture of a medicament for stimulating an immune response in a subject.

In certain embodiments, the immune response is a T cell mediated immune response.

In certain embodiments, the immune response is an immune response against a tumor (e.g., a tumor that expresses CD 73). In certain embodiments, the subject has a tumor (e.g., a tumor that expresses CD 73).

Detection application and kit

The fusion proteins of the present application are capable of specifically binding CD73 and TGF- β and thus can be used to detect the presence or level of CD73 and/or TGF- β in a sample.

Thus, in another aspect, the present application provides a kit comprising a fusion protein as described herein. In some embodiments, the fusion proteins of the present application carry a detectable label.

In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

In another aspect, the invention provides a method of detecting the presence or amount of CD73 in a sample comprising the steps of:

(1) Contacting the sample with a fusion protein of the present application;

(2) Detecting the formation of a complex between the fusion protein and CD73 or detecting the amount of the complex.

The formation of the complex indicates the presence of CD73 or cells expressing CD73.

In certain embodiments, the sample is a cell sample, i.e., a sample comprising cells (e.g., tumor cells). In such embodiments, the complex is formed between the fusion protein and CD73 expressed by cells in the sample.

In some embodiments, the antibodies or antigen binding fragments thereof of the invention also bear a detectable label. In other embodiments, in step (2), a reagent with a detectable label is used to detect the fusion protein of the application.

The method may be used for diagnostic purposes, or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient). In certain embodiments, the CD73 is human CD73, such as membrane bound and/or soluble human CD73.

In another aspect, there is provided the use of a fusion protein of the present application for determining the presence or amount of TGF- β in a sample, or for the preparation of a detection reagent for determining the presence or amount of TGF- β in a sample. In certain embodiments, the TGF- β is human TGF- β.

In another aspect, the invention provides a method of detecting the presence or amount of TGF- β in a sample comprising the steps of:

(1) Contacting the sample with a fusion protein of the present application;

(2) Detecting the formation of a complex between the fusion protein and TGF-beta or detecting the amount of the complex.

The formation of the complex indicates the presence of TGF-beta or cells expressing TGF-beta.

In certain embodiments, the sample is a cell sample, i.e., a sample comprising cells (e.g., tumor cells). In such embodiments, the complex is formed between the fusion protein and TGF- β expressed by cells in the sample.

In some embodiments, the antibodies or antigen binding fragments thereof of the invention also bear a detectable label. In other embodiments, in step (2), a reagent bearing a detectable label is used to detect the fusion protein of the present application.

The method may be used for diagnostic purposes, or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient). In certain embodiments, the TGF- β is human TGF- β.

The present application also provides the following embodiments:

1. a fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to CD73 protein, said antibody or antigen-binding fragment thereof comprising at least one CDR in the heavy chain variable region amino acid sequence as set forth in SEQ ID No. 41.

2. The fusion protein of embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises HCDR3, and the HCDR3 comprises the amino acid sequence of SEQ ID No. 3.

3. The fusion protein according to any one of embodiments 1-2, wherein the antibody or antigen-binding fragment thereof comprises HCDR2, and the HCDR2 comprises the amino acid sequence of SEQ ID No. 2.

4. The fusion protein according to any one of embodiments 1-3, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, and the HCDR1 comprises the amino acid sequence of SEQ ID No. 1.

5. The fusion protein according to any one of embodiments 1-4, wherein the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises an amino acid sequence of SEQ ID NO:1, the HCDR2 comprises an amino acid sequence of SEQ ID NO:2, and the HCDR3 comprises an amino acid sequence of SEQ ID NO: 3.

6. The fusion protein according to any one of embodiments 4-5, wherein the antibody or antigen-binding fragment thereof comprises H-FR1, the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:12 or SEQ ID NO: 27.

7. The fusion protein according to any one of embodiments 4-6, wherein the antibody or antigen-binding fragment thereof comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H-FR2 comprises the amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 13.

8. The fusion protein according to any one of embodiments 3-7, wherein the antibody or antigen-binding fragment thereof comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence of SEQ ID NO 9 or SEQ ID NO 14.

9. The fusion protein according to any one of embodiments 2-8, wherein the antibody or antigen-binding fragment thereof comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO: 15.

10. The fusion protein according to any one of embodiments 1-9, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID No. 41.

11. The fusion protein according to embodiment 10, wherein the VH comprises the amino acid sequence shown in SEQ ID NO. 11, SEQ ID NO. 16 or SEQ ID NO. 28.

12. The fusion protein according to any one of embodiments 1-11, wherein the antibody or antigen-binding fragment thereof comprises at least one CDR of the light chain variable region amino acid sequence as depicted in SEQ ID No. 42.

13. The fusion protein according to any one of embodiments 1-12, wherein the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID No. 6.

14. The fusion protein according to any one of embodiments 1-13, wherein the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID No. 5.

15. The fusion protein according to any one of embodiments 1-14, wherein the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID No. 4.

16. The fusion protein according to any one of embodiments 1-15, wherein the antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2, and LCDR3, the LCDR1 comprises the amino acid sequence of SEQ ID No. 4, the LCDR2 comprises the amino acid sequence of SEQ ID No. 5, and the LCDR3 comprises the amino acid sequence of SEQ ID No. 6.

17. The fusion protein according to any one of embodiments 15-16, wherein the antibody or antigen-binding fragment thereof comprises L-FR1, the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO: 29.

18. The fusion protein according to any one of embodiments 15-17, wherein the antibody or antigen-binding fragment thereof comprises L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO: 23.

19. The fusion protein according to any one of embodiments 14-18, wherein the antibody or antigen-binding fragment thereof comprises L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence of SEQ ID No. 19 or SEQ ID No. 24.

20. The fusion protein according to any one of embodiments 13-19, wherein the antibody or antigen-binding fragment thereof comprises L-FR4, the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence of SEQ ID NO:20 or SEQ ID NO: 25.

21. The fusion protein according to any one of embodiments 1-20, wherein the antibody or antigen-binding fragment thereof comprises an antibody light chain variable region, VL, comprising the amino acid sequence set forth in SEQ ID NO. 42.

22. The fusion protein according to embodiment 21, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO. 21, SEQ ID NO. 26 or SEQ ID NO. 30.

23. The fusion protein according to any one of embodiments 1-22, wherein the antibody or antigen-binding fragment thereof comprises a VH and a VL comprising any one of the group of amino acid sequences selected from:

24. The fusion protein according to any one of embodiments 1-23, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region.

25. The fusion protein of embodiment 24, wherein the heavy chain constant region is derived from an IgG constant region.

26. The fusion protein according to any one of embodiments 24-25, wherein the heavy chain constant region is derived from a human IgG constant region.

27. The fusion protein according to any one of embodiments 24-26, wherein the heavy chain constant region is derived from a heavy chain constant region selected from the group consisting of: igG1, igG2, igG3 and IgG4.

28. The fusion protein according to any one of embodiments 24-27, wherein the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 33 or SEQ ID NO. 34.

29. The fusion protein according to any one of embodiments 1-28, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID No. 31.

30. The fusion protein according to any one of embodiments 1-29, wherein the antibody or antigen-binding fragment thereof comprises an antibody light chain constant region.

31. The fusion protein of embodiment 30, wherein the light chain constant region is derived from igκ.

32. The fusion protein according to any one of embodiments 30-31, wherein the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 35.

33. The fusion protein according to any one of embodiments 1-32, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO. 32.

34. The fusion protein according to any one of embodiments 1-33, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 31 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 32.

35. The fusion protein according to any one of embodiments 1-34, further comprising a TGF-beta binding moiety.

36. The fusion protein according to embodiment 35, wherein the TGF-beta binding-moiety specifically binds to human TGF-beta.

37. The fusion protein according to any one of embodiments 35-36, wherein the TGF-beta binding moiety comprises an extracellular domain of a human TGF-beta receptor.

38. The fusion protein of embodiment 37, wherein the human TGF- β receptor comprises human TGF- βRII.

39. The fusion protein according to any one of embodiments 35-38, wherein the TGF-beta binding-portion comprises the amino acid sequence shown in SEQ ID NO. 37.

40. The fusion protein according to any one of embodiments 35-39, wherein the TGF-beta binding-moiety is linked to the heavy chain variable region of the antibody or antigen binding fragment thereof that specifically binds CD73 protein.

41. The fusion protein according to any one of embodiments 35-40, wherein the TGF-beta binding-moiety is directly or indirectly linked to the C-terminus of the heavy chain constant region of the antibody or antigen binding fragment thereof that specifically binds CD73 protein.

42. The fusion protein according to any one of embodiments 35-41, wherein the N-terminus of the TGF-beta binding-moiety is directly or indirectly linked to the C-terminus of the heavy chain constant region of the antibody or antigen binding fragment thereof that specifically binds CD73 protein.

43. The fusion protein according to any one of embodiments 35-42, wherein the TGF-beta binding-moiety is linked to the heavy chain variable region of the antibody or antigen binding fragment thereof that specifically binds CD73 protein through a peptide linker.

44. The fusion protein according to embodiment 43, wherein the peptide linker comprises the amino acid sequence shown in SEQ ID NO. 36.

45. The fusion protein according to any one of embodiments 1-44, comprising a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 39 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 32.

46. The fusion protein according to any one of embodiments 1-45, comprising two polypeptides each comprising the amino acid sequence set forth in SEQ ID NO. 39 and two polypeptides each comprising the amino acid sequence set forth in SEQ ID NO. 32.

47. The fusion protein according to any one of embodiments 1-46, having one or more of the following properties:

1) Capable of specifically binding to human and/or monkey CD73 protein;

2) Has inhibiting effect on CD73 enzyme activity on tumor cell surface;

3) Has inhibiting effect on soluble CD73 enzyme activity;

4) Capable of blocking the TGF- β/SMAD signaling pathway; and

5) Can relieve the inhibition of adenosine to T cells.

48. An isolated nucleic acid molecule encoding the fusion protein of any one of embodiments 1-47.

49. A vector comprising the nucleic acid molecule of embodiment 48.

50. The vector of embodiment 49, comprising a plasmid vector or a viral vector.

51. A host cell comprising the nucleic acid molecule of embodiment 48 or the vector of any one of embodiments 49-50.

52. An immunoconjugate comprising the fusion protein of any one of embodiments 1-47.

53. A multispecific molecule comprising the fusion protein of any one of embodiments 1-47.

54. The multispecific molecule of embodiment 53 that additionally specifically binds one or more other targets.

55. A chimeric antigen receptor comprising the fusion protein of any one of embodiments 1-47.

56. An immune effector cell comprising the chimeric antigen receptor of embodiment 55.

57. A method of making the fusion protein of any one of embodiments 1-47, comprising culturing the host cell of embodiment 51 under conditions such that the fusion protein is expressed.

58. A pharmaceutical composition comprising the fusion protein of any one of embodiments 1-47, the nucleic acid molecule of embodiment 48, the vector of any one of embodiments 49-50, the host cell of embodiment 51, the immunoconjugate of embodiment 52, the multispecific molecule of any one of embodiments 53-54, the chimeric antigen receptor of embodiment 55, and/or the immune effector cell of embodiment 56, and optionally a pharmaceutically acceptable carrier.

59. The pharmaceutical composition of embodiment 58, further comprising an additional pharmaceutically active ingredient.

60. A kit comprising the fusion protein of any one of embodiments 1-47, the nucleic acid molecule of embodiment 48, the vector of any one of embodiments 49-50, the host cell of embodiment 51, the immunoconjugate of embodiment 52, the multispecific molecule of any one of embodiments 53-54, the chimeric antigen receptor of embodiment 55, the immune effector cell of embodiment 56, or the pharmaceutical composition of any one of embodiments 58-59.

61. Use of the fusion protein of any one of embodiments 1-47, the nucleic acid molecule of embodiment 48, the vector of any one of embodiments 49-50, the host cell of embodiment 51, the immunoconjugate of embodiment 52, the multispecific molecule of any one of embodiments 53-54, the chimeric antigen receptor of embodiment 55, the immune effector cell of embodiment 56, the pharmaceutical composition of any one of embodiments 58-59, or the kit of embodiment 60 in the manufacture of a medicament for the prevention and/or treatment of a disease and/or disorder.

62. The use according to embodiment 61, wherein the disease and/or disorder comprises a tumor.

63. The use according to any one of embodiments 61-62, wherein the disease and/or disorder comprises a solid tumor and/or a hematological tumor.

64. The use of any one of embodiments 62-63, wherein the tumor comprises a CD 73-positive tumor.

65. The use according to any one of embodiments 62-64, wherein the tumor is selected from one or more of the following groups: colorectal cancer, renal cell carcinoma, non-small cell carcinoma, and pancreatic cancer.

66. A method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of the fusion protein of any one of embodiments 1-47 or the pharmaceutical composition of any one of embodiments 58-59.

67. A method of detecting the presence or amount of CD73 and/or TGF- β in a sample, the method comprising administering the fusion protein of any one of embodiments 1-47.

68. Use of the fusion protein of any of embodiments 1-47 in the preparation of a detection reagent for determining the presence or amount of CD73 and/or TGF- β in a sample.

Without intending to be limited by any theory, the following examples are meant to illustrate the various aspects of the present invention and are not intended to limit the scope of the present invention.

Examples

Example 1 production of murine anti-human CD73 antibody

To obtain murine anti-human CD73 antibodies, mice were immunized using different immunization strategies (table 1) (Balb/c, shanghai, ling-organism). Antigens used include: CD73 protein (i.e., recombinant expressed human CD73, its sequence is shown as SEQ ID NO: 40) and CHOS-human CD73 (i.e., CHOS cell line over-expressing CD73, expressed CD73 sequence is shown as SEQ ID NO: 40); adjuvants include: complete Freund's adjuvant CFA (InvivoGen, cat# vac-CFA-60), IFA (InvivoGen, cat# vac-IFA-60), quickAntibody (Beijing Boolon immunotechnologies Co., ltd., cat# KX 0210041); the application route comprises: intraperitoneal (ip) and subcutaneous (sc). Spleen cells of immunized mice were fused with mouse myeloma cells SP2/0 3 days after boost using polyethylene glycol to obtain B cell fusion that both expressed antibodies and were immortalized in vitro, and cultured in HAT selection medium. The fused hybridoma cells were plated in 96-well cell culture plates, and positive clones were selected for 2-3 rounds of subcloning by primary screening.

TABLE 1 immunization strategy

Primary screening: supernatants of growing clones were tested for their ability to bind to cell surface CD73 in primary screening by using tumor cells or overexpressing cell lines expressing human CD 73. Assessment of binding capacity was performed on a full field cell scan analyzer by revealing the presence of reactive antibodies in the supernatant with DyLight488 goat anti-mouse IgG (Abcam catalog No. ab 97015) (see example 2 for detailed experimental steps). Secondary screening: screening for CD73 enzyme activity blocking ability on CD73 expressing cells to assess the cell membrane surface CD73 enzyme activity blocking properties of the antibodies; screening for the ability to block the enzymatic activity of CD73 was performed using human serum soluble CD73 to evaluate the soluble CD73 enzymatic activity blocking properties of antibodies (see example 6 for detailed experimental procedures).

And separating and purifying the culture supernatant of the finally obtained positive hybridoma monoclonal cell strain to obtain the murine monoclonal antibody 13D12.

EXAMPLE 2 evaluation of antigen binding Activity of murine anti-CD 73 antibody

2.1 detection of the binding of murine antibodies to CD 73-positive cells by means of a cell scanning Analyzer

The cells used: MDA-MB-231 (endogenously expressed human CD73; a human breast cancer cell line), SK-ME-S (endogenously expressed human CD73; a human lung squamous carcinoma cell line), H2030 (endogenously expressed human CD73; a human non-small cell lung carcinoma cell line), SKLU1 (endogenously expressed human CD73; a human lung adenocarcinoma cell line), BT549 (endogenously expressed human CD73; a human breast ductal carcinoma cell line), A375 (endogenously expressed human CD73; a human melanoma cell line), calu6 (endogenously expressed human CD73; a human degenerative cancer cell line), 4T1 (endogenously expressed murine CD73; a murine breast cancer cell line), CHOS-human CD73 (transfected with human CD 73) and CHOS (CD 73 negative) cells.

Construction of CD73 expressing CHOS cells: human CD73 (SEQ ID NO: 40) was overexpressed on CHOS cells (Invitrogen) by lentiviral infection and resistance screening methods (MOI=3-10, 5. Mu.g/ml polybrene). Lentiviruses were supplied by Shanghai Ji Kai Gene chemical technology Co., ltd, and the cells were further cultured for 2-4 weeks after 72 hours of infection with the corresponding resistance, amplified and frozen for subsequent experiments.

The experimental method comprises the following steps: 10000 cells were plated in 100. Mu.L DMEM+10% FBS/well, and the supernatant was removed the next day using a flat bottom 96-well plate overnight to allow the cells to adhere to or settle to the bottom of the well. 8-point serial 3-fold dilutions were performed by diluting 1/3 of the volume (100. Mu.L) in 200. Mu.L DMEM. In each well of the cell plate100. Mu.L of diluted antibody (fusion clone supernatant or subclone supernatant was used in screening) was added, and the corresponding negative control wells were incubated with 100. Mu.L of LDMEM for 1 hour at room temperature. After removal of the supernatant, 100. Mu.L of secondary antibody (Dylight 488 goat anti-mouse IgG (Abcam catalog No. ab 97015) was added per well at a concentration of 5. Mu.g/mL (diluted in DMEM), incubated at room temperature for 0.5 hours, after completion of staining the supernatant was removed, after washing once with PBS+2% FBS, 100100. Mu.LFBS+2% FBS was added per well, and then read on the machine using a full field cell scanning analyzer (Nexcelom Co., model

Image Cytometer) for the assay reading of the experimental plate. And (3) selecting a second antibody to correspond to the fluorescent channel and the bright field channel during measurement, and simultaneously carrying out high-speed scanning imaging on cells in the hole. The imaging obtained by the fluorescent channel counts the cells combined with the antibody according to the cell morphology and the fluorescent intensity setting parameter of the fluorescent label, the imaging obtained by the bright field channel counts the adherent cells according to the cell morphology setting parameter, and then the two groups of data are divided to obtain the percentage of the cells combined with the antibody and showing fluorescence to the total number of the cells. The binding effect of the anti-CD 73 antibody to the cell line expressing CD73 was determined based on this ratio. Data analysis uses GraphPad, abscissa uses logarithm of antibody concentration, ordinate uses percentage of total number of cells and living cells that bind to CD73 antibody showing green fluorescence, and EC50 values for anti-CD 73 antibody binding on each cell are fitted according to a curve.

The EC50 values of 13D12 binding to each tumor cell are shown in tables 2-1 and 2-2, n.b. indicates no detection within the range of measured concentrations; the binding curves for a portion of the cells are shown in FIG. 1. The results show that 13D12 can bind both cells naturally expressing CD73 and CHOS cells recombinantly expressing human CD73, but these antibodies do not bind to cells that do not express CD73 (CHOS), nor to cells that express murine CD73 (4T 1).

Table 2-1 antibodies bind EC50 to tumor cells endogenously expressing human CD73

Table 2-2 antibodies bind EC50 for recombinant human CD73 expressing CHOS cells and other non-human CD73 expressing cells

2.2 binding of murine antibodies to cynomolgus T cells

Monkey blood from two donors (1132F and 1300M) was obtained from midecai. Peripheral Blood Mononuclear Cells (PBMCs) were isolated using a Ficoll density gradient centrifugation system. PBMCs were incubated with the antibodies to be tested, and the antibodies bound to the cells were stained with a fluorescent dye-labeled secondary antibody (DyLight 488 goat anti-mouse IgG, abcam catalog No. ab97015; dyLight488 goat anti-human IgG, abcam catalog No. ab 97003), and T cells were identified using fluorescent dye-labeled antibodies directed against cd3+ and cd8+. The undyed control sample and the fluorescence-compensated control sample were pooled, the samples were run on an upflow cytometer, and binding of antibodies to cynomolgus T cells was detected.

FIG. 2 shows a flow scatter plot of the binding of 13D12 to monkey 1132FCD3+CD8+T cells. The fold change in mean fluorescence intensity of murine antibody 13D12 binding is shown in the table below. The results show that 13D12 is able to bind cynomolgus monkey cd8+ T cells.

Table 3 binding of antibodies to monkey PBMCs

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EXAMPLE 3 determination of variable region sequence of murine anti-CD 73 antibody and chimeric antibody preparation

Collecting hybridoma cells by centrifugation, adding 1ml TRIzol and 0.2ml chloroform to each 5-10×106 cells, shaking vigorously for 15 seconds, standing at room temperature for 3 min, adding 0.5ml isopropanol into the water phase by centrifugation, standing at room temperature for 10 min, collecting precipitate, washing with ethanol, and drying to obtain RNA. Template RNA and a primer are added into an ice bath centrifuge tube, and the reverse transcription process is carried out after the primer and the template are correctly matched, so that PCR amplification is carried out. 2.5. Mu.l of dNTP/ddNTP mixture was added to each of the 4 microcentrifuge tubes, and the mixture was incubated at 37℃for 5min for further use. A hollow microcentrifuge tube was filled with 1pmol of PCR amplified double-stranded DNA,10pmol of sequencing primer, 2. Mu.l of 5 Xsequencing buffer, double distilled water to a total volume of 10. Mu.l, heated at 96℃for 8min, ice cooled in 1min, and centrifuged at 10000g at 4℃for 10s. Mu.l of a pre-chilled labeling mixture (dCTP, dGTP, dTTP. Mu. Mol/L each),. Alpha. -32P-dATP 5. Mu. Ci, 1. Mu.l of 0.1mol/L DDT, sequencing enzyme 2U, adding water to 15. Mu.l, mixing well and then placing on ice for 2min, labeling the newly synthesized DNA strand. 3.5 μl of the labeled reaction mixture was added to the prepared 4 microcentrifuge tubes and incubated at 37deg.C for 5min. Mu.l of stop solution was added to each tube. The sample was thermally denatured in a water bath at 80℃for 5min, 2. Mu.l of each lane was applied to a sequencing gel, and the fragments were separated by electrophoresis, and sequence information was collected.

The VH and VL sequences of murine antibody 13D12 are shown in table 4. Further, the CDR sequences of murine mab 13D12 were also determined using the method described by Kabat et al (Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, public Health Service, national institutes of health, beziida, maryland (1991), pages 647-669).

Table 4: sequence information of murine antibody

The sequences encoding the heavy chain variable region (SEQ ID NO: 11) and the light chain variable region (SEQ ID NO: 21) of the above murine antibody were ligated with the sequences encoding the heavy chain constant region (SEQ ID NO: 33) and the light chain constant region (SEQ ID NO: 35), respectively, of the human antibody, and recombinant expression was performed in HEK293 cells (ATCC), the cell supernatants grown in culture flasks containing the antibody clones were harvested, purified using protein A column, and antibody proteins were eluted using 100mM acetic acid pH 3.0. The purified antibody protein is then applied to a size exclusion chromatography column for further separation and purification. Antibody proteins corresponding to the monomers were formulated in PBS buffer, which was supplemented with 20% glycerol. Thereby obtaining the corresponding chimeric antibody ch13D12.

EXAMPLE 4 humanization of murine anti-CD 73 antibodies

To increase the sequence homology of candidate antibodies to human antibodies, to reduce the immunogenicity of antibodies to humans, murine antibodies provided in the above examples can be humanized designed and prepared using methods known in the art to insert murine CDR regions into human framework sequences (see U.S. Pat. No.5,225,539 to Winter; U.S. Pat. Nos.5,530,101 to Queen et al; 5,585,089;5,693,762 and 6,180,370; and Lo, benny, K.C., editor, in Antibody Engineering: methods and Protocols, volume 248,Humana Press,New Jersey,2004).

Specifically, the heavy and light chain CDRs of the murine antibody 13D12 were grafted onto the FR framework of the corresponding humanized template, respectively, and a series of back mutations were made to the FR region amino acid residues of the humanized template to make the humanized antibody retain as much as possible the antigen binding ability of the murine antibody. According to the above method, the inventors produced a humanized antibody of murine antibody 13D12, designated 7002-01 (the heavy chain variable region and the light chain variable region of which are shown in SEQ ID NO:16 and SEQ ID NO:26, respectively). The amino acid sequence of the heavy chain constant region of the antibody is shown as SEQ ID NO. 33, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 35.

EXAMPLE 5 evaluation of antigen binding Activity of humanized anti-CD 73 antibody

5.1 determination of antibody binding to CD73 expressing cells by flow cytometry.

500,000 CD73 expressing cells (see example 2) were placed in 100. Mu.L FACS buffer (PBS+2% FBS)/well for use, using round bottom low adsorption 96 well plates. Antibody samples were serially 3-fold diluted at 12-point at 3-fold concentration by diluting 1/2 volume (100 μl) in 200 μl FACS buffer. mu.L of diluted antibody was added to each well of the cell plate, and 100. Mu.L of FACS buffer was added to the corresponding negative control well, and incubated at 4℃for 1 hour. After centrifugation to remove the supernatant, the wells were washed twice with FACS buffer, 100. Mu.L of secondary antibody (Dylight 488 goat anti-mouse IgG, abcam catalog No. ab97015; dylight488 goat anti-human IgG, abcam catalog No. ab 97003) was added to each well, diluted in FACS buffer) and incubated at 4℃for an additional 0.5 hours. After staining was completed, the supernatant was removed by centrifugation, and after washing twice with FACS buffer, 100 μl of FACS buffer was added to each well to resuspend the cells, followed by reading on the machine. The cells in the experimental plate were read using a flow cytometer (BD company, model ACCURI C6 PLUS). In the measurement, the cell position is defined according to FCS and SSC, then the secondary antibody is selected to correspond to green fluorescence channel (FITC) and SSC for analysis, the data analysis uses GraphPad, the abscissa uses logarithm of antibody concentration, the ordinate uses average fluorescence intensity value, and the EC50 of the anti-CD 73 antibody is fitted according to the curve. Table 5 shows the binding of humanized antibody 7002-01 to both cells naturally expressing CD73 and cells recombinantly expressing human CD73, N.D. indicating no detection. The results show that humanized antibody 7002-01 has good binding activity to membrane-bound CD 73.

TABLE 5 antibody binding EC50 for human CD73 expressing tumor cells

5.2 determination of antibody binding to soluble human CD73 protein by ELISA

1. Mu.g/ml recombinant human CD73 protein (Baiying organism, recombinant human CD73 protein) was coated on ELISA plates in PBS overnight at 4 ℃. Plates were washed 3 times in wash buffer (PBS, 0.05% Tween 20) and nonspecific sites were saturated by adding 200 μl/w PBS+2% BSA block. Dose-range anti-CD 73 antibody was serially diluted 100 μl and incubated for 1h at 37 ℃ in antigen-coated ELISA plates. Plates were washed 3 times in wash buffer and HRP conjugated goat anti-human or goat anti-mouse IgG Fc fragment secondary antibody was added at room temperature for 1hr to detect bound anti-CD 73 antibody. Plates were washed 3 times in wash buffer, and bound secondary antibodies were revealed by adding TMB (HRP substrate) and incubating the plates in the dark for 5 to 10 minutes at room temperature. The enzyme reaction was terminated by adding sulfuric acid solution 1M, and the light absorption was measured at 450 nm. The graph is plotted with light absorption values on the ordinate and antibody concentration log values on the abscissa, and EC50 is calculated using graphic pad Prism (GraphPad Prism) software. As a result, as shown in FIG. 3, humanized antibody 7002-01 has a good binding activity to soluble recombinant CD73, and its EC50 is 0.0081. Mu.g/ml.

5.3 determination of affinity of humanized antibodies to recombinant human CD73 protein by Biacore

SPR measurements of antibody affinity were performed on Biacore T200 (GE) at 25 ℃. Antibodies were diluted to 1ug/ml with running buffer 1 x HBS-EP+ and captured onto the chip surface (Protein A chip, GE, cat# 29127556) at a flow rate of 10ul/min for 30s, respectively. Subsequently, a series of concentrations of CD73 protein (a hundred english organism, recombinant human CD73 protein) were injected into the corresponding antibody channels at a flow rate of 30ul/min, an association phase of 180s, followed by dissociation of 900s. 10mM pH1.5 Gly-HCl was used for regeneration. The entire sensor map set was fitted using a 1:1 dynamic binding model. Divalent affinities and dynamic binding and dissociation rate constants are shown in the table below.

Table 6 affinity constants of antibodies to recombinant CD73 binding

Antibodies to	ka(1/Ms)	kd(1/s)	KD(M)
				7002-01	2.97E+05	6.24E-05	2.102E-10

EXAMPLE 6 evaluation of inhibitory Activity of anti-CD 73 antibodies against CD73 enzymatic Activity

6.1 inhibition assay of CD73 enzymatic Activity in tumor cells

Excess AMP is known to block ATP-dependent luciferase activity. CD73, which cleaves AMP to adenosine + inorganic phosphate, restores luciferase activity and light emission by reducing AMP. Thus, antibodies that block the enzymatic activity of CD73 will reduce light emission.

Human CD73 positive cells were harvested and counted. Flat bottom 96 plates were seeded with 20,000 cells per well in 100 μl of complete medium. Antibody samples serial 3-fold dilutions were performed at 8 spots by diluting 1/3 volume (100 μl) in 200 μl DMEM, 100 μl of diluted samples were added to the corresponding wells of the plate, and the negative control was isotype control antibody (ISO). Incubate for 1 hour at 37℃and after removal of the supernatant, the cells were washed twice with PBS. A125. Mu.M AMP solution was prepared in incomplete medium, 100. Mu.L AMP was added to each well, and the plates were incubated at 37℃for two additional hours. The reaction plate was centrifuged, 50. Mu.L was added to another 96 fluorescent plate (OptiPlate-96, perkin Elmer, # 6005290) and the same volume of 50. Mu.M ATP solution was added and 50. Mu.L of CTG reagent (Promega, G7572) was added to each well, the plate was incubated at room temperature in the dark for 15 minutes, and fluorescence (Lum) was measured using a microplate reader. The data analysis uses GraphPad, the abscissa uses the logarithm of the antibody concentration, the ordinate is the inhibition rate, the inhibition rate of enzyme activity is plotted and the IC50 is calculated, the inhibition rate is calculated as follows:

Inhibition ratio = (Lum) _{Positive control} -Lum _{Antibodies to} )/(Lum _{Positive control} -Lum _{Negative control} )*100

The IC50 of antibodies in different human tumor cell lines to block endogenous cell CD73 is shown in the table below. The results show that the humanized antibody 7002-01 can significantly inhibit the enzymatic activity of CD73 on the surface of tumor cells.

Table 7 IC50 for inhibiting CD73 enzyme activity on different humanized tumor cell surfaces by antibody

6.2 test for inhibiting the Activity of serum CD73 enzyme from tumor patients

Tumor patient serum was diluted in phosphate buffer (Tris 125mM,MgCl2 25mM,NaCl125mM), and 12.5. Mu.L of each well was added for use, using a white flat bottom 96-well plate. Antibody samples were subjected to 10-point serial 2-10-fold dilutions by diluting 1/1.5 volumes (100. Mu.L) in 50. Mu.L phosphate buffer and 1/10 volumes (10. Mu.L) in 90. Mu.L phosphate buffer, 12.5. Mu.L diluted antibody was added to each well of the cell plate, 12.5. Mu.L phosphate buffer was added to the negative control, and incubated for 1.5 hours at 37℃after centrifugation. AMP was diluted to 20 μm solution using phosphate buffer, 25 μl AMP (except positive control) was added to each well, and incubated at 37 ℃ for another 1 hour after centrifugation. After completion of the reaction, 25. Mu.L of AMP was added to the positive control. Immediately add 25 μl AMP-Glo to each well ^TM Reagent I (Promega, cat# V5012), the reaction plates were centrifuged and the plates incubated for 1 hour at room temperature. To each well was added 50 μ L AMP Detection Solution (Promega Corp., cat No. V5012) and incubated for 1 hour at room temperature after centrifugation. Fluorescence (Lum) was measured using a microplate reader. The data analysis uses GraphPad, the abscissa uses the logarithm of the antibody concentration, the ordinate is the inhibition rate, the inhibition rate of enzyme activity is plotted and the IC50 is calculated, the inhibition rate is calculated as follows:

Inhibition ratio = 100- (Lum) _{Positive control} -Lum _{Antibodies to} )/(Lum _{Positive control} -Lum _{Negative control} )*100

As shown in FIGS. 4A-4B, the anti-CD 73 antibody can effectively inhibit the dephosphorylation of CD73 to AMP in serum of liver cancer patients (A) and melanoma patients (B), and inhibit the activity of CD73 enzyme.

Example 7 anti-CD 73 antibody mediated internalization of CD73

anti-CD 73 antibody mediated CD73 internalization was tested by flow cytometry. When determining the relationship between internalization by the antibody and time, the indicated cells were incubated with 10 μg/mL antibody for different times at 37 ℃. After washing several times with PBS containing 2% FBS, the cells were stained with 10. Mu.g/mL of the secondary antibody at 4℃for 30 minutes and then analyzed for CD73 expression by flow cytometry. When the comparative antibodies caused different degrees of internalization, the indicated cells were incubated with 10 μg/mL antibody for 20 hours at both temperatures, 4 ℃ and 37 ℃, in parallel. After washing several times with PBS containing 2% FBS, the cells were stained with 10. Mu.g/mL of the secondary antibody at 4℃for 30 minutes and then analyzed for CD73 expression by flow cytometry.

MFI ₃₇ MFI for samples incubated at 37 ℃; MFI (MFI) ₄ MFI for samples incubated at 4 ℃ under conditions where only binding occurs without endocytosis, MFI _Background For MFI of the secondary antibody alone, the percentage of antibody-mediated cell surface CD73 endocytosis was calculated from the following formula:

percentage of internalized CD73 = 100-100× (MFI ₄ -MFI ₃₇ )/MFI ₄

The results are shown in table 8, where these antibodies mediate varying degrees of internalization of CD73 on the surface of tumor cells.

Table 8: antibody-mediated internalization ratio of CD73 on tumor cell surface

Cells

Antibodies to

For 6 hours

4 hours

For 2 hours

1 hour

0.5 hour

0 hours

A375

7002-01

11％

3％

4％

5％

1％

0％

MDA-MB-231

7002-01

8％

4％

3％

5％

0％

H2030

7002-01

-1％

-2％

4％

1％

6％

0％

HCC44

7002-01

11％

4％

7％

0％

3％

0％

Calu6

7002-01

13％

7％

6％

3％

7％

0％

Example 8 anti-CD 73 antibody alleviating AMP-mediated inhibition of CD4+ T cells

PBMC cells were stimulated with anti-CD 3/anti-CD 28 24 hours the day before the experiment, PBMC cells were collected (fresh blood was isolated by Ficoll) and cd4+ T cells were sorted using cd4+ T Cell Isolation Kit human (meyene, cat. No. 130-096-533), the supernatant was removed by centrifugation, cd4+ T cells were resuspended in AIMV medium containing 40 μm EHNA and 120IU/ml IL2 (EHNA final concentration 20 μm, IL2 final concentration 60 IU/ml), 200,000 CD4+ T cells were placed in 100 μl/well, and 96 well low adsorption round bottom plates were used. Serial 2-10 fold dilutions at 10 spots were performed by diluting 1/3 volume (100 μl) in 200 μl of AIMV medium, adding 50 μl of diluted antibody per well, adding 50 μl of AIMV medium to the corresponding negative control well, and incubating at 37 degrees celsius for 0.5 hours. 400. Mu.M AMP (final concentration 100. Mu.M) was prepared with AIMV, and 50. Mu.L of the prepared AMP solution was added to each well (control wells were added without AMP medium). After centrifugation, the machine is set up to read. The plates were then incubated at 37℃for 72 hours and then read again on the machine. The readings were taken using a full field cell scanning analyzer (Nexcelom, model number)

Image Cytometer) was measured on the cells in the experimental plate. During measurement, a bright field channel is selected to carry out high-speed scanning imaging on cells in the hole. The anti-CD 73 antibody was judged to have an AMP-mediated cd4+ T cell inhibitory effect based on this colony size profile. Wherein, MEDI9447 (MedImmune) and BMS986179 (BMS) were used as reference antibodies, and purified by Ji Kai expression.

The cell growth on day 4 of T cell proliferation is shown in figure 5; #18 used in the figure numbered internal PBMC donor; the concentration of antibody used in the figure starts at 100. Mu.g/mL, and four-fold dilutions total nine spots. Humanized antibody 7002-01 can effectively relieve AMP-mediated CD4+ T cell inhibition, and T cell proliferation is obviously recovered, and the effect is better than that of reference antibodies MEDI9447 and BMS986179.

Example 9 anti-CD 73 anti-mediated tumor cell killing

5000A 375 cells were plated in 100. Mu.L DMEM+10% FBS/well, the cells were attached overnight using a flat bottom 96-well plate, and the supernatant was removed the next day. 10-point serial 2-10-fold dilutions were performed by diluting 1/3 of the volume (100. Mu.L) in 200. Mu.L AIMV. 50. Mu.L of diluted antibody was added to each well of the cell plate, 50. Mu.L of AIMV was added to the corresponding negative control well, and incubation was performed at 37℃for 0.5 hours. PBMC cells (isolated from fresh blood by Ficoll) were collected 24 hours prior to one day of stimulation with CD3/CD28, resuspended in AIMV containing 40. Mu.M EHNA and 120IU/ml IL2 (final EHNA concentration 20. Mu.M, final IL2 concentration 60 IU/ml) and added at 5,000/100. Mu.L per well. A400. Mu.M AMP solution was prepared with AIMV, and 50. Mu.L (100. Mu.M final AMP concentration) was added to each well. Incubation was carried out for 72 hours at 37℃after centrifugation. mu.L of CCK8 kit (Japanese Kogyo Co., ltd., cat. No. CK 04) was added to each well, and OD450 was measured using a microplate reader after incubation at 37℃for 4 hours. The OD values were converted to percent inhibition, respectively, based on control Kong Shuzhi, from which the anti-CD 73 antibody anti-mediated tumor cell killing was determined. A larger percentage represents a better anti-CD 73 anti-mediated tumor cell killing effect, whereas a smaller percentage represents a poorer anti-CD 73 anti-mediated tumor cell killing effect. Data analysis uses GraphPad, abscissa uses logarithm of antibody concentration, ordinate uses percent inhibition, and IC50 values of anti-CD 73 antibodies on a375 cells were fitted according to a curve.

The results are shown in FIG. 6, where #22 used in the figure is the number of internal PBMC donors, and the results show that humanized antibody 7002-01 is effective in restoring PBMC killing of tumor cells.

Example 10 molecular design of fusion proteins and expression plasmid preparation

By immunizing mice and humanizing the GB7002.01 antibody sequence (the amino acid sequence of VH is shown as SEQ ID NO:28, the amino acid sequence of VL is shown as SEQ ID NO: 30), the heavy chain is linked with the TGFbRII sequence (the amino acid sequence after the linkage is shown as SEQ ID NO: 39) at the C-terminal of Fc through (G4A) 4, and the light chain uses the light chain sequence of GB7002.01 antibody sequence (the amino acid sequence is shown as SEQ ID NO: 32) to construct the fusion protein capable of simultaneously targeting human CD73 and TGFb. CD73 mab GB7002.01 and Fc (CH 2-CH 3) -TGFbRII controls were also constructed.

The designed fusion protein sequence is delivered to a gene synthesis company (Tsingke) for gene synthesis, and is constructed on an expression vector of pcDNA3.1 (+) after the synthesis is completed, and a certain amount of plasmid extraction work is completed, and QC (quality control) verification such as endotoxin, sequencing comparison, enzyme digestion verification, DNA quality and the like are carried out for the next experiment.

A schematic of the fusion protein structure is shown in FIG. 7.

EXAMPLE 11 fusion protein expression and purification

Using ExpiCHO ^TM Expression System kit (from Thermo), transferring the prepared fusion Protein expression plasmid into an Expi-CHO cell, culturing the cell for 5 days according to the commodity instruction, collecting the supernatant, and purifying the target Protein by using a Protein A magnetic bead (from gold Style) sorting method. The beads were resuspended (1-4 times the volume of beads) with an appropriate volume of Binding buffer (PBS+0.1% Tween 20, pH 7.4) and added to the sample to be purified, incubated for 1 hour at room temperature, with gentle shaking. The samples were placed on a magnetic rack (purchased from beaver), the supernatant was discarded and the beads were washed 3 times with Binding buffer. Adding an Elution buffer (0.1M sodium citrate,pH 3.2) according to the volume which is 3-5 times of the volume of the magnetic beads, oscillating for 5-10min at room temperature, placing back on a magnetic frame, collecting the Elution buffer, transferring to a collecting pipe added with Neutralization buffer (1M Tris, pH 8.54), and uniformly mixing to complete the preparation.

Example 12 protein level affinity detection of fusion proteins

ForteBio affinity assays were performed according to the prior art (Estep, P et al, high throughput solution Based measurement of antibody-antigen affinity and epitope binding. MAbs,2013.5 (2): p.270-8). Briefly, the sensor was equilibrated for 30min offline in assay buffer, then online detection was performed for 60s to establish a baseline, and purified antibodies obtained as described above were loaded online onto the AHQ sensor. The sensor was then placed in 100nM MSLN antigen for 5min, after which the sensor was transferred to PBS and dissociated for 5min. Kinetic analysis was performed using a 1:1 binding model.

As shown in tables 9-11, the affinity of the fusion protein for human monkey CD73 was substantially equivalent to GB7002.01, and the affinity of the fusion protein for human TGF-beta 1 was substantially equivalent to Fc (CH 2-CH 3) -TGFbRII.

Table 9 affinity of candidate molecules for human CD73 antigen

Numbering device	KD(M)	Kon(1/Ms)	Koff(1/s)
				GB7002.01	4.7E-10	2.74E+05	1.29E-04
GB7002.01-TGFbRII	1.1E-09	2.81E+05	3.20E-04

Table 10 affinity of candidate molecules for monkey CD73 antigen

TABLE 11 affinity of candidate molecules for human TGFb1 antigen

Numbering device	KD(M)	Kon(1/Ms)	Koff(1/s)
				Fc(CH2-CH3)-TGFbRII	1.30E-07	6.27E+03	8.45E-04
GB7002.01-TGFbRII	5.80E-08	1.15E+04	6.66E-04

Example 13 detection of affinity of fusion proteins for human and monkey CD73 overexpressing cell levels

CHO cells (CHO-hMSLN cells and CHO-cynomosln cells) overexpressing human and monkey CD73 were generated by pcho1.0 vector (purchased from Invitrogen) transfected with human and monkey cdnas. Adjusting the cell density of the expanded overexpressing cells to 2X 10 ⁶ cells/ml, 100. Mu.L/well was added to a 96 well flow plate and centrifuged for use. Purified fusion protein antibody was diluted with PBS, 400nM starting 3-fold dilutionReleasing the diluted sample at 12 points, adding 100 mu L/well of the diluted sample into the 96-well flow plate with cells, incubating at 4 ℃ for 30min, and washing twice with PBS. 100. Mu.L/well of Goat F (ab') 2Anti-Human IgG-Fc (PE) (from Abcam, ab 98596) diluted 100-fold with PBS was added, incubated for 30min at 4℃and washed twice with PBS. 100 μl/well of PBS was added to resuspend cells, and the corresponding MFI was measured and calculated on a CytoFlex (Bechman) flow cytometer.

In the assay experiments described above, the results of the experiments are shown in FIG. 8 and Table 12, and all purified samples of the invention and CHO-hMSLN cells have binding activity, and the affinity of the fusion protein to human monkey CD73 over-expressed cells is substantially equivalent to that of GB 7002.01.

In the assay experiments described above, the results of the experiments are shown in FIG. 9 and Table 13, and all purified samples of the present invention and CHO-cynomoMSLN cells have binding activity, and the affinity of the fusion protein to human monkey CD73 over-expressed cells is substantially equivalent to that of GB 7002.01.

Table 12 fusion proteins affinity EC50 for human CD73 over-expressed cell level

Numbering device	EC50(nM)
		GB7002.01	5.476
GB7002.01-TGFbRII	4.764

Table 13 fusion proteins affinity EC50 for monkey CD73 over-expressed cell level

Numbering device	EC50(nM)
		GB7002.01	4.18
GB7002.01-TGFbRII	4.523

Example 14 inhibition assay of CD73 enzymatic Activity of fusion proteins on tumor cell surfaces

Human CD73 positive cells were harvested and counted. Flat bottom 96 plates were seeded with 20,000 cells per well in 100 μl of complete medium. Antibody samples serial 3-fold dilutions were performed at 8 spots by diluting 1/3 volume (100 μl) in 200 μl DMEM, 100 μl of diluted samples were added to the corresponding wells of the plate, and the negative control was isotype control antibody (ISO). Incubate for 1 hour at 37℃and after removal of the supernatant, the cells were washed twice with PBS. A125. Mu.M AMP solution was prepared in incomplete medium, 100. Mu.L AMP was added to each well, and the plates were incubated at 37℃for two additional hours. The reaction plate was centrifuged, 50. Mu.L was removed and added to another 96 fluorescent plate (OptiPlate-96, perkin Elmer, # 6005290) and the same volume of 50. Mu.M ATP solution was added and 50. Mu.L of CTG reagent (Promega, G7572) was added to each well, the plate was incubated at room temperature in the dark for 15 minutes and fluorescence (Lum) was measured using a microplate reader. The data analysis uses GraphPad, the abscissa uses the logarithm of the antibody concentration, the ordinate is the inhibition rate, the inhibition rate of enzyme activity is plotted and the IC50 is calculated, the inhibition rate is calculated as follows:

Inhibition ratio = (Lum positive control-Lum antibody)/(Lum positive control-Lum negative control) ×100

As shown in FIGS. 10 and 11, all the purified samples of the present invention have excellent inhibitory effect on the CD73 enzyme activity on the surfaces of two human tumor cell lines, and Table 14 shows that all the purified samples have substantially equivalent inhibitory IC50 for the CD73 enzyme activity on the surfaces of two human tumor cell lines.

Table 14 fusion protein inhibits the IC50 of different humanized tumor cell surface CD73 enzyme activities

EXAMPLE 15 inhibition assay of soluble CD73 antigenic enzyme Activity by fusion proteins

0.3. Mu.g/mL of soluble CD73 (purchased from Acro) was placed in 96-well plates (Costar, # 3599) at 50. Mu.L/well, and antibody at different doses of antibody concentration (400 nM, 3-fold dilution, 12 spots) was inoculated into the cell wells, 50. Mu.L/well, and incubated at 37℃for 30min. 100. Mu.M AMP (Sigma, #A2252-5G) was then added and incubated at 37℃for 30 minutes, 50. Mu.L/well. mu.L of the supernatant was placed on a white plate (Costar, # 3917) and luciferase signals were read using the AMP-Glo kit AMP Detection Solution (Promega, # V5011).

As shown in FIG. 12 and Table 15, all purified samples of the present invention had good inhibitory effect on soluble CD73 enzyme activity, and the inhibitory IC50 was substantially equivalent.

Table 15 IC50 for inhibiting soluble CD73 enzyme activity by fusion protein

Protein numbering	IC50(nM)
		GB7002.01	0.7049
GB7002.01-TGFbRII	0.9858

EXAMPLE 16 fusion protein blocking TGF-beta/SMAD Signal pathway experiments

Appropriate amount of 293-TGF-beta/SMAD effector cells were inoculated into 96-well cell culture white bottom plates and incubated overnight at 37℃in a 5% CO2 incubator. Purified Fc (CH 2-CH 3) -TGFbRII, GB7002.01-TGFbRII fusion protein, after gradient dilution, was mixed with TGF-beta 1 (acrobiosystems, TG 1-H421) and incubated at room temperature for 30min. The above mixture was added to the white bottom plate with cells for continued culture overnight. To each well was added Bio-Glo (TM) reagent (Promega) and the fluorescence signal values were read using a multifunctional microplate reader.

The results of the experiment are shown in FIG. 13 and Table 16, where all purified samples of the invention were substantially equivalent to blocking TGF-beta/SMAD signaling pathway activity.

Table 16 fusion proteins block TGF-beta/SMAD Signal pathway IC50

Protein numbering	IC50(nM)
		Fc(CH2-CH3)-TGFβRII	0.002450
GB7002.01-TGFβRII	0.001589

EXAMPLE 17 experiments on inhibition of reverse T cells by fusion proteins

PBMC cells contain adenosine pathway molecules such as CD39 and CD73, and the added ATP eventually becomes adenosine under the hydrolysis of CD39 and CD73, thereby inhibiting activation of T cells. Whereas a CD73 enzyme activity blocking antibody could theoretically block the generation of adenosine, thereby relieving the inhibition of T cells by adenosine.

According to this experimental principle, we set up the following experiment:

appropriate amounts of PBMCs (purchased from eriosema chinense) were recovered, washed by centrifugation, placed on T75 flasks, incubated on an adhesive for 2 hours to remove monocytes, the supernatant suspension cells were collected, and centrifuged to stain with 2mm CellTrace Violet (thermosusher, #c 34557). 2x 10A 5 stained cells were distributed in 96 flat bottom plates (pre-coated with 1ug/ml anti-CD 3 antibody), diluted CD73 antibody at different concentrations was added and incubated at 37℃for 1h, anti-CD28 antibody at a final concentration of 1ug/ml was added. Finally, 500uM ATP was added to inhibit T cell proliferation, and note that no ATP was added to the maximum proliferation wells. The ability of the stained diluted T cell proliferation (FACS antibody to differentiate CD4 or CD 8T cells) to assess T cell proliferation and antibody blocking adenosine immunosuppression was assessed by flow cytometry 3-5 days later.

The results of the experiment are shown in FIG. 14, where all purified samples of the present invention were substantially equivalent in terms of their inhibitory activity against reversed T cells.

The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Numerous variations of the presently exemplified embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and equivalents thereof.

Claims

1. A fusion protein comprising an antibody or antigen-binding fragment thereof that specifically binds to CD73 protein, said antibody or antigen-binding fragment thereof comprising at least one CDR of the heavy chain variable region amino acid sequence as set forth in SEQ ID No. 41, and at least one CDR of the light chain variable region amino acid sequence as set forth in SEQ ID No. 42.

2. The fusion protein of claim 1, further comprising a TGF- β binding moiety.

3. The fusion protein of any one of claims 1-2, having one or more of the following properties:

1) Capable of specifically binding to human and/or monkey CD73 protein;

2) Has inhibiting effect on CD73 enzyme activity on tumor cell surface;

3) Has inhibiting effect on soluble CD73 enzyme activity;

4) Capable of blocking the TGF- β/SMAD signaling pathway; and

5) Can relieve the inhibition of adenosine to T cells.

4. An isolated nucleic acid molecule encoding the fusion protein of any one of claims 1-3.

5. An immunoconjugate comprising the fusion protein of any one of claims 1-3.

6. A multispecific molecule comprising the fusion protein of any one of claims 1-3.

7. A chimeric antigen receptor comprising the fusion protein of any one of claims 1-3.

8. An immune effector cell comprising the chimeric antigen receptor of claim 7.

9. A pharmaceutical composition comprising the fusion protein of any one of claims 1-3, the nucleic acid molecule of claim 4, the immunoconjugate of claim 5, the multispecific molecule of claim 6, the chimeric antigen receptor of claim 7 and/or the immune effector cell of claim 8, and optionally a pharmaceutically acceptable carrier.

10. Use of the fusion protein of any one of claims 1-3, the nucleic acid molecule of claim 4, the immunoconjugate of claim 5, the multispecific molecule of claim 6, the chimeric antigen receptor of claim 7, the immune effector cell of claim 8, or the pharmaceutical composition of any one of claim 9, in the manufacture of a medicament for preventing and/or treating a disease and/or disorder.