ANTI-CD73 ANTIBODY AND USE THEREOF

Abstract

Provided in the present invention are an anti-CD73 antibody and the use thereof. Specifically, a heavy chain variable region of the antibody contains HCDR1 to HCDR3 having amino acid sequences as shown in SEQ ID NOs: 15-17, respectively. Furthermore, a light chain variable region of the antibody contains LCDR1 to LCDR3 having amino acid sequences as shown in SEQ ID NOs: 18-20, respectively.

Claims

1. An anti-CD73 (for example, human CD73) antibody or an antigen-binding fragment thereof, wherein the anti-CD73 antibody comprises: HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10; and LCDR1, LCDR2 and LCDR3 contained in a light chain variable region set forth in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14; preferably, according to an IMGT numbering system, the anti-CD73 antibody comprises: HCDR1, comprising an amino acid sequence set forth in SEQ ID NO: 15, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, HCDR2, comprising an amino acid sequence set forth in SEQ ID NO: 16, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, HCDR3, comprising an amino acid sequence set forth in SEQ ID NO: 17, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, LCDR1, comprising an amino acid sequence set forth in SEQ ID NO: 18, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, LCDR2, comprising an amino acid sequence set forth in SEQ ID NO: 19, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence or an amino acid, or a sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, and LCDR3, comprising an amino acid sequence set forth in SEQ ID NO: 20, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence.

2. The anti-CD73 antibody or the antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region of the antibody comprises the following sequences: SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10; and the light chain variable region of the antibody comprises the following sequences: SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14.

3. The anti-CD73 antibody or the antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 4; the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 8; the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 12; or the heavy chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region of the antibody comprises an amino acid sequence set forth in SEQ ID NO: 14.

4. The anti-CD73 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a heavy chain constant region having a sequence set forth in SEQ ID NO: 21, and a light chain constant region having a sequence set forth in SEQ ID NO: 22; or the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.

5. The anti-CD73 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody (preferably a heavy chain amino acid sequence set forth in SEQ ID NO: 35 and a light chain amino acid sequence set forth in SEQ ID NO: 36), a humanized antibody, a chimeric antibody and a multispecific antibody (e.g., a bispecific antibody).

6. The anti-CD73 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from Fab, Fab′, F(ab′).sub.2, Fd, Fv, dAb, Fab/c, a complementarity determining region fragment, a single chain antibody (e.g., scFv), a humanized antibody, a chimeric antibody and a bispecific antibody.

7. An isolated polypeptide, selected from the group consisting of: (1) an isolated polypeptide, comprising sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, wherein the polypeptide specifically binds to CD73 as a part of an anti-CD73 antibody, and the antibody further comprises sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20; (2) an isolated polypeptide, comprising sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, wherein the polypeptide specifically binds to CD73 as a part of an anti-CD73 antibody, and the antibody further comprises sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17; (3) an isolated polypeptide, comprising a sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to CD73 as a part of an anti-CD73 antibody, and the antibody further correspondingly comprises a sequence set forth in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; (4) an isolated polypeptide, comprising a sequence set forth in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to CD73 as a part of an anti-CD73 antibody, and the antibody further correspondingly comprises a sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; (5) an isolated polypeptide, comprising a sequence set forth in SEQ ID NO: 10, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to CD73 as a part of an anti-CD73 antibody, and the antibody further correspondingly comprises a sequence set forth in SEQ ID NO: 14, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence; and (6) an isolated polypeptide, comprising the a sequence set forth in SEQ ID NO: 14, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, wherein the polypeptide specifically binds to CD73 as a part of an anti-CD73 antibody, and the antibody further correspondingly comprises a sequence set forth in SEQ ID NO: 10, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence.

8. A nucleic acid molecule, encoding the antibody or the antigen-binding fragment thereof according to claim 1.

9. A nucleic acid molecule, encoding the isolated polypeptide according to claim 7.

10. (canceled)

11. The antibody or the antigen-binding fragment thereof according to claim 1, contained in a conjugate, a fusion protein, a multispecific antibody (preferably a bispecific antibody), or a pharmaceutical composition, wherein the conjugate further comprises a conjugated moiety, the conjugated moiety is a purification tag (for example, a His tag), a detectable label; preferably, the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme, or wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient, preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

12. (canceled)

13. A kit, comprising the antibody or the antigen-binding fragment thereof according to claim 1, wherein, preferably, the kit further comprises a secondary antibody specifically recognizing the antibody, optionally, the secondary antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; and preferably, the kit is used for detecting the presence or level of CD73 in a sample.

14. (canceled)

15. The antibody or the antigen-binding fragment thereof of claim 1 for treating and/or preventing or diagnosing tumors (such as solid tumors, preferably non-small cell lung cancer, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), triple-negative breast cancer, ovarian cancer, colorectal cancer (including colorectal cancer microsatellite stability (MSS)), gastric cancer, melanoma, head and neck cancer, renal cell carcinoma or pancreatic ductal adenocarcinoma).

16. A hybridoma cell line, selected from: LT014, deposited at China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: C2018137.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1. Detection results of affinity constants of 19F3H2L3 for HNT5E(1-552)-his. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM, respectively.

[0072] FIG. 2. Detection results of affinity constants of 19F3H2L2 for HNT5E(1-552)-his. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM, respectively.

[0073] FIG. 3. Detection results of affinity constants of MEDI9447 for HNT5E(1-552)-his. The antibody concentrations for the curve pairs from top to bottom are 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM, respectively.

[0074] FIG. 4. Binding activity of 19F3H2L3 to CD73 on the surface of MDA-MB-231 cells determined by FACS.

[0075] FIG. 5. Detection results of enzyme activity of anti-CD73 antibody added to MDA-MB-231 cells.

[0076] FIG. 6. Detection results of enzyme activity of anti-CD73 antibody added to U87-MG cells.

DETAILED DESCRIPTION

[0077] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be construed as limitations on the scope of the present invention. In the cases where the techniques or conditions are not specified, the examples were implemented according to the techniques or conditions described in the literature in the art (e.g., see, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Peitang Huang et al., 3rd Edition, Science Press) or according to the product manual. Reagents or instruments used are commercially available conventional products if the manufacturers thereof are not specified. For example, MDA-MB-231 cells and U87-MG cells can be purchased from ATCC.

[0078] In the following examples of the present invention, BALB/c mice used were purchased from Guangdong Medical Experimental Animal Center.

[0079] In the following examples of the present invention, the positive control antibody MEDI9447 (oleclumab) used was produced by Akeso Biopharma Co. Ltd., the sequence of which is as same as the antibody SEQ ID NOs: 21-24 described in the Medlmmune Limited published patent with the publication number: US20160129108A1.

[0080] In the following examples of the present invention, AD2 used was purchased from Biolegend (Cat. No. 344002).

Example 1. Preparation of Anti-CD73 Antibody 19F3

[0081] 1. Preparation of Hybridoma Cell Line LT014

[0082] The antigen used to prepare the anti-CD73 antibody was human NT5E-His (for NT5E, GenbankID: NP_002517.1, position: 1-552). Spleen cells of immunized mice were taken to fuse with myeloma cells of the mice to prepare hybridoma cells. Hybridoma cells were screened by indirect ELISA using human NT5E-Biotin (for NT5E, GenbankID: NP 002517.1, position: 1-552) as an antigen, and hybridoma cells capable of secreting an antibody specifically binding to CD73 were obtained. The hybridoma cells obtained by screening were subjected to limiting dilution to obtain a stable hybridoma cell line. The hybridoma cell line was named as hybridoma cell line LT014, and the monoclonal antibody secreted therefrom was named as 19F3.

[0083] The hybridoma cell line LT014 (also called CD73-19F3) was deposited at China Center for Type Culture Collection (CCTCC) on Jun. 21, 2018 with a collection number of CCTCC NO: C2018137 and a collection address of Wuhan University, Wuhan, China, postal code: 430072.

[0084] 2. Preparation of Anti-CD73 Antibody 19F3

[0085] The LT014 cell line prepared above was cultured with a chemical defined medium (CD medium, containing 1% Penicillin-Streptomycin) in a 5% CO2 cell incubator at 37° C. After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation and vacuum filtration through a microfiltration membrane, and purified by using a HiTrap protein A HP column to obtain an antibody 19F3.

Example 2. Sequence Analysis of Anti-CD73 Antibody 19F3

[0086] mRNA was extracted from the cell line LT014 cultured in Example 1 according to the method described in the manual of RNAprep pure Cell/Bacteria Kit (Tiangen, Cat. No. DP430).

[0087] cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR and amplified by PCR.

[0088] The PCR-amplified products were directly subjected to TA cloning according to the manual of the pEASY-T1 Cloning Kit (Transgen CT101).

[0089] The product obtained by performing TA cloning of the antibody 19F3 against CD73 on the LT014 cell line was directly sequenced. The sequencing results are as follows.

[0090] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO: 1 with a length of 363 bp.

[0091] The encoded amino acid sequence is set forth in SEQ ID NO: 2 with a length of 121 aa;

[0092] wherein the sequence of the heavy chain CDR1 is set forth in SEQ ID NO: 15, the sequence of the heavy chain CDR2 is set forth in SEQ ID NO: 16, and the sequence of the heavy chain CDR3 is set forth in SEQ ID NO: 17.

[0093] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO: 3 with a length of 339 bp.

[0094] The encoded amino acid sequence is set forth in SEQ ID NO: 4 with a length of 113 aa;

[0095] wherein the sequence of the light chain CDR1 is set forth in SEQ ID NO: 18, the sequence of the light chain CDR2 is set forth in SEQ ID NO: 19, and the sequence of the light chain CDR3 is set forth in SEQ ID NO: 20.

Example 3. Design and Preparation of Light and Heavy Chains of Humanized Anti-Human CD73 Antibodies

[0096] 1. Design of Light and Heavy Chains of Humanized Anti-Human CD73 Antibodies 19F3H1L1, 19F3H2L2 and 19F3H2L3

[0097] Based on the three-dimensional crystal structure of human CD73 protein (Hage T, Reinemer P, Sebald W., Crystals of a 1:1 Complex Between Human Interleukin-4 and the Extracellular Domain of Its Receptor Alpha Chain, Eur. J. Biochem., 1998; 258(2):831-6.) and the sequence of antibody 19F3 obtained in Example 2, the variable region sequences of antibodies 19F3H1L1, 19F3H2L2 and 19F3H2L3 were obtained by computer modeling and mutation design (antibody constant region sequences from NCBI database: the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834).

[0098] The designed variable region sequences are as follows:

[0099] (1) Sequences of heavy and light chain variable regions of humanized monoclonal antibody 19F3H1L1

[0100] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO: 5 with a length of 363 bp.

[0101] The encoded amino acid sequence is set forth in SEQ ID NO: 6 with a length of 121 aa, wherein the sequence of the heavy chain CDR1 is set forth in SEQ ID NO: 15, the sequence of the heavy chain CDR2 is set forth in SEQ ID NO: 16, and the sequence of the heavy chain CDR3 is set forth in SEQ ID NO: 17.

[0102] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO: 7 with a length of 339 bp.

[0103] The encoded amino acid sequence is set forth in SEQ ID NO: 8 with a length of 113 aa, wherein the sequence of the light chain CDR1 is set forth in SEQ ID NO: 18, the sequence of the light chain CDR2 is set forth in SEQ ID NO: 19, and the sequence of the light chain CDR3 is set forth in SEQ ID NO: 20.

[0104] (2) Sequences of heavy and light chain variable regions of humanized monoclonal antibody 19F3H2L2

[0105] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 with a length of 363 bp.

[0106] The encoded amino acid sequence is set forth in SEQ ID NO: 10 with a length of 121 aa, wherein the sequence of the heavy chain CDR1 is set forth in SEQ ID NO: 15, the sequence of the heavy chain CDR2 is set forth in SEQ ID NO: 16, and the sequence of the heavy chain CDR3 is set forth in SEQ ID NO: 17.

[0107] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO: 11 with a length of 339 bp.

[0108] The encoded amino acid sequence is set forth in SEQ ID NO: 12 with a length of 113 aa, wherein the sequence of the light chain CDR1 is set forth in SEQ ID NO: 18, the sequence of the light chain CDR2 is set forth in SEQ ID NO: 19, and the sequence of the light chain CDR3 is set forth in SEQ ID NO: 20.

[0109] (3) Sequences of heavy and light chain variable regions of humanized monoclonal antibody 19F3H2L3

[0110] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 with a length of 363 bp.

[0111] The encoded amino acid sequence is set forth in SEQ ID NO: 10 with a length of 121 aa, wherein the sequence of the heavy chain CDR1 is set forth in SEQ ID NO: 15, the sequence of the heavy chain CDR2 is set forth in SEQ ID NO: 16, and the sequence of the heavy chain CDR3 is set forth in SEQ ID NO: 17.

[0112] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO: 13 with a length of 339 bp.

[0113] The encoded amino acid sequence is set forth in SEQ ID NO: 14 with a length of 113 aa, wherein the sequence of the light chain CDR1 is set forth in SEQ ID NO: 18, the sequence of the light chain CDR2 is set forth in SEQ ID NO: 19, and the sequence of the light chain CDR3 is set forth in SEQ ID NO: 20.

[0114] 2. Preparation of Humanized Antibodies 19F3H1L1, 19F3H2L2 and 19F3H2L3

[0115] The heavy chain constant regions were all Ig gamma-1 chain C region, ACCESSION: P01857; and the light chain constant regions were all Ig kappa chain C region, ACCESSION: P01834.

[0116] The heavy chain cDNA and light chain cDNA of 19F3H1L1, the heavy chain cDNA and light chain cDNA of 19F3H2L2 and the heavy chain cDNA and light chain cDNA of 19F3H2L3 were respectively cloned into pUC57simple (provided by GenScript) vectors to obtain pUC57simple-19F3H1, pUC57simple-19F3L1, pUC57simple-19F3H2, pUC57simple-19F3L2 and pUC57simple-19F3L3, respectively. Referring to the standard technique introduced in Molecular Cloning Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into an expression vector pcDNA3.1 by an restriction enzyme (EcoRI&HindIII) through digestion to obtain expression plasmids pcDNA3.1-19F3H1, pcDNA3.1-19F3L1, pcDNA3.1-19F3H2, pcDNA3.1-19F3L2 and pcDNA3.1-19F3L3, and the heavy/light chain genes of the recombinant expression plasmids were further subjected to sequencing analysis. Then the designed gene combinations comprising corresponding light and heavy chain recombinant plasmids (pcDNA3.1-19F3H1/pcDNA3.1-19F3L1, pcDNA3.1-19F3H2/pcDNA3.1-19F3L2 and pcDNA3.1-19F3H2/pcDNA3.1-19F3L3) were respectively co-transfected into 293F cells, and the culture solutions were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression. The culture solutions were collected after 7 days, and subjected to affinity purification on a Protein A column to obtain humanized antibodies.

[0117] 3. Preparation of Humanized Antibodies 19F3H1L1(hG1DM), 19F3H2L2(hG1DM) and 19F3H2L3(hG1DM)

[0118] The light chain constant regions of the antibodies 19F3H1L1(hG1DM), 19F3H2L2(hG1DM) and 19F3H2L3(hG1DM) are the Ig kappa chain C region, ACCESSION: P01834, see SEQ ID NO: 22.

[0119] On the basis of Ig gamma-1 chain C region, ACCESSION: P01857, humanized antibodies were obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A) and a leucine-to-alanine point mutation at position 235 (L235A) in the heavy chain constant region, see SEQ ID NO: 21, and were designated as 19F3H1L1(hG1DM), 19F3H2L2(hG1DM) and 19F3H2L3(hG1DM), respectively.

[0120] The heavy chain cDNA and light chain cDNA of 19F3H1L1(hG1DM), the heavy chain cDNA and light chain cDNA of 19F3H2L2(hG1DM) and the heavy chain cDNA and light chain cDNA of 19F3H2L3(hG1DM) were respectively cloned into pUC57simple (provided by Genscript) vectors to obtain pUC57simple-19F3H1(hG1DM), pUC57simple-19F3L1, pUC57simple-19F3H2(hG1DM), pUC57simple-19F3L2 and pUC57simple-19F3L3, respectively. Referring to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into an expression vector pcDNA3.1 by an restriction enzyme (EcoRI&HindIII) through digestion to obtain expression plasmids pcDNA3.1-19F3H1(hG1DM), pcDNA3.1-19F3L1, pcDNA3.1-19F3H2(hG1DM), pcDNA3.1-19F3L2 an pcDNA3.1-19F3L3, and the heavy/light chain genes of the recombinant expression plasmids were further subjected to sequencing analysis. Then the designed gene combinations comprising corresponding light and heavy chain recombinant plasmids (pcDNA3.1-19F3H1(hG1DM)/pcDNA3.1-19F3L1, pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L2 and pcDNA3.1-19F3H2(hG1DM)/pcDNA3.1-19F3L3) were respectively co-transfected into 293F cells, and the culture solutions were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression. The culture solutions were collected after 7 days, and subjected to affinity purification on a Protein A column to obtain humanized antibodies.

Example 4. Determination of Binding Activity of Anti-CD73 Antibody to Antigen Human NT5E-Biotin by ELISA

[0121] Experimental steps: a microplate was coated with streptavidin at 2 μg/mL, and then the microplate was incubated at 4° C. for 12 hours. The antigen-coated microplate was rinsed for once with PBST after incubation, and then blocked with a PBST solution containing 1% BSA as a microplate blocking solution for 2 hours. After blocking, the microplate was washed 3 times with PBST. Then, 0.5 μg/mL antigen human NT5E-Biotin was added, and the plate was washed 3 times with PBST after incubated at 37° C. for 30 minutes. The antibody serially diluted with the PBST solution was added to the wells of the microplate. The antibody dilution gradients are shown in Table 1. The microplate containing the test antibodies was incubated at 37° C. for 30 minutes, and then washed 3 times with PBST. After the plate was washed, the secondary antibody working solution of HRP-labeled goat anti-human IgG (H+L) (Jackson, Cat. No. 109-035-088) diluted in a ratio of 1:5000 or HRP-labeled goat anti-mouse IgG (H+L) (Jackson, Cat. No. 115-035-062) diluted in a ratio of 1:5000 was added, and then the plate was incubated at 37° C. for 30 minutes. After incubation, the plate was washed 4 times with PBST, TMB (Neogen, 308177) was added in the dark for color development for 5 min, and then a stop solution was added to terminate chromogenic reaction. The microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at the light wavelength of 450 nm. The data were analyzed by SoftMax Pro 6.2.1.

[0122] OD values of doses for detecting the binding of anti-CD73 antibody to the antigen human NT5E-Biotin are shown in Table 1. The binding EC.sub.50 of the antibody was calculated by curve fitting using antibody concentration as the abscissa and absorbance value as the ordinate, and the results are shown in Table 1 below.

[0123] Experimental results show that the antibodies 19F3 H1L1, 19F3 H2L2 and 19F3 H2L3 and the murine antibody 19F3 can effectively bind to human NTSE-Biotin, and the binding efficiency is in a dose-dependent relationship. Under the basically same experimental conditions, EC.sub.50 of 19F3 H1L1 binding to human NTSE-Biotin was 0.049 nM, EC.sub.50 of 19F3 H2L2 binding to human NTSE-Biotin was 0.064 nM, EC.sub.50 of 19F3 H2L3 binding to human NTSE-Biotin was 0.061 nM, EC.sub.50 of the positive drug MEDI9447 for the same target binding to human NTSE-Biotin was 0.048 nM, and EC.sub.50 of murine antibody 19F3 binding to human NTSE-Biotin was 0.018 nM.

[0124] The above experimental results show that the binding activity of 19F3 H1L1, 19F3 H2L2, 19F3 H2L3 and murine antibody 19F3 to human NTSE-Biotin respectively is comparable to that of the positive drug MEDI9447 for the same target under the same experimental conditions, indicating that 19F3 H1L1, 19F3 H2L2 and 19F3 H2L3 have the function of effectively binding to CD73.

TABLE-US-00003 TABLE 1 Detection results of binding activity of 19F3H1L1, 19F3H2L2, 19F3H2L3 and murine antibody 19F3 to HNT5E-Biotin Antibody Antigen coating: SA (2 μg/mL) dilution Human NT5E-Biotin (0.5 μg/mL) (μg/mL) 19F3 H1L1 19F3 H2L2 19F3 H2L3 MEDI9447 19F3 0.333 2.648 2.640 2.598 2.688 2.623 2.588 2.548 2.527 2.706 2.743 1:3  2.601 2.697 2.578 2.618 2.581 2.582 2.573 2.604 2.736 2.763 1:9  2.407 2.332 2.163 2.330 2.186 2.257 2.268 2.284 2.566 2.641 1:27  1.821 1.820 1.579 1.680 1.626 1.649 1.774 1.742 2.330 2.361 1:81  1.044 1.035 0.870 0.933 0.918 0.931 1.058 1.030 1.693 1.769 1:243 0.525 0.516 0.434 0.454 0.450 0.457 0.536 0.528 1.001 1.000 1:729 0.260 0.273 0.239 0.247 0.241 0.252 0.272 0.275 0.446 0.470 0    0.125 0.123 0.119 0.123 0.120 0.123 0.121 0.116 0.060 0.062 HRP-labeled goat anti-mouse Secondary IgG (H + L) antibody HRP-labeled goat anti-human IgG (H + L) (1:5000) (1:5000) EC.sub.50 (nM) 0.049 0.064 0.061 0.048 0.018

Example 5. Kinetic Parameters for Binding of Humanized Antibodies 19F3H2L3, 19F3H2L2 and MEDI9447 to the Antigen Human HNT5E (1-552)-his Determined Using Fortebio Molecular Interaction Instrument

[0125] The sample dilution buffer was PBST, pH 7.4. 5 μg/mL antibody was immobilized at on the Protein A sensor for 15 s. The sensor was equilibrated in the buffer for 120 s. The binding of the immobilized antibody on the sensor to the antigen human NT5E(1-552)-his at concentrations of 3.125-200 nM (two-fold dilution) was determined for 120 s. The antigen-antibody was dissociated in the buffer for 600 s. The sensor was refreshed with 10 mM glycine solution (pH 1.5). The detection temperature was 37° C., the detection frequency was 0.6 Hz, and the sample plate shaking rate was 1000 rpm. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0126] The results of measuring the affinity constants of humanized antibodies 19F3H2L3, 19F3H2L2 and MEDI9447 (as control antibodies) and human CD73 are shown in Table 2, and the detection results are shown in FIGS. 1-3.

[0127] As shown in Table 2 and FIGS. 1-3, the affinity constants of humanized antibodies 19F3H2L3, 19F3H2L2 and MEDI9447 and human CD73 were 1.04E-10 M, 2.36E-10 M, 2.59E-10 M and 1.04E-10 M sequentially.

[0128] The above experimental results show that the binding ability of 19F3H2L3 and 19F3H2L2 to HNT5E(1-552)-his is comparable, indicating that the humanized antibodies 19F3H2L3 and 19F3H2L2 have strong binding capacity to human CD73.

TABLE-US-00004 TABLE 2 Determination of affinity constants of 19F3H2L3, 19F3H2L2 and HNT5E(1-552)-his Test KD kon S E kdis S E Rmax antibodies (M) (1/Ms) (kon) (1/s) (kdis) (nm) 19F3 H2L3 2.36E−10 4.82E+05 7.74E+03 1.14E−04 5.87E−06 0.34-0.41 19F3 H2L2 2.59E−10 4.80E+05 6.44E+03 1.24E−04 4.86E−06 0.35-0.39 MEDI9447 1.04E−10 2.34E+05 3.20E+03 2.44E−05 5.02E−06 0.59-0.82 K.sub.D is the affinity constant; K.sub.D = kdis/kon

Example 6. Binding Ability of 19F3H2L3 Antibody Determined by FACS

[0129] MDA-MB-231 cells in logarithmic phase were digested with conventional trypsin, centrifuged, and resuspended in PBSA. The cells were filled into 1.5 mL centrifuge tubes (0.3 million cells in each tube) and centrifuged at 5600 rpm for 5 min to remove the supernatant. 100 μL of CD73 antibodies diluted by PBSA with the final concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.05 nM were added, respectively. The system was mixed gently and uniformly, and then was incubated on ice for 1 h. Then 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. The 500-fold diluted FITC labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-095-098) was added to resuspend cell precipitates, and the mixture was incubated on ice in the dark for 0.5 h. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 min to remove the supernatant. At last, 200 μL of PBSA was added to resuspend cell precipitates, and the mixture was transferred to a flow tube for detection by a flow cytometer (BD FACSCalibur™).

[0130] Results are shown in Table 3 and FIG. 4, the binding ability of 19F3H2L3 to CD73 endogenously expressed in MDA-MB-231 is comparable to that of the positive drug MEDI9447 for the same target, and the binding is in a dose-dependent relationship. Under the same experimental conditions, EC.sub.50 of 19F3H2L3 binding to CD73 endogenously expressed in MDA-MB-231 is 3.423 nM, and EC.sub.50 of MEDI9447 binding to CD73 endogenously expressed in MDA-MB-231 is 1.433 nM.

[0131] The above experimental results show that the binding activity of 19F3H2L3 to CD73 endogenously expressed in MDA-MB-231 is comparable to that of the positive drug MEDI9447 for the same target under the same experimental conditions, indicating that 19F3H2L3 has the function of effectively binding to CD73.

TABLE-US-00005 TABLE 3 Binding ability of 19F3H2L3 to CD73 on the surface of MDA-MB-231 cells determined by FACS Absorbance (nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC.sub.50 MEDI9447 28.38 43.18 90.96 266.72 454.45 518.94 545.63 580.76 1.433 19F3H2L3 20.04 31.49 61.09 135.31 264.86 407.09 485.32 480.42 3.423

Example 7. Determination of Inhibitory Activity of Anti-CD73 Antibody on Enzyme Activity of CD73 Endogenously Expressed in Cells

[0132] 1. Detection of Inhibitory Activity of Anti-CD73 Antibody on Enzyme Activity of CD73 Endogenously Expressed in MDA-MB-231 Cells

[0133] The experimental procedures were as follows. MDA-MB-231 cells in logarithmic phase in good condition were taken, resuspended in a serum-free RPMI-1640 culture solution, and then counted. The MDA-MB-231 cells were seeded into a 96-well plate at 3><10.sup.4 cells/100 μL/well. The antibody was diluted with the serum-free RPMI-1640 culture solution at an initial concentration of 200 μg/mL (serial 2.5-fold dilution). The antibody was added to the 96-well plate at 50 μL/well, and the plate was incubated at 37° C. for 1 hour. After 1 hour, 50 μL of RPMI-1640-diluted 600 μM AMP was added to each well. After 3 hours, 25 μL of cell culture supernatant was taken and transferred to a new 96-well plate, and 25 μL of 100 μM ATP was added to each well. 50 μL of CTG (CellTiter-Glo® One Solution Assay, promega, Cat. No. G8461) color developing solution was added to each well for color development, and data were read by a multi-label microplate tester (PerkinElmer 2140-0020).

[0134] Experimental results: as shown in FIG. 5, both 19F3H2L3 and the positive control drug MEDI9447 for the same target showed dose-dependent inhibition of the activity of CD73 endogenously expressed in MDA-MB-231 converting AMP into adenosine A through enzyme catalysis, thereby reducing the produced mean fluorescence intensity RLU produced in a dose-dependent manner.

[0135] The above experimental results show that the added AMP can be subjected to enzyme catalysis of CD73 endogenously expressed on the cell surface by MDA-MB-231 and then converted into adenosine A under the condition of no CD73 antibody treatment, so that the inhibition of luciferase activity is relieved. However, after addition of the antibody, as CD73 was bound by the antibody, its enzymatic activity was reduced, so that AMP could not be converted into adenosine. It is suggested that the anti-CD73 antibody effectively inhibits the enzyme activity reaction of CD73 in a non-substrate competition mode and reduces the production of adenosine.

[0136] 2. Determination of Enzyme Activity by Addition of Anti-CD73 Antibody to U87-MG Cells

[0137] The experimental procedures were as follows. U87-MG cells in logarithmic phase in good condition were taken, resuspended in a serum-free RPMI-1640 culture solution, and then counted. The U87-MG cells were seeded into a 96-well plate at 3×10.sup.4 cells/100 μL/well. The antibody was diluted with the serum-free RPMI-1640 culture solution at an initial concentration of 200 μg/mL (serial 2.5-fold dilution). The antibody was added to the 96-well plate at 50 μL/well, and the plate was incubated at 37° C. for 1 hour. After 1 hour, 50 μL of RPMI-1640-diluted 600 μM AMP was added to each well. After 3 hours, 25 μL of cell culture supernatant was taken and transferred to a new 96-well plate, and 25 μL of 100 μM ATP was added to each well. 50 μL of CTG (CellTiter-Glo® One Solution Assay, promega, Cat. No. G8461) color developing solution was added to each well for color development, and data were read by a multi-label microplate tester (PerkinElmer 2140-0020).

[0138] Experimental results: as shown in FIG. 6, both 19F3H2L3 and the positive control drug MEDI9447 for the same target showed dose-dependent inhibition of the activity of CD73 endogenously expressed in MDA-MB-231 converting AMP into adenosine through enzyme catalysis, thereby reducing the produced mean fluorescence intensity RLU produced in a dose-dependent manner.

[0139] The above experimental results show that the added AMP can be subjected to enzyme catalysis of CD73 endogenously expressed on the cell surface by U87-MG and then converted into adenosine under the condition of no CD73 antibody treatment, so that the inhibition of luciferase activity is relieved. However, after addition of the antibody, as CD73 was bound by the antibody, its enzymatic activity was reduced, so that AMP could not be converted into adenosine. It is suggested that the anti-CD73 antibody effectively inhibits the enzyme activity reaction of CD73 in a non-substrate competition mode and reduces the production of adenosine.

Example 8. Dynamic Affinity Determination of Anti-CD73 Antibodies and FcγRIIIa Mediating Antibody-Dependent Cytotoxic Effects

[0140] The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 0.5 μg/mL FcγRIIIa (from Sino Biological, also known as CD16a) was immobilized on the SA sensor for 120 s. The sensor was equilibrated in a buffer for 60 s, and the binding of the immobilized CD16a on the sensor to the antibodies at concentrations of 31.3-500 nM (serial two-fold dilution) was determined for 60 s. The antibody-antigen was dissociated in buffer for 60 s. The sensor was refreshed with 10 mM NaOH. The detection temperature was 30° C. and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants. As shown in Table 4, 19F3H2L3(hG1DM) did not bind to FcγRIIIa, whereas MEDI9447 had binding activity to FcγRIIIa, indicating that 19F3H2L3(hG1DM) had a lower risk of causing antibody-dependent cytotoxic effects in CD73-expressing cells, whereas MEDI9447 had a risk of causing antibody-dependent cytotoxic effects.

[0141] CD73 is abundantly expressed in vascular endothelium and normal tissues, and binds to FcγRIIIa to mediate antibody-dependent cytotoxic effects, resulting in damage to vascular endothelial cells and normal tissues, which has significant effect on the safety of antibody drugs.

TABLE-US-00006 TABLE 4 Dynamic affinity determination of anti-CD73 antibodies and FcγRIIIa mediating antibody-dependent cytotoxic effects Standard Standard KD kon error Kdis error (M) (1/Ms) (kon) (1/s) (kdis) 19F3H2L3(hG1DM) N/A N/A N/A N/A N/A MEDI9447 1.41E−07 2.43E+05 4.36E+04 3.42E−02 2.08E−03 Wild-type IgG1 antibody 1.25E−07 1.76E+05 2.22E+04 2.20E−02 1.11E−03 KD = kdis/kon

SEQUENCE LISTING

[0142]

TABLE-US-00007 The nucleotide sequence of the heavy chain variable region of 19F3 with CDR sequences underlined: (SEQ ID NO: 1) GAGGTGCAGCTGCAGCAGTCCGGACCAGAGCTGGTGAAGCCTGGCGCC TCCATGCGGATGTCTTGTAAGGCCTCTGGCTACAGCTTCACCGGCTATA CAATGAACTGGGTGAAGCAGTCTCACGGCAAGAATCTGGAGTGGATCG GCCTGATCAACCCTTACAATGCCGGCACCAGCTATAACCAGAAGTTTA AGGGCAAGGCCACCCTGACAGTGGACAAGAGCTCCTCTACCGCCTACA TGGAGCTGCTGTCCCTGACATCTGAGGATAGCGCCGTGTACTATTGCGC CCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCA GGGCACCACACTGACAGTGAGCTCC The amino acid sequence of the heavy chain variable region of 19F3 with CDR sequences underlined: (SEQ ID NO: 2) EVQLQQSGPELVKPGASMRMSCKASGYSFTGYTMNWVKQSHGKNLEWI GLINPYNAGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSEYRYGGDYFDYWGQGTTLTVSS The nucleotide sequence of the light chain variable region of 19F3 with CDR sequences underlined: (SEQ ID NO: 3) GACATCGTGATGACCCAGTCTCCAAGCTCCCTGGCAATGAGCGTGGGA CAGAAGGTGACAATGTCTTGTAAGTCTAGCCAGAGCCTGCTGAACTCCT CTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCAGGCCAGT CTCCCAAGCTGCTGGTGTACTTTGCCAGCACCAGGGAGTCCGGAGTGCC TGACAGATTCATCGGCTCCGGCTCTGGCACAGACTTCACCCTGACAATC AGCTCCGTGCAGGCAGAGGACCTGGCAGATTATTTCTGCCAGCAGCAC TACGACACCCCTTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG The amino acid sequence of the light chain variable region of 19F3 with CDR sequences underlined: (SEQ ID NO: 4) DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSP KLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYDT PYTFGGGTKLEIK The nucleotide sequence of the heavy chain variable region of 19F3H1L1 with CDR sequences underlined: (SEQ ID NO: 5) CAGGTGCAGCTGCAGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCC TCTATGAAGATGAGCTGTAAGGCCAGCGGCTACTCCTTCACCGGCTATA CAATGAACTGGGTGAAGCAGGCCCACGGCCAGAATCTGGAGTGGATCG GCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTCA GGGCAAGGCCACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACAT GGAGCTGAGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGC CCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCA GGGCACCACACTGACCGTGTCTAGC The amino acid sequence of the heavy chain variable region of 19F3H1L1 with CDR sequences underlined: (SEQ ID NO: 6) QVQLQQSGAEVVKPGASMKMSCKASGYSFTGYTMNWVKQAHGQNLEWI GLINPYNAGTSYNQKFQGKATLTVDKSTSTAYMELSSLRSEDTAVYYC ARSEYRYGGDYFDYWGQGTTLTVSS The nucleotide sequence of the light chain variable region of 19F3H1L1 with CDR sequences underlined: (SEQ ID NO: 7) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCAATGTCTGTGGGA GAGAGGGTGACAATGTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCT CTAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGG CCCCTAAGCTGCTGGTGTACTTTGCCTCTACCAGGGAGAGCGGAGTGCC AGACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAAT CAGCTCCGTGCAGGCAGAGGACCTGGCAGATTATTTCTGCCAGCAGCA CTACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAA G The amino acid sequence of the light  chain variable region of 19F3H1L1 with CDR sequences underlined: (SEQ ID NO: 8) DIVMTQSPSSLAMSVGERVTMSCKSSQSLLNSSNOKNYLAWYOOKPG QAPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVOAEDLADYFC QQHYDTPYTFGGGTKLEIK The nucleotide sequence of the heavy chain variable regions of 19F3H2L2 and 19F3H2L3 with CDR sequences underlined: (SEQ ID NO: 9) CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGGTGAAGCCAGGAGCC TCTGTGAAGGTGAGCTGTAAGGCCAGCGGCTACTCCTTCACCGGCTATA CAATGAACTGGGTGAGGCAGGCACCAGGACAGAATCTGGAGTGGATCG GCCTGATCAACCCTTACAATGCCGGCACCTCTTATAACCAGAAGTTTCA GGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTACAT GGAGCTGAGCTCCCTGCGGAGCGAGGATACAGCCGTGTACTATTGCGC CCGGTCCGAGTACAGATATGGCGGCGACTACTTTGATTATTGGGGCCA GGGCACCACACTGACCGTGTCTAGC The amino acid sequence of the heavy chain variable regions of 19F3H2L2 and 19F3H2L3 with CDR sequences underlined: (SEQ ID NO: 10) QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWI GLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC ARSEYRYGGDYFDYWGQGTTLTVSS The nucleotide sequence of the light chain variable region of 19F3H2L2 with CDR sequences underlined: (SEQ ID NO: 11) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAG AGCGGGTGACAATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTC TAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGC CCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCA GACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATC AGCTCCGTGCAGGCAGAGGACGTGGCAGATTACTATTGCCAGCAGCAC TACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG The amino acid sequence of the light chain variable region of 19F3H2L2 with CDR sequences underlined: (SEQ ID NO: 12) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQ APKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYC QQHYDTPYTFGGGTKLEIK The nucleotide sequence of the light chain variable region of 19F3H2L3:  (SEQ ID NO: 13) GACATCGTGATGACCCAGTCCCCAAGCTCCCTGGCCGTGTCTGTGGGAG AGCGGGTGACAATCTCCTGTAAGTCTAGCCAGTCTCTGCTGAACTCCTC TAATCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGC CCCTAAGCTGCTGATCTACTTCGCCTCTACCAGGGAGAGCGGAGTGCCA GACAGATTCTCTGGCAGCGGCTCCGGCACAGACTTCACCCTGACAATC AGCTCCCTGCAGGCAGAGGACGTGGCCGTGTACTATTGCCAGCAGCAC TACGATACCCCCTATACATTTGGCGGCGGCACCAAGCTGGAGATCAAG The amino acid sequence of the light chain variable region of 19F3H2L3 with CDR sequences underlined: (SEQ ID NO: 14) DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQ APKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC FQQHYDTPYTGGGTKLEIK The CDR regions of 19F3, 19F3H1L1, 19F3H2L2 and 19F3H2L3 HCDR1: (SEQ ID NO: 15) GYSFTGYT  HCDR2: (SEQ ID NO: 16) INPYNAGT  HCDR3: (SEQ ID NO: 17) ARSEYRYGGDYFDY LCDR1: (SEQ ID NO: 18) QSLLNSSNQKNY LCDR2: (SEQ ID NO: 19) FAS LCDR3: (SEQ ID NO: 20) QQHYDTPYT The sequence of the heavy chain constant regions (330 aa, mutation sites are underlined in bold) of 19F3H1L1(hG1DM), 19F3H2L2(hG1DM) and 19F3H2L3(hG1DM): (SEQ ID NO: 21) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK The sequence of the light chain constant regions (107 aa) of 19F3H1L1(hG1DM), 19F3H2L2(hG1DM) and 19F3H2L3(hG1DM): (SEQ ID NO: 22) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC