CN115368458A

CN115368458A - anti-CD 73 antibodies and uses thereof

Info

Publication number: CN115368458A
Application number: CN202210550186.2A
Authority: CN
Inventors: 黄贤明; 岳睿; 张慧; 陈振埕; 梁世德
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2021-05-21
Filing date: 2022-05-20
Publication date: 2022-11-22
Also published as: WO2022242758A1

Abstract

The invention provides anti-CD 73 antibodies and uses thereof, the antibodies of the invention can specifically bind to CD73 and block the interaction between CD73 and its ligand. The antibodies of the invention are useful for the immune system to eliminate tumor cells and also for the diagnosis and prognosis of tumors or cancers.

Description

anti-CD 73 antibodies and uses thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an anti-CD 73 antibody and application thereof.

Background

CD73 (cluster of differentiation 73), also known as extracellular 5' -nucleotidase, is encoded by the NT5E gene in humans. CD73 is a glycoprotein immobilized on the cell surface via glycosylphosphatidylinositol, and has enzymatic activity to convert AMP into adenosine. CD73 is expressed on a variety of cell types, such as endothelial cells, subpopulations of lymphocytes, stromal cells and tumor cells.

In the tumor microenvironment, CD73 catalyzes the adenosine produced by AMP to bind to adenosine receptors on the surface of T cells and inhibit the expansion of T cells and their immune activity, thereby forming an immunosuppressive microenvironment. In animal models, the knockout of CD73 or the targeted inhibition of the activity of CD73 enzyme can show a certain antitumor effect; meanwhile, the anti-tumor effect of monoclonal antibodies such as PD-L1/PD1, CTLA4 or 4-1BB can be remarkably improved by targeted inhibition of CD 73. Targeting CD73 inhibits the enzyme activity thereof, and relieves the adenosine-mediated immunosuppression microenvironment, so as to achieve the anti-tumor effect. Research shows that certain anti-CD 73 antibodies can improve the activity of B cells after being combined with the B cells, and contribute to the generation of antibodies and the formation of immunological memory.

CD73 is a potential target for cancer therapy.

Disclosure of Invention

The invention provides anti-CD 73 antibodies or antigen binding fragments, which can specifically bind to CD73, block an immunosuppressive signal path downstream of CD73 and help the immune system to remove tumor cells.

Some embodiments provide an antibody or antigen-binding fragment that specifically binds CD73 and comprises one or more of the following amino acid sequences:

(a) HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence substantially identical to SEQ ID NO:1 or consists of an amino acid sequence having a single site substitution, deletion or insertion;

(b) HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence substantially identical to SEQ ID NO:2 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion;

(c) HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:3 or an amino acid sequence corresponding to SEQ ID NO:3 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion;

(d) LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence substantially identical to SEQ ID NO:4 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion;

(e) LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence corresponding to SEQ ID NO:5 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion;

(f) LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:6-9 or a sequence substantially identical to the amino acid sequence shown in any one of SEQ ID NOs: 6-9, or consists of an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence set forth in any one of claims 6-9.

In some embodiments, the antibody or antigen binding fragment specifically binds CD73 and comprises:

(b) HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence substantially identical to SEQ ID NO:2 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion; and

(c) HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:3 or an amino acid sequence corresponding to SEQ ID NO:3 compared to, or consisting of an amino acid sequence having a single site substitution, deletion or insertion.

In some embodiments, the HCDR1 comprises SEQ ID NO:1, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 or consists thereof.

(e) LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence corresponding to SEQ ID NO:5 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion; and

In some embodiments, LCDR1 comprises SEQ ID NO:4, and LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6-9 or consisting of an amino acid sequence as set forth in any one of claims 6 to 9. In some embodiments, LCDR1 comprises SEQ ID NO:4, and LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6 or consists thereof. In some embodiments, LCDR1 comprises SEQ ID NO:4, and LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:7 or consists thereof. In some embodiments, LCDR1 comprises SEQ ID NO:4, and LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or consists thereof. In some embodiments, LCDR1 comprises SEQ ID NO:4, and LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9 or consists thereof.

(b) HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence corresponding to SEQ ID NO:2 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion;

(c) HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:3 or an amino acid sequence substantially identical to SEQ ID NO:3 compared to an amino acid sequence having a single site substitution, deletion or insertion;

(e) LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence substantially identical to SEQ ID NO:5 compared to, or consisting of, an amino acid sequence having a single site substitution, deletion or insertion; and

(f) LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO:6-9 or a sequence identical to the amino acid sequence shown in any one of SEQ ID NOs: 6-9, or consists of an amino acid sequence having a single site substitution, deletion or insertion compared to the sequence set forth in any one of claims 6-9.

In some embodiments, the substitution is a conservative amino acid substitution.

In some embodiments, the antibody or antigen-binding fragment comprises the amino acid sequence set forth as SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown in FIG. 6.

In some embodiments, the antibody or antigen-binding fragment comprises the amino acid sequence set forth as SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and the LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 7.

In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 8.

In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence as set forth in SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and the LCDR2 as set forth in SEQ ID NO: LCDR3 shown at 9.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, and the amino acid sequence shown in SEQ ID NO:10, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:10, or consists of an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in seq id no; and/or

The light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:11-14, and the amino acid sequence shown in SEQ ID NO:11-14, or a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs: 11-14, or consists of an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in any one of claims 11-14.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, and/or the light chain variable region of the antibody or antigen binding fragment comprises the sequence set forth in SEQ ID NO: 11-14.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:11, and (c) the sequence shown in fig. 11. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:12, or a fragment thereof. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:13, or a fragment thereof. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:14, or a sequence shown in fig. 14.

In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or antigen-binding fragment is of one of the isotypes IgG, igM, igA, igE, or IgD. In some embodiments, the isotype is IgG1, igG2, igG3, or IgG4. In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, relative to the parent Fc region. In some embodiments, the amino acid modification of the Fc region alters effector function activity relative to the activity of the parent Fc region. In some embodiments, a variant Fc region may have altered (i.e., increased or decreased) Antibody Dependent Cellular Cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In some embodiments, the Fc region amino acid modification may alter the affinity of the variant Fc region for an fcyr (fey receptor) relative to a parent Fc region. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A, L234A or L235A (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering).

In some embodiments, the antibody or antigen binding fragment is an scFv, fab', or F (ab) ₂ . In some embodiments of the present invention, the substrate is,the antibody or antigen binding fragment is a monoclonal antibody.

In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:15 or 16, or a sequence substantially identical to SEQ ID NO:15 or 16, or a sequence having at least 80% identity to a sequence as set forth in SEQ ID NO:15 or 16 with one or more conservative amino acid substitutions; and/or

The light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:17, or a sequence substantially identical to SEQ ID NO:17, or a sequence having at least 80% identity to a sequence set forth in SEQ ID NO:17 represents or consists of an amino acid sequence having one or more conservative amino acid substitutions relative to the sequence.

In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:15, the light chain constant region of said antibody or antigen-binding fragment comprises a sequence having an amino acid sequence set forth in SEQ ID NO:17, and (b) is the sequence shown in (17). In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:16, the light chain constant region of said antibody or antigen binding fragment comprises a sequence having an amino acid sequence set forth in SEQ ID NO:17, and (b) is the sequence shown in 17.

In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18 or 19, and SEQ ID NO:18 or 19, or a sequence having at least 80% identity to a sequence as set forth in SEQ ID NO:18 or 19, or consists of an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in seq id no; and/or

The light chain of the antibody comprises an amino acid sequence shown in SEQ ID NO:20-23, and SEQ ID NO:20-23, or a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs: 20-23, or consists of an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in any one of claims 20-23.

In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18 or 19, or consists thereof; and/or

The light chain of the antibody comprises an amino acid sequence shown in SEQ ID NO:20-23, or consisting of the same.

In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18 and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:20, or a sequence shown in fig. 20. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18, and the light chain of the antibody comprises an amino acid sequence shown as SEQ ID NO:21, and (b) the sequence shown in (21). In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18 and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:22, or a sequence shown in fig. 22. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18 and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:23, or a sequence as shown in seq id no. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19, and the light chain of the antibody comprises an amino acid sequence shown in SEQ ID NO:20, or a sequence as shown in figure 20. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19 and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:21, and (b) the sequence shown in figure 21. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19, and the light chain of the antibody comprises an amino acid sequence shown in SEQ ID NO:22, or a sequence shown in fig. 22. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19 and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:23, or a sequence as shown in seq id no.

In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody (including full-length monoclonal antibodies), a multispecific antibody, or antigen-binding fragment (e.g., a bispecific antibody or antigen-binding fragment thereof).

In some embodiments, the antibody has two heavy chains of identical sequence and two light chains of identical sequence, with the Fc regions paired to form disulfide bonds. In some embodiments, the antigen binding fragment is a Fab, fab', fv, or scFv.

In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

The invention also provides nucleic acids encoding the antibodies or antigen-binding fragments. In some embodiments, the nucleic acid is an isolated nucleic acid. In some embodiments, the nucleic acid sequence is selected from the group consisting of the nucleic acid sequences listed in table 6.

The invention also provides vectors comprising said nucleic acids. In some embodiments, the vector comprising the nucleic acid is a nucleic acid fragment, a plasmid, a phage, or a virus. In some embodiments, the vector is an isolated vector.

The invention also provides a host cell comprising the nucleic acid or vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, a HEK cell (e.g., HEK293F cell), a BHK cell, a Cos1 cell, a Cos7 cell, a CV1 cell, or a murine L cell.

The invention also provides methods of making the antibodies or antigen-binding fragments comprising culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment in a culture medium. In some embodiments, the method further comprises purifying the antibody or antigen-binding fragment. Purification can be carried out by conventional methods, for example, by first centrifuging the cell suspension and collecting the supernatant, and then centrifuging again to further remove impurities. Protein A affinity column and ion exchange column can be used for purifying antibody Protein.

The invention also provides a pharmaceutical composition comprising the antibody or antigen binding fragment and a pharmaceutically acceptable excipient.

The invention also provides methods and uses for preventing or treating tumors, cancers or infections. In some embodiments, a method for treating or ameliorating a tumor, cancer, or infection is provided, the method comprising administering to a patient an effective dose of the antibody or antigen-binding fragment. In some embodiments, there is provided the use of the antibody or antigen binding fragment for the treatment or amelioration of a tumor, cancer, or infection. In some embodiments, there is provided the use of the antibody or antigen-binding fragment in the manufacture of a medicament for the treatment or amelioration of a tumor, cancer, or infection.

In some embodiments, the cancers and tumors include, but are not limited to, breast, endocrine, neuroendocrine, eye, genitourinary, germ cell, gynecological, head and neck, hematologic/hematologic, musculoskeletal, neurological, respiratory/thoracic, bladder, colon, rectal, lung, endometrial, kidney, pancreatic, liver, stomach, testicular, esophageal, prostate, brain, cervical, ovarian, and thyroid cancers. In some embodiments, cancers and tumors include, but are not limited to, leukemia, melanoma, and lymphoma. In some embodiments, the leukemia includes, but is not limited to, a lymphocytic leukemia or a myeloid leukemia, such as Acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), acute myeloid (myelogenous) leukemia (AML), chronic Myelogenous Leukemia (CML), hairy cell leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, or adult T-cell leukemia. In some embodiments, the lymphoma includes, but is not limited to, histiocytic lymphoma, follicular lymphoma, and hodgkin's lymphoma. In some embodiments, the infection includes, but is not limited to, chronic infectious diseases such as HIV, HBV, HCV, COVID-19, HSV, and the like.

The invention also provides diagnostic methods and uses. In some embodiments, methods are provided for detecting CD73 expression in a sample by contacting the sample with the antibody or antigen-binding fragment such that the antibody or antigen-binding fragment binds to CD73 and detecting its binding, i.e., the amount of CD73 in the sample. In some embodiments, there is provided the use of the antibody or antigen-binding fragment in the preparation of a kit for the diagnosis or prognosis of cancer or a tumour. In some embodiments, a diagnostic or prognostic kit is provided comprising the antibody or antigen-binding fragment; optionally, the kit further comprises a second antibody that specifically recognizes the anti-CD 73 antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme; optionally, the kit is for detecting the presence or level of CD73 in a sample; optionally, the kit further comprises antibodies or antigen-binding fragments against other antigens, and/or cytotoxic agents, and optionally, instructions for use.

The invention provides an anti-CD 73 antibody and application thereof, and the antibody or an antigen binding fragment can be specifically bound with CD73, block an immunosuppressive signal path at the downstream of the CD73 and is beneficial to an immune system to eliminate tumor cells. The antibodies or antigen-binding fragments of the invention are useful for treating or ameliorating tumors, cancers or infections, and also for the diagnosis and prognosis of cancers or tumors.

Drawings

FIG. 1 shows scFv binding to the antigen hCD 73; where BLK represents the control.

FIG. 2 shows that anti-CD 73 antibodies inhibit the enzymatic activity of cell membrane CD 73.

Fig. 3A shows the flow detection results of CD73 (PE label) in peripheral blood T cells, wherein a represents the peak area.

Fig. 3B shows the flow detection results of CD73 (PE label) in peripheral blood B cells, wherein a represents the peak area.

FIG. 4 shows that anti-CD 73 antibody abrogates the inhibition of T cell proliferation by AMPs.

FIG. 5 shows that anti-CD 73 antibody upregulates expression of B cell activation marker (CD 69); where the abscissa BLK represents the control and the ordinate represents the mean fluorescence intensity of CD69 in B cells.

Term(s)

Unless otherwise specified, each of the following terms shall have the meaning set forth below.

Definition of

It should be noted that the term "an" entity refers to one or more of the entities, e.g., "an antibody" should be understood to mean one or more antibodies, and thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

The term "comprising" or "comprises" as used herein means that the compositions and methods and the like include the recited elements, such as components or steps, but not excluding others. "consisting essentially of" means that the compositions and methods exclude other elements that have a fundamental effect on the characteristics of the combination, but do not exclude elements that do not materially affect the composition or method. "consisting of" means excluding elements not specifically recited.

The term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule formed of monomers of amino acids linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids and does not refer to a particular length of the product. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of, or in alternation with, any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modification. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, and it may be produced in any manner, including chemical synthesis.

"amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an alpha-amino acid, which may be encoded by a nucleic acid, either directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons and is referred to as "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three letter code: ala, one letter code: a), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

"conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of classes of amino acids containing chemically similar side chains include: 1) Aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) Aliphatic hydroxyl side chain: serine and threonine; 3) Side chain of the acylamide: asparagine and glutamine; 4) Aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) Basic side chain: lysine, arginine and histidine; 6) Acidic side chain: aspartic acid and glutamic acid.

The number of amino acids for "conservative amino acid substitutions of VL, VH" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive) or any value therein. The number of amino acids that are "conservative amino acid substitutions for a heavy chain constant region, a light chain constant region, a heavy chain, or a light chain" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (including the endpoints) or any value therein.

The term "isolated" as used herein with respect to a cell, nucleic acid, polypeptide, antibody, etc., e.g., "isolated" DNA, RNA, polypeptide, antibody, refers to a molecule that is separated from one or more other components, e.g., DNA or RNA, respectively, in the natural environment of the cell. The term "isolated" as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture media when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur in nature, and which do not occur in nature. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides. Isolated polypeptides, antibodies, and the like are typically prepared by at least one purification step. In some embodiments, an isolated nucleic acid, polypeptide, antibody, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or ranges between any two of these values (including the endpoints) or any value therein.

The term "recombinant" refers to a polypeptide or polynucleotide, and means a form of a polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may include combinations that produce polynucleotides or polypeptides that do not normally occur.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

"at least 80% identity" is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (including endpoints) or any value therein.

A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "identity or sequence identity" with another sequence, meaning that the percentage of bases (or amino acids) in the two sequences being compared are identical when the sequences are aligned. This alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as the software program described in Current Protocols in Molecular Biology (2007). Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: geneticcode = standard; filter = none; strand = booth; cutoff =60; expect =10; matrix = BLOSUM62; descriptions =50sequences; sortby = HIGHSCORE; databases = non-redundant; genBank + EMBL + DDBJ + PDB + GenBank CDStranslations + SwissProtein + Spupdate + PIR. A biologically equivalent polynucleotide is a polynucleotide having the above specified percentage of identity and encoding a polypeptide having the same or similar biological activity.

A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or thymine when the polynucleotide is RNA is exchanged for uracil (U). A "polynucleotide sequence" can be represented by the letters of a polynucleotide molecule. The alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as for functional genomics and homology searches.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are examples of non-limiting polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, a messenger RNA (mRNA), a transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, structural modifications to the nucleotide can be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include a double-stranded form and each of two complementary single-stranded forms known or predicted to comprise the double-stranded form.

The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which polypeptide and/or fragments thereof can be produced by transcription and/or translation in its native state or when manipulated by methods well known to those skilled in the art.

The antibodies, antigen binding fragments disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric, single chain antibodies, epitope binding fragments (e.g., fab ', and F (ab') ₂ Single chain Fv (scFv)).

"antibody," "antigen-binding fragment," refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The antibody may be a whole antibody and any antigen binding fragment thereof or a single chain thereof. The term "antibody" thus includes any protein or peptide in a molecule that contains at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, complementarity Determining Regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions (CH), light chain constant regions (CL), framework Regions (FR), or any portion thereof of a heavy or light chain or ligand-binding portion thereof, or at least a portion of a binding protein. CDR regions include the CDR regions of the light chain (LCDR 1-3) and the CDR regions of the heavy chain (HCDR 1-3). Antibodies and antigen-binding fragments can specifically recognize and bind to one or more (e.g., two) polypeptides or polypeptide complexes of antigens. An antibody or antigen-binding fragment that specifically recognizes and binds multiple (e.g., two) antigens may be referred to as a multispecific (e.g., bispecific) antibody or antigen-binding fragment.

The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, and the constituent form of an antibody fragment of the invention may resemble F (ab') ₂ 、F(ab) ₂ Fab', fab, fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen that is recognized by an intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a particular antigen.

"Single chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are linked to a short linker peptide of 10 to about 25 amino acids. The linker may be glycine rich to increase flexibility and serine or threonine rich to increase solubility and may link the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has the constant region removed and the linker introduced, it retains the specificity of the original immunoglobulin. scFv molecules are generally known in the art and are described in relation to, for example, U.S. Pat. No. 5,892,019.

The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the class of heavy chains includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ 1- γ 4). The nature of this chain determines the "class" of antibodies as IgG, igM, igA, igG or IgE, respectively. The immunoglobulin subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, igG5, etc., have been well characterized and the functional specificity conferred is also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is an IgG class.

Light chains can be classified as kappa (. Kappa.) or lambda (. Lamda.). Each heavy chain may be associated with a kappa or lambda light chain. In general, when an immunoglobulin is produced by a hybridoma, B cell or genetically engineered host cell, its light and heavy chains are joined by covalent bonds and the "tail" portions of the two heavy chains are joined by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus of the bottom of each chain. The immunoglobulin kappa light chain variable region is Vkappa; immunoglobulin lambda light chain variable region is V _λ 。

The terms "constant" and "variable" are used in accordance with function. The Variable Light (VL) and Variable Heavy (VH) regions determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, placental movement, fc receptor binding, complement fixation, and the like. By convention, the numbering of constant regions increases as they become further away from the antigen binding site or amino terminus of the antibody. The N-terminal part is a variable region and the C-terminal part is a constant region; the CH3 and CL domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that form the antigen binding domain that specifically bind to an antigen, assuming the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining other amino acids in the antigen binding domain, referred to as the "framework" region, show less intermolecular variability. The framework regions largely adopt a beta-sheet conformation, with the CDRs forming a loop structure connected to, or in some cases forming part of, the beta-sheet structure. Thus, the framework regions allow the CDRs to be positioned in the correct orientation by forming a scaffold via interchain non-covalent interactions. The antigen binding domain with the CDRs at a particular location forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of an antibody to its antigenic epitope. Amino acids comprising CDRs and framework regions can be identified by known methods by those of ordinary skill in the art for a given heavy or light chain variable region (see Kabat, e., et al, u.s.department of Health and Human Services, sequences of Proteins of Immunological Interest, (1983) and Chothia and leave, j.mol.biol.,196, 901-917 (1987)).

Where two or more definitions are provided for a term used and/or accepted in the art, the definition of the term as used herein includes all such meanings unless explicitly stated to the contrary. One specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al, U.S. Dept. Of Health and Human Services, sequences of Proteins of Immunological Interest (1983) and Chothia et al, J.mol.biol.196:901-917 (1987), which is incorporated herein by reference in its entirety, is described.

CDRs defined according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof. The exact residue number comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues a CDR contains based on the amino acid sequence of the variable region of an antibody.

Kabat et al also defines a numbering system for the variable region sequences applicable to any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on other experimental data beyond the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al, U.S. Dept. Of Health and Human Services at "Sequence of Proteins of Immunological Interest" (1983). Antibodies may also be used with the EU or Chothia numbering system.

The antibodies disclosed herein may be derived from any animal, including birds and mammals. Preferably, the antibody is human, murine, donkey, rabbit, goat, camel, llama, horse or chicken. In another embodiment, the variable region may be of chondrocyclic fish (condricthoid) origin (e.g. from sharks).

The "heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant region of an antibody may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a heavy chain constant region derived from an IgG ₁ CH1 Domain of a molecule and derived from IgG ₃ The hinge region of the molecule. In another embodiment, the heavy chain constant region may comprise a portion derived from an IgG ₁ Molecules and moieties derived from IgG ₃ The hinge region of the molecule. In another embodiment, a portion of the heavy chain may comprise a portion derived from IgG ₁ Molecules and moieties derived from IgG ₄ A chimeric hinge region of the molecule.

A "light chain constant region" comprises a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain. The four chains are linked by disulfide bonds in a "Y" configuration, in which the light chain begins at the "Y" opening and continues through the variable region surrounding the heavy chain.

The "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain, and the "CH1 domain" comprises the first (largely amino-terminal) constant region of an immunoglobulin heavy chain. One branched carbohydrate chain was attached to each of N297 in the two CH2 domains of the intact native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues. The "hinge region" includes a portion of the heavy chain region that connects the CH1 domain and the CH2 domain. The hinge region comprises about 25 residues and is flexible, thereby enabling independent movement of the two N-terminal antigen-binding regions. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (rouxetal, j.immunol 161.

"disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of cysteine may form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds.

"chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be intact, partial, or modified) are derived from a second species. In certain embodiments, the variable region is from a non-human source (e.g., mouse or primate) and the constant region is from a human source.

"specifically binds" or "is specific to" typically means that the antibody or antigen-binding fragment binds to a particular antigen through its antigen-binding domain complementary to an epitope to form a relatively stable complex. "specificity" can be expressed in terms of the relative affinity of an antibody or antigen-binding fragment for binding to a particular antigen or epitope. For example, an antibody "a" can be considered to have a higher specificity for the same antigen than an antibody "B" if antibody "a" has a greater relative affinity for the antigen than antibody "B". Specific binding may be measured by the equilibrium dissociation constant (K) _D ) To describe, smaller K _D Meaning a tighter bond. Methods of determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, biofilm layer optical interferometry, and the like. An antibody that "specifically binds" to antigen a includes an equilibrium dissociation constant K for antigen a _D Less than or equal to about 100nM, less than or equal to about 10nM, less than or equal to about 5nM, less than or equal to about 1nM.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, with the object of preventing, slowing, ameliorating, or halting undesirable physiological changes or disorders, such as the progression of a disease, including, but not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration, palliation, alleviation or abolition (whether partial or total) of disease state, extending the expected life span when not treated, and the like, whether detectable or undetectable. Patients in need of treatment include patients already with a condition or disorder, patients susceptible to a condition or disorder, or patients in need of prevention of the condition or disorder, patients who may or are expected to benefit from administration of the antibodies or pharmaceutical compositions disclosed herein for detection, diagnostic procedures, and/or treatment.

"patient" refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, rabbits, mice, horses, cattle, etc.

"about" refers to the conventional error range for corresponding numerical values as would be readily understood by one of ordinary skill in the relevant art. In some embodiments, reference herein to "about" refers to the numerical values recited and ranges of ± 10%, ± 5%, or ± 1% thereof.

“EC ₅₀ "i.e., the half maximum effect concentration (concentration for 50%; of maximum effect ₅₀ ) Refers to a concentration that causes 50% of the maximum effect.

“IC ₅₀ "means the 50% inhibitory concentration, i.e., the concentration of drug or inhibitor required to inhibit half of a given biological process.

The "parent Fc region" in the present invention may be a naturally occurring Fc region, and the gene encoding the Fc region may be derived from human, mouse, rabbit, camel, monkey, preferably human and mouse; for example, the parent Fc region is SEQ ID NO:18 or 19.

The relevant descriptions of the publications mentioned herein are incorporated herein by reference in their entirety.

anti-CD 73 antibodies

The present invention provides antibodies or antigen-binding fragments with high affinity for CD73 protein. The anti-CD 73 antibodies or antigen-binding fragments of the invention exhibit potent binding activity, biological activity, and are useful in therapy and diagnosis. For example, these antibodies or antigen binding fragments effectively block inhibitory immune checkpoints, activate lymphocytes to release cytokines, and are useful for treating various types of cancer, tumors, or infection related diseases.

In some embodiments, the antigen-binding fragment is an scFv.

In some embodiments, the VH in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:10, VL in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:11, and (c) the sequence shown in figure 11. In some embodiments, the VH in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:10, VL in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:12, or a sequence shown in figure 12. In some embodiments, the VH in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:10, VL in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:13, or a sequence shown in figure 13. In some embodiments, the VH in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:10, VL in the scFv comprises an amino acid sequence as set forth in SEQ ID NO:14, or a sequence shown in fig. 14.

In some embodiments, the linker connecting the heavy chain variable region and the light chain variable region in the scFv fragment is (G) ₄ S) _n . In some embodiments, n is 1, 2, 3, 4, or 5.

In some embodiments, the antibody is an IgG1 or an IgG4.

In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18, and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:20, or a sequence shown in fig. 20. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18 and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:21, and (b) the sequence shown in figure 21. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence set forth in SEQ ID NO:18 and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:22, or a sequence shown in fig. 22. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:18, and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:23, or a sequence shown in seq id no. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19 and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:20, or a sequence shown in fig. 20. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19 and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:21, and (b) the sequence shown in figure 21. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19 and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:22, or a sequence shown in fig. 22. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as set forth in SEQ ID NO:19 and the light chain of the antibody comprises the amino acid sequence shown as SEQ ID NO:23, or a sequence shown in seq id no.

In some embodiments, an antibody of the invention comprises two heavy chains of identical sequence and two light chains of identical sequence.

It will also be understood by those of ordinary skill in the art that the sequences of the disclosed antibodies or antigen-binding fragments may be substituted with substitutions having an amino acid sequence that differs from the naturally occurring amino acid sequence of the antibody. For example, the substituted amino acid sequence can be similar to the starting sequence, e.g., has a certain proportion of identity to the starting sequence, e.g., it can be about 80%, about 85%, about 90%, about 95%, about 98%, about 99% identical to the starting sequence, or a range between any two of these values (including the endpoints), or any value therein.

In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence having one or more modification groups. For example, the antibodies or antigen-binding fragments disclosed herein may comprise a flexible linker sequence, or may be modified to add functional groups (e.g., PEG, drugs, toxins, or tags).

The antibodies, antigen-binding fragments disclosed herein include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody or antigen-binding fragment, wherein the covalent attachment does not prevent the antibody or antigen-binding fragment from binding to an epitope. Including, but not limited to, examples where the antibody or antigen-binding fragment may be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to a cellular ligand or other protein, and the like. Any of a number of chemical modifications may be made by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. The antibodies, antigen-binding fragments, and modified derivatives thereof disclosed herein include salts thereof with acids and/or bases.

In some embodiments, the antibody or antigen-binding fragment may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, agent, or PEG.

The antibody or antigen-binding fragment may be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-labeled antibody or antigen-binding fragment is then determined by detecting the luminescence that occurs during the course of the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

Antibody and method for preparing polynucleotide encoding antibody

Also disclosed are polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the invention. The polynucleotides disclosed herein may encode a heavy chain variable region, a light chain variable region, an Fc region, a portion of a heavy chain variable region, a portion of a light chain variable region, a heavy chain or a light chain, and the like. Methods of making antibodies are well known in the art and are described in the present invention.

In certain embodiments, the antibodies are prepared so as not to elicit an adverse immune response in the animal (e.g., human) to be treated. In some embodiments, the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified using art-recognized techniques to reduce their immunogenicity. For example, the antibody may be humanized, primatized, deimmunized or a chimeric antibody may be prepared. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen binding properties of the parent antibody but are less immunogenic in humans. This can be achieved by a variety of methods, including (a) grafting the entire variable region of non-human origin to a constant region of human origin to produce a chimeric antibody; (b) Grafting at least a portion of one or more non-human Complementarity Determining Regions (CDRs) into a framework and constant region of human origin, with or without retention of critical framework residues; or (c) transplanting the entire variable regions of non-human origin, but "hiding" them by replacing surface residues with portions of human-like origin. Typically, framework residues in the human framework region will be substituted with corresponding residues from the CDR donor antibody, such as residues capable of improving antigen binding. These framework substitutions can be identified by methods well known in the art, for example, by modeling the interaction of the CDRs and framework residues to identify framework residues that play an important role in antigen binding and by sequence alignment to identify framework residues that are aberrant at a particular position. (see U.S. Pat. Nos. 5,585,089, riechmann et al, nature 332 (1988); incorporated herein by reference in their entirety). Antibodies can be humanized using a variety of techniques well known in the art, such as CDR grafting (EP 239,400, WO 91/09967; U.S. Pat. Nos. 5,225,539,5,530,101 and 5,585,089), repair or resurfacing (EP 592,106; EP519,596; padlan, et al, molecular Immunology 28 (4/5): 489-498 (1991); studnicka et al, protein Engineering 7 (6): 805-814 (1994); roguska, et al, proc. Natl. Sci. USA 91 969-973 (1994)), and chain rearrangement (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

Deimmunization may also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering antibodies to modify T cell epitopes (see, e.g., WO/9852976 A1 and WO/0034317 A2). For example, the heavy and light chain variable region sequences from the starting antibody are analyzed and a human T cell epitope "map" is generated from each variable region, showing the position of the epitope relative to the Complementarity Determining Regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions with lower risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding polypeptides. The genes comprising the modified variable regions and the complete heavy and light chains of the human constant regions were then cloned into expression vectors, and the plasmids were subsequently transferred into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological experiments to identify the best antibody.

The binding specificity of the antibodies or antigen-binding fragments disclosed herein can be detected by in vitro assays, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

scFv can be prepared by techniques for the production of single-stranded units (U.S. Pat. Nos. 4,694,778, bird, science242 (1988), huston et al, proc. Natl. Acad. Sci.USA 55. The heavy and light chain fragments that bridge the Fv region by amino acids form a single chain unit, resulting in a single chain fusion peptide. Techniques for assembling functional Fv fragments in E.coli can also be used (Skerra et al, science 242.

Examples of techniques that can be used to produce single chain Fv (scFv) and antibodies include, for example, U.S. Pat. nos. 4,946,778 and 5,258,498, and Huston et al, methods in Enzymology 203:46-88 (1991), shu et al, proc. Natl. Sci. USA 90:1995-1999 (1993) and Skerra et al, science 240:1038-1040 (1988). For certain uses, including the use of antibodies in humans and in vitro detection assays, chimeric, humanized or fully human antibodies may be used. Chimeric antibodies are a class of molecules in which different portions of the antibody are derived from different animal species, such as antibodies having the variable regions of murine monoclonal antibodies and human immunoglobulin constant regions. Methods for producing chimeric antibodies are known in the art, see Morrison, science 229:1202 (1985); oi et al, bio Techniques 4:214 (1986); gilles et al, j.immunol.methods 125:191-202 (1989); neuberger et al, nature372:604-608 (1984); takeda et al, nature 314:452-454 (1985); and U.S. Pat. nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.

Furthermore, in Newman, biotechnology 10:1455-1460 (1992), the entire contents of which are incorporated herein by reference, discloses another efficient method for producing recombinant antibodies, and in particular, techniques that produce primate antibodies comprising monkey variable region and human constant region sequences. In addition, this technique is also mentioned in U.S. Pat. nos. 5,658,570, 5,693,780, and 5,756,096, the entire contents of each of which are incorporated herein by reference.

Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. Reference may also be made to U.S. Pat. Nos. 4,444,887 and 4,716,111, and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each of which is incorporated herein by reference in its entirety.

In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E.coli cells, simian COS cells, chinese Hamster Ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. Isolated DNA (which may be synthetic as described herein) may also be used to prepare the sequences of the constant and variable regions of antibodies, as described in U.S. Pat. No. 5,658,570, which is incorporated herein by reference in its entirety. This method extracts RNA from selected cells and converts it to cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Pat. No. 5,658,570.

In addition, using conventional recombinant DNA techniques, one or more CDRs of an antibody of the invention can be inserted into a framework region, e.g., into a human framework region, to construct a humanized non-fully human antibody. The framework regions can be naturally occurring or shared framework regions, preferably human framework regions (see Chothia et al, J.mol.biol.278:457-479 (1998) which lists a series of human framework regions). Some polynucleotides may encode antibodies produced by the framework region and CDR combination that specifically bind to at least one epitope of an antigen of interest. One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected which improve binding of the antibody to its antigen. Alternatively, substitution or deletion of cysteine residues in one or more of the variable regions involved in interchain disulfide bond formation can be performed in this manner, thereby producing an antibody molecule lacking one or more interchain disulfide bonds. Other variations of polynucleotides within the skill of the art are also encompassed by the present invention.

Antibodies can be prepared by using conventional recombinant DNA techniques. Vectors and cell lines for producing antibodies can be selected, constructed and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as the Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammarian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences,2017, the entire contents of which, including the supplements, are incorporated by Reference in their entirety.

In some embodiments, DNA encoding the antibody can be synthesized according to the antibody amino acid sequence design described herein by conventional methods, placed into an expression vector, and then transfected into a host cell, and the transfected host cell cultured in a medium to produce the monoclonal antibody. In some embodiments, the expression antibody vector comprises at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, kozak sequences and donor and acceptor sites for RNA splicing on both sides of the insert. Efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI and the early promoters of cytomegalovirus, and other cellular promoters such as actin can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1 (+/-), pcDNA/Zeo (+/-), pcDNA3.1/Hygro (+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, among others. Commonly used mammalian cells include 293 cells, cos1 cells, cos7 cells, CV1 cells, murine L cells, CHO cells, and the like.

In some embodiments, the inserted gene fragment contains a selection marker, and common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and the like, so as to facilitate the selection and isolation of cells that are transfected successfully. The constructed plasmid is transfected to host cells without the genes, and the cells successfully transfected grow in large quantities through selective culture medium culture to produce the target protein to be obtained.

In addition, standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequences encoding the antibodies of the present invention, including but not limited to site-directed mutations resulting in amino acid substitutions and PCR-mediated mutations. Variants (including derivatives) encode substitutions of less than 50 amino acids, substitutions of less than 40 amino acids, substitutions of less than 30 amino acids, substitutions of less than 25 amino acids, substitutions of less than 20 amino acids, substitutions of less than 15 amino acids, substitutions of less than 10 amino acids, substitutions of less than 5 amino acids, substitutions of less than 4 amino acids, substitutions of less than 3 amino acids or substitutions of less than 2 amino acids relative to the original heavy chain variable region and light chain variable region. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

Method of treatment

The invention also provides methods of treatment and uses. In some embodiments, methods are provided for treating or ameliorating various types of cancer, tumor, or infection, among other related diseases, comprising administering to a patient in need thereof an effective dose of an anti-CD 73 antibody or antigen-binding fragment. In some embodiments, there is provided the use of an anti-CD 73 antibody or antigen-binding fragment for the treatment or amelioration of a cancer, tumor, or infection, among other related diseases. In some embodiments, there is provided a use of the anti-CD 73 antibody or antigen-binding fragment in the preparation of a medicament for treating or ameliorating a cancer, a tumor, or an infection, or a related disease.

The specific dose and treatment regimen for any particular patient will depend upon a variety of factors including the particular antibody or derivative used, the age and weight of the patient, general health, sex and diet, and the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are judged by a medical caregiver who is within the purview of one of ordinary skill in the art. The dosage will also depend upon the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound employed, the severity of the disease and the effect desired. The dosage employed can be determined by pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the antibodies of the invention are administered to the patient at a dose of 0.01mg/kg to 100mg/kg of patient body weight per administration. In some embodiments, the administration is once every 1 week, 2 weeks, 3 weeks, or monthly. In some embodiments, the antibodies or antigen-binding fragments of the invention are administered to a patient at a dose of 0.01mg/kg to 100mg/kg of patient body weight, or 0.1mg/kg to 20mg/kg of patient body weight. A second or more doses of the antibody or antigen-binding fragment may be administered subsequently after the initial dose, at about the same or less dose as the initial dose, wherein the subsequent doses may be separated by at least 1 to 3 days; or at least one week. The dosage and frequency of administration of the antibodies or antigen-binding fragments of the invention may be reduced by enhancing the uptake and tissue penetration (e.g., into the brain) of the antibodies or antigen-binding fragments by modifications such as lipidation.

In some embodiments, an effective dose of an anti-CD 73 antibody (e.g., antibody P59-L17 or antibody P59-L17') administered is about 15mg to 1200mg per dose. In some embodiments, the effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or antibody P59-L17') administered is about 15mg to 1200mg per treatment cycle. In some embodiments, a treatment cycle is about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values (including the endpoints) or any value therein.

In some embodiments, an effective amount of an anti-CD 73 antibody administered to a patient per treatment cycle is about 15mg, about 18mg, about 90mg, about 120mg, about 160mg, about 180mg, about 200mg, about 230mg, about 250mg, about 280mg, about 300mg, about 310mg, about 334mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 500mg, about 600mg, about 720mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, or a range between any two of these values (including endpoints) or any value therein, or a formulation containing such a dose of anti-CD 73 antibody. In some embodiments, one treatment cycle is administered 1 time from 1 week to 7 weeks. In some embodiments, an effective amount of the anti-CD 73 antibody is about 100mg to 300mg, or a formulation containing such a dose, per treatment cycle; wherein a treatment cycle is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or a range between any two of these values (inclusive of the endpoints), or any value therein. In some embodiments, the patient is administered an effective amount of the anti-CD 73 antibody of about 300mg to 600mg, or a formulation containing such a dose of anti-CD 73 antibody, per treatment cycle; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, an effective amount of anti-CD 73 antibody is about 700mg to 1100mg, or a formulation containing such a dose of anti-CD 73 antibody, administered per treatment cycle; wherein a treatment cycle is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or a range between any two of these values (inclusive of the endpoints), or any value therein. In some embodiments, the effective amount of the anti-CD 73 antibody administered to the patient per treatment cycle is about 60mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 360mg, about 420mg, about 720mg, about 1080mg, or a range between any two of these values (including the endpoints) or any value therein, or a formulation comprising such a dose of the anti-CD 73 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In some embodiments, the patient is administered an effective amount of the anti-CD 73 antibody of about 50mg to 80mg, or a formulation containing such a dose of anti-CD 73 antibody, per treatment cycle; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the patient is administered an effective amount of the anti-CD 73 antibody of about 60mg, or a formulation containing such a dose of the anti-CD 73 antibody, per treatment cycle; such as about 60mg, for 1 administration.

In some embodiments, the patient is administered an effective amount of the anti-CD 73 antibody of about 150mg to 200mg, or a formulation containing such a dose of anti-CD 73 antibody, per treatment cycle; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 180mg, or a formulation containing such a dose of anti-CD 73 antibody; such as about 180mg, for 1 administration.

In some embodiments, the patient is administered an effective amount of the anti-CD 73 antibody of about 345mg to 380mg, or a formulation containing such a dose of anti-CD 73 antibody, per treatment cycle; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 360mg, or a formulation containing such a dose of anti-CD 73 antibody; such as about 360mg, 1 time.

In some embodiments, the patient is administered an effective amount of the anti-CD 73 antibody of about 693mg to 730mg, or a formulation containing such a dose of anti-CD 73 antibody, per treatment cycle; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 720mg, or a formulation containing such a dose of anti-CD 73 antibody; such as about 720mg, for 1 administration.

In some embodiments, each administration of the anti-CD 73 antibody (e.g., antibody P59-L17 or antibody P59-L17') is about 0.3mg/kg, about 1mg/kg, about 1.2mg/kg, about 2mg/kg, about 2.4mg/kg, about 3mg/kg, about 3.6mg/kg, about 4mg/kg, about 4.8mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.9mg/kg, about 7mg/kg, about 8.4mg/kg, about 9mg/kg, about 11mg/kg, about 12mg/kg, about 15mg/kg, about 18mg/kg, or a range between any two of these values (inclusive) or any value therein, or a formulation comprising this dose of the anti-CD 73 antibody.

In some embodiments, an effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or antibody P59-L17') is administered at about 0.3mg/kg to 18mg/kg every 2 weeks or 1 time every 3 weeks. In some embodiments, an effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17) is administered at about 0.3mg/kg, about 1mg/kg, about 3mg/kg, about 5mg/kg, about 6mg/kg, about 12mg/kg, about 13mg/kg, or about 18mg/kg 1 time every 2 weeks or every 3 weeks. In some embodiments, an effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or antibody P59-L17') is administered at about 1 time every 2 weeks of about 5mg/kg, 1 time every 2 weeks of about 6mg/kg, 1 time every 2 weeks of about 10mg/kg, 1 time every 3 weeks of about 3mg/kg, 1 time every 3 weeks of about 5mg/kg, 1 time every 3 weeks of about 6mg/kg, 1 time every 3 weeks of about 7.5mg/kg, 1 time every 3 weeks of about 12mg/kg, 1 time every 3 weeks of about 15mg/kg, 1 time every 3 weeks of about 18mg/kg, or 1 time every 3 weeks of about 3 weeks.

In some embodiments, the patient is administered the anti-CD 73 antibody once per treatment cycle. In some embodiments, the anti-CD 73 antibody is administered multiple times, e.g., 2, 3, 4, or 5 times, per treatment cycle. In some embodiments, the patient is administered only 1 or 4 times per treatment cycle.

In some embodiments, the patient receives one treatment cycle of treatment. In some embodiments, the patient is treated with multiple (e.g., 2, 3, or 4) treatment cycles. In some embodiments, the patient receives treatment until the condition is alleviated and no treatment is required.

Methods of administration of the antibodies, antigen-binding fragments or derivatives thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral administration. The pharmaceutical compositions may be administered by any convenient route, for example by infusion or bolus injection, absorbed through epithelial or cutaneous mucosa (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other biologically active agents. Thus, a pharmaceutical composition comprising an antibody, antigen-binding fragment or derivative thereof of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), orally, or by oral or nasal spray.

The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The mode of administration may be systemic or local.

In some embodiments, the compositions of the invention comprise a nucleic acid or polynucleotide encoding an antibody or antigen-binding fragment, which can be administered in vivo by constructing it as part of a suitable nucleic acid expression vector to facilitate expression of the protein it encodes, and then administering that part of the vector to become intracellular, for example by using a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microprojectile bombardment (e.g., gene gun; biolistic, dupont), or coating with lipid or cell surface receptors or transfection reagents, or by ligation with a homologous heterologous cassette peptide known to enter the nucleus (see, e.g., joliot et al, 1991, proc.Natl.Acad.Sci.USA 88). Alternatively, the nucleic acid may be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.

Various known delivery systems may be used to administer the antibodies, antigen-binding fragments or derivatives thereof, or polynucleotides encoding same, of the invention, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., wu and Wu,1987, j.biol.chem.262 4429-4432), construction of nucleic acids as part of a retrovirus or other vector, and the like.

Combination therapy

In some embodiments, the anti-CD 73 antibodies or antigen-binding fragments of the invention may be used in combination with other therapeutic or prophylactic regimens, including the administration of one or more antibodies or antigen-binding fragments of the invention, together with one or more other therapeutic agents or methods. In some embodiments, other treatment regimens include, but are not limited to, radiation therapy, chemotherapy, hormonal therapy, surgical therapy, and the like. For combination therapy, the antibody may be administered simultaneously or separately with the other therapeutic agent. When administered separately, the antibody or antigen-binding fragment of the invention may be administered before or after administration of another additional therapeutic agent.

In some embodiments, the antibodies or antigen-binding fragments of the invention may be used in combination with chemotherapeutic agents for the treatment of cancer or tumors, including but not limited to: camptothecin (camptothecin, CPT-11), 5-fluorouracil (5-FU), cisplatin (cispinin), doxorubicin (doxorubicin), irinotecan (irinotecan), paclitaxel (paclitaxel), gemcitabine (gemcitabine), cisplatin, carboplatin, proteasome inhibitors (e.g., bortezomib or MG 132), bcl-2 inhibitors (e.g., BH3I-2' (Bcl-xl inhibitors), indoleamine dioxygenase-1 (IDO 1) inhibitors (e.g., INCB 24360), AT-101 (R- (-) -gossypol derivatives), ABT-263 (small molecule), GX-15-070 (obacla (obaclax)), MCL-1 (myeloid leukemia cell differentiation protein-1) antagonists, iAP antagonists (e.g., ac7, smac4, small molecule smac mimetics, synthetic smac peptides), HDAC (HDAC) inhibitors (HDAC) and anti-angiogenic agents (e.g., VEGF-receptor antagonists), such as anti-peroxisome proliferator other receptor antagonists (e.g., sorafenib) agonists), as inhibitors of the receptor kinase (e.g., sorafer).

In some embodiments, the antibodies or antigen binding fragments of the invention may be used in combination with cytotoxic agents for the treatment of cancer or tumors, including but not limited to: uracil mustard, mechlorethamine (Chlormetine), cyclophosphamide (CYTOXANTM), ifosfamide, melphalan (Melphalan), chlorambucil, hemagglutin (Piporroman), triethylenemelamine, triethylenethiophosphoramide, busulfan (Busufan), carmustine (Carmustine), lomustine (Lomustine), streptozotocin (Streptozocin), dacarbazine, and Temozolomide (Temozolomide).

In some embodiments, the antibodies or antigen-binding fragments of the invention can be used in combination with antimetabolites for the treatment of cancer or tumors, including but not limited to: methotrexate, 5-fluorouracil, floxuridine, cytarabine (Cytarabine), 6-mercaptopurine, 6-thioguanine, fludarabine phosphate (Fludarabine phosphate), pentostatin (pentastatin), and gemcitabine.

In some embodiments, the antibodies or antigen-binding fragments of the invention may be used in combination with anti-proliferative agents for the treatment of cancer or tumors, including but not limited to: docetaxel (docetaxel), discodermolide (DDM), dicystatin (DCT), pelorubide (Peloruside) A, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furaetheromycin D, deoxyepothilone B1, discodermolide, paclitaxel (EPO-906), X-651 (tasedotin hydrochloride), halichondrin (Halichondrin) B, eribulin mesylate (Eribulin mesylate, E-7389), hemiasterlin (HTI-286), sartostatin (Cyrtophycin), LY-355703, maytansinoid immunoconjugates (maytansine-1), leucinolone (β -17-acetyloxytetracycline), acetolide-2-5-oxolide (epothilone-5-D), labyrin (IL5-5-epoxylide), and labyrin (epothilone-5, 5-epoxydolastatin, respectively).

In some embodiments, the antibodies or antigen binding fragments of the invention may be used in combination with agonists of co-stimulatory receptors and/or antagonists of inhibitory signals on T cells for the treatment of cancer or tumors. Targets for agonists or antagonists include, but are not limited to, CTLA-4, PD-1, PD-L2, LAG-3, TIM-3, anti-galectin 9 antibodies, VEGF, BTLA, CD69, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-4, CD39, B7-1, B7-2, CD28, 4-1BB, 4-1BBL, GITR, GITRL, OX40L, CD70, CD27, CD40, DR3, and CD28H, and the like. In some embodiments, the antibody that may be administered with the antibody or antigen-binding fragment of the invention is rituximab, trastuzumab, tositumomab (tositumomab), ibritumomab (ibritumomab), alemtuzumab, epratuzumab (eprtuzumab), bevacizumab (bevacizumab), ipilimumab (ipilimumab), galiximab (galiximab), lucamumab (lucidumab), molomab (Muromonab). In some embodiments, an antibody or antigen-binding fragment of the invention can be administered with an anti-PD-1 antibody, such as Nivolumab

Pembrolizumab

Toripalimab

Sintilimab

Camrelizumab

Tislelizumab

Penpulimab

Zimberelimab

Serplulimab

Or an anti-PD-1 antibody disclosed in WO2020207432, and the like.

Pharmaceutical composition

The invention also provides a pharmaceutical composition. Such compositions comprise an effective amount of an anti-CD 73 antibody or antigen-binding fragment, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1% to 90% of the anti-CD 73 antibody or antigen-binding fragment. In some embodiments, the pharmaceutical composition further comprises an anti-cancer agent (e.g., an immune checkpoint inhibitor).

In some embodiments, the term "pharmaceutically acceptable" refers to substances approved by a governmental regulatory agency or listed in generally recognized pharmacopeia for use in animals, particularly in humans. Furthermore, "pharmaceutically acceptable adjuvant" refers generally to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid, and the like.

The term "adjuvant" refers to a diluent, adjuvant, excipient, or carrier with which the active ingredient may be administered to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal or vegetable origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated into suppository with conventional binder and carrier such as triglyceride. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences of e.w. martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with appropriate amounts of excipients to provide a form of administration suitable for the patient. The formulation should be suitable for the mode of administration. The parent formulation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous injection into a human. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. The composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the site of injection. Generally, the active ingredients are provided in unit dosage forms, either separately or in admixture, e.g., as a dry lyophilized powder or as an anhydrous concentrate, in a sealed container (e.g., ampoule or sachet) which is indicative of the serving size of the active agent. In the case of administration of the composition by infusion, the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water or saline for injection may be used so that the effective ingredients may be mixed before administration.

The compounds of the invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts with anions derived from, for example, hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and salts with cations derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications or adaptations of the concepts of the present invention made by others are within the scope of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 phage selection

The antigen hCD73-biotin (ACROBIOSystems, cat. CD3-H82E 3) was screened using a fully human scFv phage display library. The screening method comprises the following steps: SA-magnetic beads (Dynabeads MyOneTM Streptavidin T1, cat # 65602, thermo fisher Scientific) were combined with hCD73-biotin for 2 hours at room temperature or overnight at 4 ℃; adding the blocked phage display library into the magnetic beads washed by PBS (phosphate buffer solution), and screening at room temperature for 2 hours or overnight at 4 ℃; PBST (0.05% Tween-20 in PBS buffer) 15 times, washing away non-specific binding of phage; digesting with pancreatin at room temperature for 30min, adding 1/10 volume of FBS (fetal bovine serum) to terminate the digestion reaction, infecting the digested phage into TG1 strain again, amplifying and collecting the amplified phage weightRepeating the steps for 3-5 times, wherein the concentration of the antigen in the first round is 10 mug/ml, the concentration of the antigen in the second round is 5 mug/ml, and the concentration of the antigen in the third round is 1 mug/ml; detecting the binding of the finally enriched phage clones to the antigen hCD73 by ELISA, thereby obtaining phage clones with high affinity and specific binding to the antigen CD73, and finally screening 4 phage clones (shown in figure 1) such as scFv (P59-L1), scFv (P59-L6), scFv (P59-L12) and scFv (P59-L17); the DNA sequence of the phage clone, i.e. the nucleic acid sequence of the anti-human CD73 antibody (scFv), was then obtained by sequencing techniques. Wherein the VH and VL sequences in the scFv are shown in Table 1 via a linker (G) ₄ S) ₃ And (4) connecting.

The construction method of the fully human scFv phage display library comprises the following steps: amplifying VH genes and VL genes in nonimmunized human Peripheral Blood Lymphocytes (PBLs) by Polymerase Chain Reaction (PCR), and randomly combining heavy chain VH and light chain VL into single chain antibodies (single chain Fv, scFv) by Overlapping polymerase chain reaction (OVAP PCR); scFv is inserted into phagemid vector through enzyme cutting site to construct phage library (the library capacity is more than 10) ¹¹ ) Displayed on the surface of phage for antibody screening (see SHEETS et al (1998) Cell biology.95: 6157-6162).

TABLE 1 scFv compositions

scFv numbering	VH SEQ ID NO (SEQ ID NO)	VL SEQ ID NO
			scFv(P59-L1)	10	11
scFv(P59-L6)	10	12
			scFv(P59-L12)	10	13
scFv(P59-L17)	10	14

EXAMPLE 2 method for producing antibody

Constructing IgG1 monoclonal antibody based on the selected scFv (P59-L1), scFv (P59-L6), scFv (P59-L12), and scFv (P59-L17): designing primers to carry out PCR on DNA fragments of VH and VL in the selected scFv, carrying out double enzyme digestion on PCR products (restriction site endonuclease adopted by VH is ApaI/MfeI, restriction site endonuclease adopted by VL is HindIII/BsiWI), respectively linking the fragments to a pcDNA3.1 vector (purchased from Invitrogen company, V79020) containing an IgG1 heavy chain constant region and a kappa light chain constant region, carrying out sequence optimization on heavy chain and light chain nucleic acid sequences (the optimized nucleic acid sequences are shown in a table 6) after sequencing confirms that the sequences are correct, respectively linking the optimized heavy chain and light chain nucleic acid sequences to an expression vector, and obtaining a light chain plasmid and an antibody heavy chain plasmid for a protein expression antibody after sequencing confirms that the sequences are correct. The corresponding light and heavy chain plasmids were transiently expressed by PEI (polyetherimide) co-transfected HEK293 cells (purchased from ATCC), and after 7 days of culture, the supernatant was collected and then purified by Immobilized Metal Affinity Chromatography (IMAC) using Protein a column (GE Healthcare), with the purity of the purified antibody Protein > 95%.

IgG1-Fc (i.e., igG1 constant region, including CH1, hinge region, CH2 and CH 3) was prepared by: pcDNA3.1 vector containing IgG1-Fc (purchased from Invitrogen, V79020) was transiently expressed by PEI cotransfected HEK293 cells, and after 7 days of culture, the supernatant was collected and purified.

The irrelevant antibody NR is an antibody specifically binding to the new coronavirus, and the preparation method comprises the following steps: the corresponding light chain plasmid and heavy chain plasmid were co-transfected into HEK293 cells by PEI for transient expression, and the supernatants were collected for purification after 7 days of culture.

Heavy chain amino acid sequence of antibody NR (SEQ ID NO: 30):

EVQLVQSGPEVKKPGTSVKVSCKASGFTASLSAVQWVRQARGQRLEWIGWIMPGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAAPYCSTTLCNDGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

light chain amino acid sequence of antibody NR (SEQ ID NO: 31):

DIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGISDRfSGSGSGTDFTLTISRLEPEDFATYYCQQYASSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the anti-CD 73 antibody prepared in the embodiment comprises antibodies P59-L1, P59-L6, P59-L12 and P59-L17, and the amino acid sequences and the nucleic acid sequences related to the antibody examples are shown in tables 2-6; the Fc region of the heavy chain in table 5 is single underlined. Sequencing the purified antibody, wherein the sequencing result is the same as the designed sequence. The purified antibody is used for affinity detection, bioactivity identification and the like.

TABLE 2 composition of the antibodies

TABLE 3 CDR regions of anti-CD 73 antibodies

TABLE 4 variable and constant regions of anti-CD 73 antibodies

TABLE 5 heavy and light chains of anti-CD 73 antibodies

TABLE 6 nucleic acid sequences related to anti-CD 73 antibodies

EXAMPLE 3 determination of antibody affinity

Determination of the affinity constant (K) of the antibodies by means of Biacore T200 surface plasmon resonance _D ) The main experimental procedure is as follows (see standard protocol of Biacore T200): hCD73-His (Catalog 10904-H08H, beijing Yiqiao Shenzhou science Co., ltd.) sample was diluted in HBS-EP buffer (150mM NaCl,3mM EDTA,0.005% (v/v) surfactant P-20, and 10mM HEPES, pH 7.4) in gradient (initial concentration 32nM, then 2-fold dilution); the antibodies were each diluted to a final concentration of about 20. Mu.g/ml with a sodium acetate solution (10mM, pH 5.5); make it possible toThe assay was performed using Biacore T200, protein A chip (GE healthcare, cat # 29127556) with the following instrument settings: the antibody capture binding time (contact time) was 180s, and the flow rate was 30. Mu.l/min; the sample binding time (contact time) was 120s, the dissociation time (dissociation time) was 300s, and the flow rate was 30. Mu.l/min; the regeneration (regeneration) conditions were: glycine-HCl (pH 2.5), regeneration time 60s, flow rate 30. Mu.l/min. Analyzing the test result by adopting data analysis software Evaluation software3.1, carrying out reference flow path and sample blank double deduction on the sensing signal acquired by the sample experiment flow path, and fitting by adopting a dynamics model of 1: 1 to obtain a dynamics parameter (K) _a To the rate of binding, K _d Is the dissociation rate; k _D As the equilibrium constant for dissociation of binding).

As shown in Table 7, the antibodies (P59-L1, P59-L6, P59-L12, P59-L17) have good binding ability to hCD 73-His; in particular the antibody P59-L17.

TABLE 7 affinity constants for binding of anti-CD 73 antibodies to hCD73-His

Antibodies	K _a (1/Ms)	K _d (1/s)	K _D (M)
				P59-L1	7.26E+05	5.57E-03	7.67E-09
P59-L6	1.07E+05	1.98E-04	1.85-09
				P59-L12	8.27E+05	6.8E-03	8.25E-09
P59-L17	2.21E+05	1.26E-04	5.69E-10

Example 4 detection of binding of antibodies to CD73 of different species

Detection is carried out by adopting an ELISA method: coating a 96-hole ELISA plate with 2 mu g/ml of human hCD73-His (Catalog 10904-H08H), cynomolgus monkey cCD73-His (a nearshore organism, catalog CD3-C52H 9) or mouse mCD73-His (a nearshore organism, catalog CD3-M52H 9) and standing at 4 ℃ overnight; blocking with PBS buffer containing 5% BSA (bovine serum albumin); after blocking, washing with PBS containing 0.05% TW-20, followed by addition of antibody solution diluted with PBS containing 0.05% TW-20 and 0.5% BSA (initial concentration of antibody was 4. Mu.g/ml, 3-fold dilution, 10 gradients); incubation at 37 ℃ for 1 hour; after washing the plate, adding a secondary antibody anti-h-Kappa-HRP (purchased from Sigma under the code of A7164) marked by HRP (horse radish peroxidase) for incubation for 1h; the plate was washed, and OD450 was read by adding TMB (tetramethylbenzidine) color developing solution for color development.

As shown in table 8, antibody P59-L17 bound well to human, cynomolgus monkey CD73 and did not bind to mouse CD 73.

TABLE 8 EC for binding of antibody P59-L17 to CD73-His ₅₀ (ng/ml)

Antibodies	hCD73-His	cCD73-His	mCD73-His
				EC ₅₀	33.9	42.7	NA

NA means no or substantially no binding.

Example 5 detection of binding of antibodies to CD 73-expressing cells

MDA-MB-231 cells are human breast cancer cells, and the surface of the cell membrane highly expresses CD73 protein (https:// www.proteinalas.org/ENSG 00000101017-CD 40/cell). This example uses MDA-MB-231 cells for testing the binding ability of anti-CD 73 antibodies to CD73 molecules on the cell membrane surface.

The test method comprises the following steps: MDA-MB-231 cells with good culture state are collected, centrifuged and incubated with isotype control IgG1-Fc (with the concentration of 10ug/ml, single concentration point) or anti-CD 73 antibody diluted in gradient (with the initial concentration of 10ug/ml, 2-fold dilution, 8 concentration gradient); after 30min, centrifuging to remove supernatant, washing once with PBS buffer, and resuspending in PBS buffer and adding anti-human IgG flow antibody anti-humanFc-PE (purchased from Invitrogen, with the product number of 12-4998-82); after incubation for 30min, the supernatant was washed off by centrifugation, washed twice with PBS buffer, flow-based assay was performed after the cells were resuspended in PBS buffer, MFI was counted and the data were processed with SoftMax Pro.

As shown in Table 9, antibody P59-L17 bound MDA-MB-231 cells, while negative control IgG1-Fc did not bind MDA-MB-231 cells.

TABLE 9 EC for antibody binding to MDA-MB-231 cells ₅₀ (ng/ml)

Antibodies	P59-L17	IgG1-Fc
			EC ₅₀	438.1	NA

Example 6 inhibition of CD73 Activity on cell membranes by antibodies

The detection principle of the detection kit (purchased from promega, with the product number of G7570) is as follows: excess AMP inhibits ATP-dependence

The detection reagent fluoresces, and the anti-CD 73 antibody which can decompose AMP by the cell membrane CD73 so as to relieve the inhibition effect of AMP can inhibit the activity of CD73 enzyme so as to keep the inhibition of AMP on the fluorescence process, and the detection reagent is finally expressed as follows: as the concentration of antibody increases, the fluorescence intensity decreases accordingly. This example tests the inhibition of cell membrane CD73 enzyme activity by anti-CD 73 antibodies.

The test method comprises the following steps: MDA-MB-231 cells in good growth status were cultured in DMEM medium containing 10% FBS at 2.5X 10 ⁴ cells/well are laid on a 96-well cell culture plate and placed in a cell culture box at 37 ℃ for culture overnight; removing the next dayAdding serum-free DMEM (DMEM) gradient diluted antibody solution (initial concentration is 5ug/ml,2 times dilution, 8 concentration gradients) and AMP with final concentration of 600 mu M into the culture medium supernatant, and placing the mixture in a cell culture box at 37 ℃ for reaction for 3 hours; after the reaction, 50. Mu.l of the supernatant was put in a 96-well white plate, 50. Mu.l of ATP at a concentration of 200. Mu.M was added thereto, and finally 100. Mu.l of the detection reagent was added thereto, and the fluorescence value was immediately read in a microplate reader.

As shown in FIG. 2, as the concentration of the antibody P59-L17 increases, the fluorescence intensity decreases accordingly, i.e., the concentration gradient of the antibody P59-L17 inhibits the enzyme activity of CD73, the IC of which is dependent on the concentration of the antibody P59-L17 ₅₀ 126.8ng/ml; while the negative control IgG1-Fc had essentially no effect on CD73 enzyme activity.

Example 7 flow assay for expression of CD73 in T-cells and B-cells of peripheral blood

Some T and B cells express CD73, and CD73 is involved in lymphocyte activity. In this example, flow assay was performed to investigate the expression of CD73 in T cells and B cells in peripheral blood.

The test method comprises the following steps: peripheral blood of healthy adults was collected and fresh PBMC (peripheral blood mononuclear cells) was isolated from a lymph separation medium (purchased from Biotechnology Ltd., beijing Dada, inc., cat. 7912011); blocking the Fc receptor of PMBC for 30min on ice with 1mg/ml irrelevant antibody IgG1 (antibody NR); centrifuging to remove blocking solution, adding 10 μ g/ml biotin-labeled anti-CD 73 antibody or biotin-labeled IgG1-Fc, and incubating on ice for 30min; centrifuging to remove supernatant, adding fluorescent labeled streptavidin SA-PE (purchased from Invitrogen, cat # 21627) and anti-CD3-APC (purchased from Elapscience, cat # FW 2671) or anti-CD19-APC (purchased from Elapscience, cat # FW 1075) flow-type secondary antibody, incubating on ice for 30min, centrifuging to wash cells twice, and performing flow-type detection on CD3 ⁺ T cells and CD19 ⁺ CD73 expression on B cells.

As shown in fig. 3A, the positive rate of CD 73-expressing T cells reached 47%; as shown in fig. 3B, CD 73-expressing B cells were 80% positive.

Example 8 antibody abrogation of inhibition of T cell proliferation by AMP

CD73 on the cell membrane of CD73 positive T cells can breakdown extracellular AMP into adenosine, which mediates immunosuppression by binding to adenosine receptors on T cells, inhibiting T cell proliferation and cytokine secretion. This example examined the ability of anti-CD 73 antibodies to abrogate the inhibition of T cell proliferation by AMPs.

The test method comprises the following steps: extracting peripheral blood of healthy adult, separating with lymph separation solution to obtain fresh PBMC (peripheral blood mononuclear cells), adding CD3/CD28 antibody coupled magnetic beads (TL-601, beijing Hoodia Biotech Co., ltd.) into RPMI-1640 medium (purchased from Gibco) containing 10% FBS, and stimulating to culture PBMC; adding 100IU/ML IL2 to perform amplification culture in the next day, and obtaining CD3 with higher purity after 6 days of amplification culture ⁺ Removing magnetic beads in a culture medium by using a magnetic frame and centrifugally washing the cells twice; t cells were cultured at 2.5X 10 ⁴ cells/well were plated in 96-well cell culture plates, a gradient diluted anti-CD 73 antibody solution or negative control IgG1-Fc (starting at 1ug/ml, 3-fold dilution, 7 concentration gradients) was added, AMP was added to each well to a final concentration of 300 μ M, and the 96-well plates were placed in an incubator for 72h; centrifuging the cell culture plate after cell culture, discarding culture supernatant, and adding

The detection reagent detects the proliferation condition of the T cells.

As shown in FIG. 4, antibody P59-L17 released the inhibition of T cell proliferation by AMP in a concentration gradient-dependent manner.

Example 9 anti-CD 73 antibodies upregulate expression of B cell activated CD69

In addition to blocking adenosine receptor-mediated immunosuppression by inhibiting CD73 enzymatic activity, anti-CD 73 antibodies have the function of upregulating B-cell activated marker expression. This example examines the ability of anti-CD 73 antibodies to upregulate B-cell activated marker CD69 expression.

The test method comprises the following steps: extracting peripheral blood of healthy adult, separating with lymph separation solution to obtain fresh PBMC, culturing PBMC with RPMI-1640 medium containing 10% FBS, adding anti-CD 73 antibody or IgG1-Fc, and culturing overnight; the next dayThe cells were centrifuged to remove the supernatant, and CD19-APC (purchased from Elabscience, cat # FW 1075) and CD69-PE (purchased from Elabscience, cat # E-AB-F1138D) flow-type secondary antibodies were added thereto, and after incubating for 30min on ice, detection was performed, and CD19 was assayed ⁺ Expression of CD69 on B cells was analyzed.

As shown in fig. 5, antibody P59-17 significantly increased the expression of CD69 expressed by B cell activation.

Sequence listing

<110> Baiotai biopharmaceutical GmbH

<120> anti-CD 73 antibody and use thereof

<150> PCT/CN2021/095287

<151> 2021-05-21

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

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Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

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Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

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Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

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Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

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Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

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Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

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Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

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Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

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Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

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Leu Ser Leu Ser Leu Gly Ala

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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp Arg Tyr Ala Tyr Leu Val Asp Gly Trp Phe Asp Tyr Trp

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Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

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Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

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His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

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Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

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His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

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Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

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Ser Pro Gly Lys

450

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asp Thr Tyr

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Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ser Ala Ile Ser Gly Asp Thr Gly Val Thr Gly Tyr Ala Asp Ser Val

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

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195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

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245 250 255

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Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

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Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

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Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

Ala

<210> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ser Tyr Thr Gly Asp

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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180 185 190

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

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

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<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Tyr Asp Phe Ser Phe Tyr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 22

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Thr Ser Asp Phe Phe Pro Asn

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 23

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Thr Trp Ser Gly Trp Pro Asp

85 90 95

Tyr Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 24

<211> 1356

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gaggttcagt tggtagagtc cggcggaggg ttggtgcagc ctggggggtc gcttcgctta 60

agctgcgcgg cgtcagggtt ttcgtttgat acatatgcta tgagctgggt tcgacaggca 120

ccgggtaagg gtctggagtg ggtaagcgcc ataagcggtg acacaggagt gacaggttac 180

gcagatagcg tgaagggacg atttacgatc tctagagaca attcaaaaaa taccctatac 240

ttgcagatga attcgttgcg agcagaggat acggcggtat actattgtgc aagagaccgc 300

tatgcatatt tggttgatgg gtggttcgat tactggggac aagggactct cgttaccgtt 360

tctagcgcat cgacaaaagg accgagtgtt ttcccgctcg cgcccagttc gaagtccaca 420

tctggtggta cagccgcact tggttgtctg gttaaagact acttccccga accagtcact 480

gtcagttgga atagtggtgc actcactagc ggagtccata cctttcctgc agtattgcag 540

tcgtccgggt tgtactccct gtcctcggtt gtaacggttc cgtctagtag tttgggaaca 600

caaacttaca tttgtaatgt caatcacaaa ccaagtaaca caaaggtaga caaaaaggtt 660

gaaccgaagt cctgtgacaa aacgcataca tgccctccgt gccccgctcc tgagcttctc 720

ggcgggccgt ccgtgttcct ttttccaccc aaaccaaaag atacgttaat gatcagccgg 780

acaccagaag taacatgcgt agtggtagac gtgagccacg aagatccgga agtcaagttt 840

aactggtacg tcgacggggt tgaggtgcac aatgctaaga cgaaaccacg tgaagaacag 900

tataactcca cctaccgtgt ggtatcggta ctgacggttt tacatcagga ttggctgaac 960

ggaaaggagt ataaatgcaa agtcagtaac aaagcgcttc cagctcccat tgagaagact 1020

atttccaagg ctaaaggaca gccgcgagag cctcaggtgt atacgctgcc accatcccga 1080

gacgagctta ctaagaatca agttagcctg acatgcctcg tgaaaggttt ttatccaagc 1140

gatatcgcgg ttgagtggga gtcgaacggc cagccagaga ataattacaa aacaacgcct 1200

ccggttcttg attcggatgg ctcgttcttt ctgtattcga aattgactgt tgataaaagt 1260

cgttggcagc aggggaatgt cttcagttgc agcgtaatgc atgaagccct acacaaccac 1320

tacacacaaa agagtttatc gttatctcca ggtaag 1356

<210> 25

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gaggtgcagc tggtggagag cggcggcgga ctggtgcagc ctggaggaag cctgcggctg 60

tcctgcgctg ctagcggctt cagctttgac acctacgcca tgagctgggt gcggcaggcc 120

cctggaaagg gcctggagtg ggtgtccgcc atctccggcg acaccggcgt gaccggatat 180

gctgactccg tgaagggcag gtttaccatc agccgggata actccaagaa caccctgtat 240

ctgcagatga acagcctgag ggccgaggat accgccgtgt attactgcgc tagggacagg 300

tacgcctatc tggtggacgg ctggttcgac tactggggcc agggcaccct ggtgaccgtg 360

agctccgcca gcaccaaggg cccttccgtg tttcccctgg ctccctgctc caggtccacc 420

agcgagtcca ccgccgccct gggatgtctg gtgaaggatt atttccctga gcccgtgacc 480

gtgtcctgga atagcggcgc tctgacctcc ggcgtgcaca ccttccctgc cgtgctgcag 540

tcctccggcc tgtattccct gagctccgtg gtgaccgtgc ccagcagctc cctgggcacc 600

aagacctaca cctgcaacgt ggatcacaag cctagcaaca ccaaggtgga caagagggtg 660

gagagcaagt acggcccccc ctgcccccca tgtcctgctc ctgagttcct gggcggcccc 720

agcgtgttcc tgttcccccc taagcccaag gataccctga tgatcagcag gacccccgag 780

gtgacctgcg tggtggtgga cgtgagccag gaggatcccg aggtgcagtt taactggtac 840

gtggatggcg tggaggtgca caacgccaag accaagcccc gggaggagca gtttaacagc 900

acctacaggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa tggcaaggag 960

tataagtgca aggtgagcaa caagggcctg cccagcagca tcgagaagac catcagcaag 1020

gccaagggcc agccccggga gcctcaggtt tataccctgc ccccttccca ggaggagatg 1080

accaagaacc aggtgtccct gacctgtctg gtgaaaggct tctacccctc cgatatcgcc 1140

gtggagtggg agtccaacgg ccagcccgag aataattaca agaccacccc tcctgtgctg 1200

gattccgatg gctccttctt cctgtactcc cggctgaccg tggataagag caggtggcag 1260

gagggcaacg tgtttagctg cagcgtgatg cacgaggctc tgcacaacca ctacacccag 1320

aagagcctga gcctgtccct gggcgct 1347

<210> 26

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gatatacaaa tgactcagag tccaagttcg cttagtgcaa gcgttggcga tagggtcact 60

atcacctgca gagcctcaca gggcatttct agttacttag cgtggtacca gcagaagccg 120

ggcaaagcac caaagttgct tatctacgca gctagttccc tgcaatctgg agtaccgtca 180

cggtttagcg gtagcggttc tgggacggac ttcacactca cgatctcttc tctacagcct 240

gaagacttcg cgacttatta ttgccaacag gcatcataca caggtgatcc tgttacgttc 300

ggccagggca cgaaagtaga aatcaagcgg acagttgccg ccccttccgt tttcatattt 360

cccccttcag acgagcagct aaaatcgggg actgcctcgg ttgtgtgtct tctaaataat 420

ttttatccca gagaggcaaa ggttcagtgg aaagtggata acgcgctcca gtccggaaac 480

agtcaggaat cagttacaga acaggacagt aaggactcaa catattcgct cagttccacg 540

ttgacattgt cgaaggcaga ttacgagaag cataaagtgt acgcatgcga ggtgacccac 600

caagggctct cctcccctgt gaccaaaagc tttaacaggg gagaatgt 648

<210> 27

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gatattcaga tgacgcaaag cccgtcatct ctcagcgcaa gtgtcggcga ccgcgtcact 60

attacatgtc gtgcttctca gggtataagc tcatatctcg cctggtatca acagaagcct 120

ggcaaagcac cgaagctact aatttatgca gcttcgagtt tacagtctgg cgtccccagt 180

cggttctcag gttccggctc gggtacagat tttactttga cgatttcttc gctccagcca 240

gaagatttcg ccacgtatta ttgcgcccaa tacgacttta gcttctacgg tgtcaccttc 300

ggccaaggaa caaaagtaga aattaagcgc acggtcgccg ccccttcagt atttattttc 360

cccccaagcg atgagcaact aaaaagcggt actgcctctg ttgtatgcct tctcaataat 420

ttctatccta gggaggctaa ggtgcagtgg aaagtagata atgcactcca gtcgggtaac 480

tcccaagaat ctgtcactga gcaggattcc aaagattcta cttattccct ctctagcaca 540

ctgacactga gtaaagcgga ctacgagaaa cacaaggttt atgcctgcga agtcactcat 600

caaggcctgt cgagtcctgt cactaagtct tttaataggg gcgaatgc 648

<210> 28

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gatatccaga tgacccagtc cccgagcagt ctgtccgcga gcgtaggaga ccgcgtgacc 60

attacatgca gggccagtca gggcattagt tcatatctag catggtatca gcaaaaacca 120

ggtaaggccc caaaattgct tatttacgct gcttcaagtc tacaaagcgg agtgccgagc 180

aggttttccg ggtcaggatc gggaacagac ttcaccctca cgataagttc cttgcaacct 240

gaggactttg ccacgtatta ctgccaaaca agcgattttt ttccaaatcc tgtaacgttt 300

ggtcaaggaa ccaaagtaga aatcaaacgg actgtagccg cgccctcagt gttcatcttt 360

cctccgtcag atgagcaact caagtctggg acggcgtcag tcgtgtgttt actaaataac 420

ttctacccaa gagaagcgaa agtgcaatgg aaagttgata atgccctcca gtctggtaat 480

tcccaagagt ccgtcaccga acaagactca aaggactcta catacagtct gtcttctact 540

ctaacgttgt ctaaagctga ctacgaaaag cacaaggtgt atgcatgtga agttacgcat 600

caaggactgt cttctcccgt aacaaagtca ttcaacaggg gcgaatgt 648

<210> 29

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gatattcaaa tgacgcagtc cccgagctct ctgtcggcgt ctgtcggcga ccgtgtaacc 60

atcacttgcc gggcgtctca aggaatcagt tcctacctcg cctggtacca gcaaaaaccg 120

ggtaaagctc ctaagttgct tatttatgcc gcctcttcct tacagtctgg cgtaccctcg 180

cgattctctg gaagcggatc cggcacagac tttactctaa caatcagttc tctgcaaccg 240

gaggacttcg caacctacta ttgtcagacc tggtcgggat ggcctgacta tctcacgttt 300

ggtcaaggaa ctaaggtaga aataaaaagg acagtcgcag caccttcggt gtttatcttt 360

cctccgtcgg acgaacagtt aaagagtgga accgctagcg tggtctgtct tttgaataat 420

ttctacccga gggaagctaa agtccagtgg aaagtggaca acgcactcca atcgggtaat 480

tcacaagagt cagtgaccga acaggactct aaggactcaa catattccct ctcctcgact 540

ttgactttat ctaaagcaga ttatgagaaa cacaaggtgt atgcctgcga ggtgactcat 600

caaggtctct ctagccctgt taccaaaagt tttaacaggg gagagtgt 648

<210> 30

<211> 453

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Ala Ser Leu Ser

20 25 30

Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Met Pro Gly Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Pro Tyr Cys Ser Thr Thr Leu Cys Asn Asp Gly Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

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

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 31

<211> 215

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Ser Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ala Ser Ser Pro

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

Claims

1. An antibody or antigen-binding fragment that specifically binds to CD73, the antibody or antigen-binding fragment comprising an amino acid sequence set forth in SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO:6-9 of one or more of the LCDR3 shown in any one of claims 6.

2. The antibody or antigen-binding fragment of claim 1, comprising an amino acid sequence as set forth in SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3 HCDR3 as shown in fig. 3.

3. The antibody or antigen-binding fragment of claim 1 or 2, comprising an amino acid sequence as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO:6-9 of an LCDR3.

4. An antibody or antigen-binding fragment that specifically binds to CD73, comprising the amino acid sequence set forth in SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, as shown in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO: LCDR3 as shown in 6; or

The antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:1, as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO:7, LCDR3; or

The antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO: LCDR3 shown as 8; or

The antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR1 as shown in SEQ ID NO:2, as shown in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:4, as shown in SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO: LCDR3 shown at 9.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:10, and the amino acid sequence shown in SEQ ID NO:10, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:10, or consists of an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in seq id no; and/or

The light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:11-14, and the amino acid sequence shown in SEQ ID NO:11-14, or a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs: 11-14 with one or more conservative amino acid substitutions as compared to the sequence.

6. An antibody or antigen-binding fragment that specifically binds to CD73, the heavy chain variable region of which comprises the amino acid sequence of SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 11; or

The heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 12; or

The heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:13, and (c) a sequence set forth in (c); or

The heavy chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO:10, the light chain variable region of said antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:14, or a sequence shown in fig. 14.

7. The antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:15 or 16, or a sequence substantially identical to SEQ ID NO:15 or 16, or a sequence having at least 80% identity to a sequence as set forth in SEQ ID NO:15 or 16 with one or more conservative amino acid substitutions; and/or

The light chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID NO:17, or a sequence corresponding to SEQ ID NO:17, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:17, or consists of an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in claim 17.

8. An antibody that specifically binds to CD73, the heavy chain of which comprises an amino acid sequence set forth in SEQ ID NO:18 or 19, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 20; or

The heavy chain of the antibody comprises an amino acid sequence shown as SEQ ID NO:18 or 19, and the light chain of the antibody comprises a sequence with an amino acid sequence shown as SEQ ID NO: 21; or

The heavy chain of the antibody comprises an amino acid sequence shown as SEQ ID NO:18 or 19, and the light chain of the antibody comprises a sequence with an amino acid sequence shown as SEQ ID NO: 22; or

The heavy chain of the antibody comprises an amino acid sequence shown in SEQ ID NO:18 or 19, and the light chain of the antibody comprises a sequence with an amino acid sequence shown as SEQ ID NO:23, or a sequence shown in seq id no.

9. A biomaterial is prepared from

(1) A nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-7 or the antibody of claim 8;

(2) A vector comprising a nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-7 or the antibody of claim 8; or

(3) A host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1-7 or the antibody of claim 8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-7, or the antibody of claim 8; or, further comprises pharmaceutically acceptable auxiliary materials.

11. A diagnostic or prognostic kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 7 or the antibody of claim 8; optionally, the kit further comprises a second antibody that specifically recognizes the anti-CD 73 antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme; optionally, the kit is for detecting the presence or level of CD73 in a sample; optionally, the kit further comprises antibodies or antigen-binding fragments against other antigens, and/or cytotoxic agents, and optionally, instructions for use.

12. A method and use for preventing or treating a disease comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-7, the antibody of claim 8, or the pharmaceutical composition of claim 9, wherein the disease is a tumor, cancer, or infection.