ANTI-CD22 ANTIBODY MOLECULE OR ANTIGEN-BINDING FRAGMENT AND USE THEREOF

Abstract

Provided in the present invention is an isolated anti-CD22 antibody or an antigen-binding fragment thereof, and further provided is the use thereof. The antibody or the antigen binding fragment thereof shows strong identification capability for and a high affinity to CD22 recombinant protein and a CD22 positive cell line, a strong internalization capability to enter the CD22 positive cell line, and an effective growth inhibition and cell apoptosis effect on the CD22 positive cell line after fusion with bacterial exotoxin.

Claims

1. An isolated anti-CD22 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise respectively: (1) H-CDR1 having an amino acid sequence as shown in SEQ ID NO: 19, H-CDR2 having an amino acid sequence as shown in SEQ ID NO: 25, and H-CDR3 having an amino acid sequence as shown in SEQ ID NO: 21; and, L-CDR1 having an amino acid sequence as shown in SEQ ID NO: 22, L-CDR2 having an amino acid sequence as shown in SEQ ID NO: 23, and L-CDR3 having an amino acid sequence as shown in SEQ ID NO: 24; or (2) H-CDR1 having an amino acid sequence as shown in SEQ ID NO: 19, H-CDR2 having an amino acid sequence as shown in SEQ ID NO: 26, and H-CDR3 having an amino acid sequence as shown in SEQ ID NO: 21; and, L-CDR1 having an amino acid sequence as shown in SEQ ID NO: 22, L-CDR2 having an amino acid sequence as shown in SEQ ID NO: 23, and L-CDR3 having an amino acid sequence as shown in SEQ ID NO: 24.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 5, or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 5; and/or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8, or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region comprise respectively: (1) an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 4; and, an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 2; (2) an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 4; and, an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 7; (3) an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 3; and, an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 7; (4) an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 3; and, an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 8; (5) an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 6; and, an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 8; or (6) an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 5; and, an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 8.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a murine antibody, a chimeric antibody or a fully or partially humanized antibody; alternatively, the antibody is in the form of a scFv, dsFv, (dsFv).sub.2, Fab, Fab′, F(ab′).sub.2 or Fv antibody; preferably, the antibody is a monoclonal antibody or a single chain antibody; preferably, the antibody comprises a human or murine constant region, preferably a human or murine heavy chain constant region and/or light chain constant region; preferably, the antibody comprises a heavy chain and a light chain; more preferably, the antibody comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE and/or a kappa or lambda type light chain constant region.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the light chain of the monoclonal antibody is of a kappa type; more preferably, the monoclonal antibody is an IgG1 antibody.

6. A nucleic acid molecule comprising a nucleotide sequence encoding the anti-CD22 antibody or antigen-binding fragment thereof according to claim 1, or encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in the anti-CD22 antibody or antigen-binding fragment thereof.

7. A vector comprising the nucleic acid molecule according to claim 6.

8. A host cell comprising the nucleic acid molecule according to claim 6 and/or a vector comprising the nucleic acid molecule, or transformed or transfected with the nucleic acid molecule and/or the vector.

9. A composition comprising the antibody or antigen-binding fragment thereof according to claim 1.

10. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to claim 1 and a partner molecule which is a therapeutic agent.

11. (canceled)

12. A kit comprising the antibody or antigen-binding fragment thereof according to claim 1.

13. A method for treating a disease, the method comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof according to claim 1.

14. A composition comprising: the nucleic acid molecule according to claim 6, and/or a vector comprising the nucleic acid molecule, and/or a host cell transformed or transfected with the nucleic acid molecule and/or the vector.

15. A kit comprising: the nucleic acid molecule according to claim 6, and/or a vector comprising the nucleic acid molecule; and/or a host cell transformed or transfected with the nucleic acid molecule and/or the vector; and/or a composition comprising the nucleic acid molecule, and/or the vector, and/or the host cell; and/or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a partner molecule which is a therapeutic agent.

16. A method for treating a disease, the method comprising administering to a subject in need thereof: the nucleic acid molecule according to claim 6, and/or a vector comprising the nucleic acid molecule; and/or a host cell transformed or transfected with the nucleic acid molecule and/or the vector; and/or a composition comprising the nucleic acid molecule, and/or the vector, and/or the host cell; and/or an immunoconjugate comprising the antibody or antigen-binding fragment thereof and a partner molecule which is a therapeutic agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Embodiments of the present invention are described in detail below with reference to the attached figures, in which:

[0042] FIG. 1 shows detection results of binding of the antibodies provided by the present disclosure to CD22 by ELISA.

[0043] FIG. 2 shows detection results of binding kinetics of the antibodies provided by the present disclosure to CD22 by SPR using 1:1 binding model, in which panel 2A: HA22 mAb; panel 2B: mAb-1; panel 2C: mAb-2; panel 2D: mAb-3; panel 2E: mAb-4; panel 2F: mAb-5; and panel 2G: mAb-6.

[0044] FIG. 3 shows detection results of binding of the antibodies provided by the present disclosure to CD22 on cell membrane by FACS, in which panel 3A: Raji cells; panel 3B: REH cells; and panel 3C: CA46 cells.

[0045] FIG. 4 shows detection results of endocytosis of the antibodies provided by the present disclosure by FACS, in which panel 4A: Raji cells; panel 4B: CA46 cells; and panel 4C: REH cells.

[0046] FIG. 5 shows detection results of ADCC activity of the antibodies provided by the present disclosure, in which panel 5A: CA46 cells; panel 5B: Raji cells; and panel 5C: REH cells.

[0047] FIG. 6 shows detection results of ADCC activity of the antibodies provided by the present disclosure at a high dose of 50 μg/mL, in which panel 6A: CA46 cells; panel 6B: Raji cells; and panel 6C: REH cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0048] The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the invention and do not limit the scope of the invention in any way.

[0049] Experimental procedures in the following examples are all conventional ones, unless otherwise specified. Raw materials and reagents used in the following examples are all commercially available products, unless otherwise specified.

Example 1 Construction and Preparation of the Antibodies

[0050] In this Example, an IgG1-type monoclonal antibody was reconstructed by obtaining VH and VL region sequences and fusing them to constant regions.

1.1 Humanization Design

[0051] A framework shuffling strategy was used to alter amino acids in the framework regions of heavy chain variable region (V.sub.H) and light chain variable region (V.sub.L) comprised in the recombinant immunotoxin HA22 from the original murine to human ones. All the CDRs, which were determined using both the Kabat and Chothia numbering systems and the online tool (http://www.bioinf.org.uk/abs/), were left unchanged. Each of the framework and J regions in both the VH and VL of HA22 was aligned to the human antibody germline sequences in the international ImMunoGeneTics information System® (IMGT: http://www.imgt.org) for the closest human antibody matches. Murine residues that were deemed as Vernier residues or important for VH/VL interactions were not changed to preserve the specificity and affinity towards CD22.

[0052] A panel of humanization designs were prepared and evaluated to select the final candidate that possessed maximum humanization while maintaining optimal affinity and efficacy profile.

1.2 Gene Sequence Determination

[0053] Amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) contained in HA22 and humanized versions of the antibody are shown in Table 1. Elbow regions of the heavy and light chains were determined through defining antibody variable regions according to Kabat nomenclature, for subsequent mAb reconstruction; and CDR1, CDR2, and CDR3 were identified and underlined.

TABLE-US-00001 TABLE 1 V region sequences in the heavy and light chains (CDRs are underlined) Name Sequence HA22 VH EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTP EKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQ MSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSA (SEQ ID NO: 1) HA22 VL DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPD GTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQED FATYFCQQGNTLPWTFGCGTKLEIK (SEQ ID NO: 2) VH1 EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQAP GKCLEWVAYISSGGGTTYYPDSVKGRFTISRENAKNSLYLQ MNSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTMVTVSS (SEQ ID NO: 3) VH2 EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQTP EKCLEWVAYISSGGGTTYYPDSVKGRFTISRENAKNSLYLQ MNSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTMVTVSS (SEQ ID NO: 4) VH4 EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWIRQAP GKCLEWVAYISSGGGTTYYPGSVKGRFTISRENAKNSLYLQ MNSLRAGDTAVYYCARHSGYGTHWGVLFAYWGQGTMVTVSS (SEQ ID NO: 5) VH5 EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWIRQAP GKCLEWVAYISSGGGTTYYPGSVKGRFTISRENAKNSLYLQ MNSLRSEDTAVYYCARHSGYGTHWGVLFAYWGQGTMVTVSS (SEQ ID NO: 6) VL-2 DIQMTQSTSTLSASVGDRVTITCRASQDISNYLNWYQQRPD GSVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQDED FATYFCQQGNTLPWTFGCGTKVEIK (SEQ ID NO: 7) VL-4 DIQMTQSPSTLSASVGDRVTITCRASQDISNYLNWFQQRPG QSPRLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQGNTLPWTFGCGTKVEIK (SEQ ID NO: 8)

1.3 Amino Acid Sequences of Constant Regions of IgG1

[0054] The antibody was determined to have a kappa-type light chain upon an analysis of the variable regions, so kappa-type light chain constant region and IgG1 heavy chain constant region were selected for the reconstruction of IgG antibodies. The constant region sequences are shown in Table 2.

TABLE-US-00002 TABLE 2 Constant region sequences Name Sequence CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9) CH1 + Fc ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 10)

1.4 Reconstructed IgG Monoclonal Antibodies

[0055] Complete heavy and light chains shown in Table 3 were reconstructed through fusing the V region sequences provided in Table 1 to the constant region sequences provided in Table 2.

TABLE-US-00003 TABLE 3 Amino acid sequences of the reconstructed IgG antibodies (CDRs are underlined and constant regions are in italics) Name Combination Sequence HA22 mAb HA22 HC EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQ TPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNT LYLQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQ GTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 11) HA22 LC DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQK PDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQ EDFATYFCQQGNTLPWTFGCGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 12) mAb-1 V2 HC EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQ TPEKCLEWVAYISSGGGTTYYPDSVKGRFTISRENAKNSL YLQMNSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 13) HA22 LC DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQK PDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQ EDFATYFCQQGNTLPWTFGCGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 12) mAb-2 V2 HC EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQ TPEKCLEWVAYISSGGGTTYYPDSVKGRFTISRENAKNSL YLQMNSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 13) V2 LC DIQMTQSTSTLSASVGDRVTITCRASQDISNYLNWYQQR PDGSVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQD EDFATYFCQQGNTLPWTFGCGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 14) mAb-3 V1 HC EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQ APGKCLEWVAYISSGGGTTYYPDSVKGRFTISRENAKNS LYLQMNSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 15) V2 LC DIQMTQSTSTLSASVGDRVTITCRASQDISNYLNWYQQR PDGSVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQD EDFATYFCQQGNTLPWTFGCGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 14) mAb-4 V1 HC EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQ APGKCLEWVAYISSGGGTTYYPDSVKGRFTISRENAKNS LYLQMNSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 15) V4 LC DIQMTQSPSTLSASVGDRVTITCRASQDISNYLNWFQQRP GQSPRLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGCGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 16) mAb-5 V5 HC EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWIRQ APGKCLEWVAYISSGGGTTYYPGSVKGRFTISRENAKNS LYLQMNSLRSEDTAVYYCARHSGYGTHWGVLFAYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 17) V4 LC DIQMTQSPSTLSASVGDRVTITCRASQDISNYLNWFQQRP GQSPRLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGCGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 16) mAb-6 V4 HC EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWIRQ APGKCLEWVAYISSGGGTTYYPGSVKGRFTISRENAKNS LYLQMNSLRAGDTAVYYCARHSGYGTHWGVLFAYWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 18) V4 LC DIQMTQSPSTLSASVGDRVTITCRASQDISNYLNWFQQRP GQSPRLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPE DFATYYCQQGNTLPWTFGCGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 16)

1.5 Preparation of the Monoclonal Antibodies

[0056] pCDNA 3.4 vector was used as the expression vector for the mAbs. After codon optimization, full genes were synthesized and inserted into the pCDNA 3.4 vector and expression vectors for transfection were prepared using endotoxin-free plasmid preparation kit. ExpicCHO cell strain was used as a host for transient expression host. After transient transfection, cell culture supernatants were harvested, and antibodies were obtained through affinity purification, and then identified and stored at −80° C.

Example 2 Detection of Binding of the Antibodies to CD22 Antigen

[0057] In this Example, ELISA plates were coated with CD22 antigen to capture antibodies to be evaluated, and the color developed through an HRP-labeled secondary antibody capable of specifically recognizing Fd region, and the binding (EC.sub.50) of each mAb to the CD22 antigen was detected.

[0058] ELISA plates were coated with 200 ng/mL CD22 antigen (Biotinylated Human Siglec-2/CD22 Protein, Fc, Avitag™, ACROBiosystems (ARCO)) at 100 μl per well, at 4° C. overnight. Next day, the plates were washed with PBST, then blocked by the added blocking buffer (PBST+3% milk) at 37° C. for 1-2 hours, and then washed again with PBST.

[0059] Each antibody to be tested was diluted with PBST+1% BSA in gradient, to obtain antibody dilutions at concentrations of 0.01 ng/mL, 0.05 ng/mL, 0.25 ng/mL, 1.28 ng/mL, 6.40 ng/mL, 32 ng/mL, 160 ng/mL, 800 ng/mL, 4000 ng/mL, 20000 ng/mL, respectively; and 100 μl of each of the dilutions was added to each well of the ELISA plates which were then incubated at 37° C. for 1 hour.

[0060] After incubation, the plates were washed with PBST. 100 μl of 1:10000 diluted secondary antibody (Mouse anti-human IgG Fd secondary antibody, HRP; REF: SA5-10190, Invitrogen, USA) was added to each well of the ELISA plates which were then incubated at 37° C. for 1 hour. Thereafter, the plates were washed with PBST.

[0061] 100 μl of TMB solution (TMB Single-Component Substrate solution, Cat #PR1200, Beijing Solarbio Science & Technology Co., Ltd.) was added to each well of the plates which were then incubated at room temperature for 15 minutes. Then 50 μl of stop buffer (ELISA stop buffer (10×), Cat #C1058, Beijing Solarbio Science & Technology Co., Ltd.) was added thereto, and the plates were shaken. Absorbance values at 450 nm were detected. Results are shown in FIG. 1 and Table 4.

[0062] As shown in FIG. 1 and Table 4, the OD450 value of the isotype control was below 0.1, indicating no interference on the results due to non-specific binding; the fitting degree of 4-parameter non-linear fitting was greater than 0.99, an excellent fitting degree, indicating the results can be analyzed objectively. The Top values showed mAb-6 exhibited a remarkable high signal value, reaching 2.435-2.564, which was significantly higher than the value 2.147-2.283 of HA22 mAb, indicating that mAb-6 has a higher plateau of saturation binding; the Top value of mAb-1 was 1.726-1.795, significantly lower than 2.147-2.283 of HA22 mAb; and the Top values of the remaining mAb-2, mAb-3, mAb-4, and mAb-5 were comparable to or slightly higher than that of HA22 mAb.

[0063] EC.sub.50 value of the positive antibody HA22 mAb was 20.28 ng/mL, and EC.sub.50 values of mAb-1 to mAb-6 were all lower than that of HA22 mAb, and the EC.sub.50 values can be ranked as follows: mAb-2<mAb-6<mAb-3<mAb-4<mAb-1<mAb-5<HA22 mAb.

TABLE-US-00004 TABLE 4 Detection results of binding of the antibodies to CD22 antigen by ELISA EC.sub.50 (ng/mL) R2 Top HA22 mAb 20.28 HA22 mAb 0.9935 HA22 mAb 2.147-2.283 mAb-1 15.40 mAb-1 0.9964 mAb-1 1.726-1.795 mAb-2 9.057 mAb-2 0.9961 mAb-2 2.202-2.293 mAb-3 13.91 mAb-3 0.9954 mAb-3 2.176-2.278 mAb-4 13.92 mAb-4 0.9927 mAb-4 2.191-2.329 mAb-5 16.35 mAb-5 0.9962 mAb-5 2.221-2.325 mAb-6 10.44 mAb-6 0.9942 mAb-6 2.435-2.564 Isotype control 0.1545 Isotype control 0.1733 Isotype control 0.05848-0.06417

Example 3 Study on Binding Kinetics of the Antibodies to CD22 Antigen

[0064] In this Example, the binding kinetics of the antibodies to the antigen was detected by SPR.

[0065] Following reagents and materials were mainly used: [0066] Antigen to be immobilized: CD22-Fc-biotinylated antigen: ACROBiosystems (ARCO)) SI2-H82F8; [0067] Antibodies to be tested: Anti-CD22 mAbs; [0068] Chip SA; [0069] Immobilization buffer: (1) 1 M NaCl/50 mM NaOH; [0070] (2) 1 M NaCl/50 mM NaOH/50% isopropanol; [0071] Running buffer: 1×HBS-EP buffer; [0072] Regeneration buffer: Glycine, pH 1.5.

[0073] Experimental steps were as follows: [0074] 1) A sensor chip SA was taken and its surface was cleaned with ultrapure water; and after dried under nitrogen, was inserted into the slot. The immobilization buffers (1) and (2) were added to the columns A1 and A2 in a 96 well plate, and 3.85 nM CD22-Fc-biotinylated antigen were added at 200 μL per well into corresponding wells in column A3, respectively. The program was set to immobilize the antigen, and the immobilization was stopped when delta RU of about 70 RU was achieved. [0075] 2) Program “binding test” was set, including association time of 250 s and dissociation time of 600 s, and a test was performed using HA22 mAb, a dissociation degree of 4-5% was shown. [0076] 3) The HA22 mAb was diluted to 1000 nM and subsequently to 100 nM, and further 2-fold diluted starting from 100 nM for 8 times, at a final volume of about 250 μL per well. The program was set up to detect the binding capacity of that antibody No. 1 to the antigen, with association time of 250 s and dissociation time of 600 s. The analysis software showed the binding of the HA22 mAb was fitted normally. [0077] 4) All the antibodies, i.e., mAb-1 to mAb-6, were diluted to 1000 nM and subsequently to 100 nM, and further 2-fold diluted starting from 100 nM for 8 times, at a final volume of about 250 μL per well. The same program as that in step 3 was set up to detect affinity in batches. Results were analyzed using Biacore 8K evaluation.

[0078] The results are shown in FIG. 2 and Table 5.

TABLE-US-00005 TABLE 5 Results of SPR analysis of antibody affinity Quality Kinetics 1:1 Binding Antibody Chi.sup.2 (RU.sup.2) Ka (1/Ms) Kd (1/s) KD (M) HA22 mAb 1.91E−02 3.70E+05 8.93E−05 2.42E−10 mAb-1 2.26E−02 4.34E+05 2.62E−04 6.04E−10 mAb-2 2.30E−02 3.76E+05 1.77E−04 4.70E−10 mAb-3 3.15E−02 5.67E+05 4.04E−04 7.13E−10 mAb-4 5.75E−02 6.85E+05 9.91E−05 1.45E−10 mAb-5 3.76E−02 1.08E+06 4.11E−04 3.82E−10 mAb-6 3.10E−02 6.99E+05 2.20E−04 3.15E−10

Example 4 Study on Binding of the Antibodies to CD22-Positive Cell Lines

[0079] Raji cells (having a relatively high expression of CD22), CA46 cells (having a relatively high expression of CD22), and REH cells (having a relatively medium expression of CD22) were selected, and the cell binding ability of the antibodies was evaluated by curves of mean fluorescence intensity versus concentration obtained through FACS.

[0080] Reagents: [0081] FACS Buffer: 2% FBS+PBS; [0082] Goat anti-Human IgG (H+L) Secondary Antibody, FITC: Invitrogen, Catalogue number 31529; 4% PFA; [0083] Allophycayanin (APC) AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, Fcγ fragment specific, Jackson Immunoresearch, Catalogue number: 109-136-170.

[0084] Experimental steps were as follows: [0085] 1) Raji, CA46 and REH cells in logarithmic growth phase were harvested, and centrifuged at 1,000 rpm for 3 min. [0086] 2) The cells were washed twice with FACS Buffer (2% FBS+PBS), by centrifugation at 1,000 rpm for 3 min. [0087] 3) The cells were resuspended in FACS Buffer, and the cell density was adjusted to 2E6/mL. [0088] 4) The cell suspensions were added into 96 well round-bottom plates, at 50 μL per well, which were then placed on ice. [0089] 5) Each of the antibodies to be detected were 5-fold diluted serially to 8 concentrations: 100 μg/mL, 20 μg/mL, 4 μg/mL, 0.8 μg/mL, 0.16 μg/mL, 0.032 μg/mL, 0.0064 μg/mL and 0.00128 μg/mL. [0090] 6) The diluted antibodies were added into the wells containing cells at a 1:1 ratio by volume, to obtain final concentrations of 50 μg/mL, 10 μg/mL, 2 μg/mL, 0.4 μg/mL, 0.08 μg/mL, 0.016 μg/mL, 0.0032 μg/mL, 0.00064 μg/mL, and mixed with the cell suspensions, and the plates were then placed on ice in a 37° C. refrigerator and incubated for 30 min. [0091] 7) After the incubation, the cells were taken out and centrifuged at 2,000 rpm for 3 min. [0092] 8) The cells were washed twice with FACS Buffer, by centrifugation at 2,000 rpm for 3 min. [0093] 9) The cells were fixed with 2% PFA for 20 min at room temperature. [0094] 10) The cells were washed twice with FACS Buffer, by centrifugation at 2,000 rpm for 3 min. [0095] 11) The secondary antibodies (Goat anti-Human IgG (H+L) Secondary Antibody, FITC was used for Raji cells and REH cells; and Allophycayanin (APC) AffiniPure F(ab′).sub.2 Fragment Goat Anti-Human IgG, Fcγ fragment specific was used for CA46 cells) were formulated at 1:800 in buffers, and 100 μL of each were added into the cells, and the plates were then placed on ice in a 4° C. refrigerator and incubated for 30 min. [0096] 12) After the incubation, the cells were centrifuged at 2,000 rpm for 3 min. [0097] 13) Supernatants were discarded, and the cells were washed twice with FACS Buffer, by centrifugation at 2,000 rpm for 3 min. [0098] 14) The cells were suspended in 200 μL of FACS Buffer, and fluorescence intensity of FITC and fluorescence intensity of APC were detected.

[0099] Results are shown in FIG. 3 and Tables 6-1 to 6-3.

TABLE-US-00006 TABLE 6-1 Detection results of binding of the antibodies to CD22 on the cell membrane of Raji cells by FACS R2 Top EC.sub.50 (ng/mL) HA22 mAb 0.9888 HA22 mAb 7506-8131 HA22 mAb 152.0 mAb-1 0.9777 mAb-1 7594-8558 mAb-1 151.6 mAb-2 0.9546 mAb-2  5072-19663 mAb-2 276.2 mAb-3 0.9659 mAb-3 (very wide) mAb-3 ~1.355e+022 mAb-4 0.9572 mAb-4  8407-13897 mAb-4 206.3 mAb-5 0.9467 mAb-5 (very wide) mAb-5 ~1.362e+017 mAb-6 0.9646 mAb-6 (very wide) mAb-6 ~5.621e+013 isotype control 0.9878 isotype control 458.1-1079  isotype control 32619

[0100] As shown in panel 3A and Table 4, all the fitting parameters R2 reached above 0.95; the EC.sub.50 value of the reference antibody HA22 mAb was 152 ng/mL, and the maximum fluorescence signal of the antibody was between 7506 and 8131 and had entered a plateau phase. The maximum fluorescence signal of mAb-1 was comparable to that of the reference antibody HA22 mAb, while the maximum fluorescence signals of the remaining mAbs were all higher than that of the reference antibody HA22 mAb, and the maximum fluorescence signals can be ranked as: mAb-6>mAb-5>mAb-3>mAb-4>mAb-2>mAb-1>HA22 mAb. Higher fluorescence intensity indicates that the humanized mAbs have higher binding potential on Raji cells than HA22 mAb. At a concentration of 10 ng/mL, a fluorescence signal already could be detected, indicating that binding of each antibody to Raji cells was achieved at 10 ng/mL. Antibodies mAb-3, mAb-5, and mAb-6 still did not reach a fluorescence signal plateau at 50 μg/mL, thereby resulting in inaccurate EC.sub.50 values, but from the overall linearity evaluation, it can be determined that mAb-1 through mAb-6 had comparable or even stronger binding capacity to Raji cells.

TABLE-US-00007 TABLE 6-2 Detection results of binding of the antibodies to CD22 on the cell membrane of REH cells by FACS R2 Top EC.sub.50 (ng/mL) HA22 mAb 0.9707 HA22 mAb 2364-2868 HA22 mAb 172.3 mAb-1 0.9520 mAb-1 1847-6579 mAb-1 811.9 mAb-2 0.9471 mAb-2 (very wide) mAb-2 ~3.944e+025 mAb-3 0.9494 mAb-3 (very wide) mAb-3 ~2.2033+012 mAb-4 0.9472 mAb-4 (very wide) mAb-4 ~5.759e+013 mAb-5 0.9855 mAb-5 (very wide) mAb-5 ~4.166e+010 mAb-6 0.9709 mAb-6 (very wide) mAb-6 ~1.114e+099 isotype control 0.9250 isotype control (very wide) isotype control ~8.250e+006

[0101] As shown in panel 3B and Table 6-2, on REH cells with moderate expression of CD22, mAb-1 through mAb-6 had comparable or even stronger binding capacity to REH cells compared to HA22 mAb.

TABLE-US-00008 TABLE 6-3 Detection results of binding of the antibodies to CD22 on the cell membrane of CA46 cells by FACS R2 Top EC.sub.50 (ng/mL) HA22 mAb 0.9935 HA22 mAb 5724-6118 HA22 mAb 221.4 mAb-1 0.9866 mAb-1 6789-8271 mAb-1 520.9 mAb-2 0.9872 mAb-2 5247-5846 mAb-2 229.7 mAb-3 0.9551 mAb-3  2309-17864 mAb-3 1611 mAb-4 0.9804 mAb-4 5170-5899 mAb-4 152.5 mAb-5 0.9662 mAb-5 (very wide) mAb-5 ~2.015e+007 mAb-6 0.9622 mAb-6  3087-20780 mAb-6 2752 isotype control 0.8198 isotype control (very wide) isotype control ~4.590e+006

[0102] As shown in panel 3C and Table 6-3, on CA46 cells with high expression of CD22, mAb-1 through mAb-6 retained similar binding properties to that of HA22 mAb, all of which achieved high CA46 binding. According to the results of detection on all the three CD22 positive cell lines, humanized mAb-1 to mAb-6 can retain cell binding capacity comparable to or even superior to that of HA22 mAb.

Example 5 Study on Internalization Efficiency of the Antibodies

[0103] CD22 is an efficient target for internalization, so the internalization efficiency of an antibody against CD22 is often the most important index of evaluation for the development of immunotoxins and ADCs. To evaluate the internalization efficiency of the humanized antibodies i.e. mAb-1 to mAb-6, three CD22 positive cell lines (Raji, CA46 and REH cells) were selected for the evaluation and the internalization efficiency of the antibodies was compared with that of the reference antibody HA22 mAb.

[0104] Reagents: [0105] FACS Buffer: 2% FBS+PBS; [0106] Goat anti-Human IgG (H+L) Secondary Antibody, FITC: Invitrogen, Catalogue number 31529; 4% PFA; [0107] Allophycayanin (APC) AffiniPure F(ab′).sub.2 Fragment Goat Anti-Human IgG, Fcγ fragment specific, Jackson Immunoresearch, Catalogue number: 109-136-170.

[0108] Experimental steps were as follows: [0109] 1) Raji, CA46 and REH cells in logarithmic growth phase were harvested, and centrifuged at 1,000 rpm for 3 min. [0110] 2) The cells were washed twice with FACS Buffer, by centrifugation at 1,000 rpm for 3 min. [0111] 3) The cells were resuspended in FACS Buffer, and the cell density was adjusted to 5E6/mL. [0112] 4) The cell suspensions were added into 96 well round-bottom plates, at 100 μL per well, which were then placed on ice. [0113] 5) 100 μg/mL stock solution of an antibody to be tested was prepared, and the solution was added to the wells of the plates, at 100 μL per well, to obtain an antibody concentration of 50 μg/mL; the cells in the plates and the solution were mixed, and then the plates were placed on ice in a 4° C. refrigerator and incubated for 30 min. [0114] 6) After the incubation, the plates were taken out and the cells were centrifuged at 2,000 rpm at 4° C. for 3 min. [0115] 7) The cells were washed twice with FACS Buffer, by centrifugation at 2,000 rpm at 4° C. for 3 min. [0116] 8) One plate was used as the “0 h plate”: the cells therein were fixed with 2% PFA for 20 min at room temperature, and then were washed twice with FACS Buffer (2,000 rpm, 3 min), suspended in FACS Buffer, and placed at 4° C. for subsequent secondary antibody labeling. [0117] 9) Other plates were used as the “0.5 h plate”, the “1 h plate”, the “2 h plate”, and the “4 h plate”: the cells in each well of the plates were suspended in 100 μL of FACS Buffer, and the plates were placed at a 37° C. constant temperature incubator for stationary culture and the culture time was recorded. [0118] 10) The plates were taken out at 0.5 h, 1 h, 2 h and 4 h respectively. [0119] 11) All the plates were centrifuged at 2,000 rpm at 4° C. for 3 min. [0120] 12) The secondary antibodies were formulated in buffers; and 100 μL of each were added into the cells and the plates were then placed on ice in a 4° C. refrigerator and incubated for 30 min. [0121] 13) After the incubation, the cells were centrifuged at 2,000 rpm 4° C. for 3 min. [0122] 14) Supernatants were discarded, and the cells were washed twice with FACS Buffer by centrifugation at 2,000 rpm 4° C. for 3 min. [0123] 15) The cells were suspended in 200 μL of FACS Buffer, and were detected.

[0124] Results are shown in FIG. 4.

[0125] At 0 h, no endocytosis occurred, the antibodies were 100% concentrated on the cell surface; and the antibodies were endocytosed into the cells over time and the amounts of the antibodies on the membrane surface which could be detected decreased. The lower antibody signal is detected, the more endocytosis occurs. As shown in FIG. 4, no significant difference in the endocytosis rates was found between mAb-1 to mAb-6 and HA22 mAb when endocytosis percentages of the antibodies into the Raji, CA46 and REH cells were evaluated, with maximal endocytosis efficiency achieved at 0.5 h on Raji and CA46 cells and at 1 h on REH cells. Although the 7 antibodies showed comparable endocytosis percentages, a large difference in the number of endocytosed molecules could be found by analysis of the mean fluorescence intensity values. On the CA46 cells, the antibodies achieved essentially identical number of endocytosed molecules, while on the Raji and REH cells, the numbers of endocytosed molecules of the humanized antibodies mAb-1 to mAb-6 were significantly higher than that of the reference antibody HA22 mAb, and could be ranked from high to low as follows: mAb-6>mAb-5>mAb-4>mAb-3>mAb-2=mAb-1=HA22 mAb.

Example 6 Detection of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of the Antibodies

[0126] To evaluate whether the candidate antibodies have antibody-dependent cell-mediated cytotoxicity, three tumor cell lines, i.e., Raji, CA46 and REH cell lines having different CD22 expression levels were selected as target cells and PBMC as effector cells for ADCC assay.

[0127] Reagent: [0128] R-10 medium: phenol red-free RPMI 1640+10% inactivated FBS+2 mM Glutamax; [0129] R-2 medium: phenol red-free RPMI 1640+2% inactivated FBS+2 mM Glutamax; [0130] Cytotox Glo™ Cytotoxity Assay kit (Promega, Catalog number: G9291).

[0131] Experimental steps were as follows: [0132] 1) PBMCs were thawed one day in advance. [0133] 2) On the day of the experiment, the thawed PBMCs were harvested, centrifuged at 200 g for 15 min, and the supernatant was discarded. The cells were resuspended in 2 mL of fresh R-10 medium, counted, and the cell density was adjusted to 5E6 cells/mL. The cell suspension was added into plates, at 50 μL per well to obtain 250000 cells per well, or R-10 medium was added. [0134] 3) An antibody to be detected was diluted to 200 μg/mL, 40 μg/mL, 8 μg/mL, 1.6 μg/mL, 0.32 μg/mL, 0.064 μg/mL, 0.0128 μg/mL, and 0.00256 μg/mL with R-2 medium, and added into the plates, at 25 μL per well to obtain final concentrations of 50 μg/mL, 10 μg/mL, 2 μg/mL, 0.4 μg/mL, 0.08 μg/mL, 0.016 μg/mL, 0.0032 μg/mL, and 0.00064 μg/mL. [0135] 4) The Raji, CA46 and REH cells were diluted to 2E5 cells/mL with R-10 medium, and added into the plates, at 25 μL per well, to achieve an effector-to-target ratio of 50:1. [0136] 5) The plates were incubated at 37° C., 5% CO.sub.2 for 4 h, and the color developed and was detected according to the instructions in the Cytotox Glo™ Cytotoxity Assay kit.

[0137] Results are shown in FIGS. 5 and 6.

[0138] As shown in FIG. 5, all the 7 mabs had no significant ADCC effects on the tumor cells with different CD22 expression levels, and a dose-response dependence curve could not be fitted; while they exhibited slight ADCC effects at the high dose of 50 μg/mL.

[0139] The cytotoxicities observed at the dose of 50 μg/mL were analyzed, and as shown in FIG. 6, mAb-3 showed a relatively low cytotoxicity, having no difference from an isotype control. Antibodies mAb-1, mAb-2, mAb-4, mAb-5, and mAb-6 showed no different cytotoxicities from HA22 on the Raji and CA46 cells having high CD22 expression levels. However, mAb-2 showed a slightly more potent cytotoxicity than HA22 mAb on REH cells having a lower CD22 expression level. In conclusion, no significant ADCC effect was shown by the 7 IgG1 antibodies.

[0140] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications to the present invention without departing from the spirit of the present invention, which should fall within the scope of the appended claims.