ANTI-CLL1 ANTIBODY AND USE THEREOF

Abstract

Provided is an anti-CLL1 antibody and an application thereof. The variable region of the anti-CLL1 antibody includes the CDRs of SEQ ID NO: 1 to 6, SEQ ID NO: 5, and SEQ ID NO: 7 to 11 or SEQ ID NO: 12 to 17. The anti-CLL1 antibody of the present application has significant binding ability to both free and cell surface CLL1. After humanization, the affinity of the antibody to CLL1 is further improved, and it has important application prospect in the clinical diagnosis and/or treatment of tumors.

Claims

1. An anti-CLL1 antibody, comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region includes CDR3 shown in SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 14; and the light chain variable region includes CDR3 shown in SEQ ID NO: 6, SEQ ID NO: 11 or SEQ ID NO: 17.

2. The anti-CLL1 antibody according to claim 1, wherein the heavy chain variable region further comprises CDR1 shown in SEQ ID NO: 1, SEQ ID NO: 7 or SEQ ID NO: 12.

3. The anti-CLL1 antibody according to claim 1, wherein the heavy chain variable region further comprises CDR2 shown in SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 13.

4. The anti-CLL1 antibody according to claim 1, wherein the light chain variable region further comprises CDR1 shown in SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 15.

5. The anti-CLL1 antibody according to claim 1, wherein the light chain variable region further comprises CDR2 shown in SEQ ID NO: 5 or SEQ ID NO: 16.

6. The anti-CLL1 antibody according to claim 1, wherein, the heavy chain variable region of the anti-CLL1 antibody includes CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3; and the light chain variable region of the anti-CLL1 antibody includes CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6; or the heavy chain variable region of the anti-CLL1 antibody includes CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9; and the light chain variable region of the anti-CLL1 antibody includes CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 11; or the heavy chain variable region of the anti-CLL1 antibody includes CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 14; and the light chain variable region of the anti-CLL1 antibody includes CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 17.

7. The anti-CLL1 antibody according to claim 1, wherein, the heavy chain variable region of the anti-CLL1 antibody includes the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 19; or the heavy chain variable region of the anti-CLL1 antibody includes the amino acid sequence shown in SEQ ID NO: 20, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 21; or the heavy chain variable region of the anti-CLL1 antibody includes the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 23; or the heavy chain variable region of the anti-CLL1 antibody includes the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.

8. The anti-CLL1 antibody according to claim 1, wherein the heavy chain variable region and the light chain variable region of the anti-CLL1 antibody are connected by an interchain disulfide bond; optionally, the heavy chain variable regions of the anti-CLL1 antibody are connected by an interchain disulfide bond; optionally, the anti-CLL1 antibody further includes a constant region; optionally, the anti-CLL1 antibody is modified with a glycosylation group.

9. A nucleic acid molecule comprising a DNA fragment encoding the anti-CLL1 antibody of claim 1.

10. An expression vector comprising the nucleic acid molecule of claim 9.

11. A recombinant cell expressing the anti-CLL1 antibody of claim 1.

12. The recombinant cell according to claim 11, wherein a nucleic acid molecule comprising a DNA fragment encoding the anti-CLL1 antibody is integrated into the genome of the recombinant cell.

13. The recombinant cell according to claim 11, wherein the recombinant cell comprises an expression vector comprising a nucleic acid molecule comprising a DNA fragment encoding the anti-CLL1 antibody.

14. A pharmaceutical composition comprising the anti-CLL1 antibody of claim 1; optionally, the pharmaceutical composition further includes an anti-tumor drug; optionally, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

15. Use of the anti-CLL1 antibody of claim 1 in the preparation of a reagent for detecting a disease and/or a medicine for treating a disease; optionally, the disease includes acute myeloid leukemia.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0052] FIG. 1 shows the ForteBIO evaluation results of chimeric antibodies ch23D7, ch27H4 and ch19C1;

[0053] FIG. 2 shows the flow cytometric detection of the binding of chimeric antibodies ch23D7, ch27H4, ch19C1 to CLL1 antigen;

[0054] FIG. 3 shows the flow cytometric detection of the binding of the humanized antibody hz27H4 to the CLL1 antigen;

[0055] FIG. 4 shows the flow cytometric detection of the binding ability of the antibody to 293T cells transiently transfected by CLL1;

[0056] FIG. 5A shows the MPA test result of the binding ability of 23D7-scFv-hFc to CLL1 antigen, FIG. 5B shows the MPA test result of the binding ability of 27H4-scFv-hFc to CLL1 antigen, FIG. 5C shows the MPA test result of the binding ability of 19C1-scFv-hFc and CLL1 antigen, and FIG. 5D shows the MPA test result of the binding ability of Hz27H4-scFv-hFc and CLL1 antigen;

[0057] FIG. 6A shows a graph of the binding kinetics of recombinant human CLL-1; FIG. 6B shows a graph of the binding kinetics of recombinant cynomolgus monkey CLL-1.

DETAILED DESCRIPTION

[0058] In order to further illustrate the technical means adopted by the present application and effects thereof, the application will be further described below in conjunction with examples and drawings. It can be understood that the specific implementations described here are only used to explain the application, but not to limit the application.

[0059] If the specific technology or conditions are not indicated in the examples, it shall be carried out according to the technology or conditions described in the literature in the field or according to the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased through formal channels.

Example 1 Acquisition of Antibodies

[0060] 10 healthy female BALB/C mice aged 7-8 weeks were selected and immunized with the immunogen CLL1-mFc (a fusion protein of the extracellular segment of CLL1 and the Fc segment of mouse IgG1, the amino acid sequence of the extracellular segment of CLL1 as shown in SEQ ID NO: 28); two weeks after the second immunization, blood was collected from the tail vein of the mouse to obtain the serum, and the antibody titer was detected by ELISA; two mice with antibody titers meeting the requirements for fusion were selected, and 50 μg/100 μL of antigen was injected intraperitoneally three days before fusion for rush immunization; at the same time, myeloma cells SP2/0 were resuscitated one week before the titer test and cultured them in a 37° C., 5% CO.sub.2 incubator, and myeloma cells were passaged or replaced with fresh medium one day before fusion to keep the cells in the best condition;

TABLE-US-00008 SEQ ID NO: 28: HVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQTI ATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACA AQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINS SAWVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGST YFREA;

[0061] The blood was taken from the mice that had received rush immunization by breaking their necks, and after 10 min of disinfection with 75% alcohol, the spleen was taken; connective tissue was removed and spleen cell suspension was prepared; the suspension of spleen cells was transferred to a 50 mL centrifuge tube, RPMI 1640 was added to 30 mL, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and RPMI 1640 was added to 30 mL for cell counting; and the myeloma cells in good growth condition (the number of living cells>95%) were transferred to a 50 mL centrifuge tube, RPMI 1640 was added to 30 mL, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and RPMI 1640 was added to 30 mL for cell counting;

[0062] Spleen cells and myeloma cells were mixed in a 4:1 ratio, centrifuged at 1000 rpm for 5 min, discarded the supernatant, and the precipitated cells were flick into paste and placed in a water bath at 37° C., 1 mL of fusion agent was added in 1 min and stirred evenly, placed in a water bath at 37° C. for 45-60 s, added RPMI1640 within 1 min to stop the fusion effect of the fusion agent, centrifuged at 1000 rpm for 5 min, and discard the supernatant;

[0063] The cells were gently and evenly flicked and slowly added into the complete culture medium containing HAT, then the cell suspension was added to the prepared complete medium; the suspension was dropped into 96-well plates with 150-200 μL per well by a multi-channel pipette, and the cells were cultured in a 37° C., CO.sub.2 incubator and observed;

[0064] From the first day after cell fusion, the growth status of the cells was observed, and the culture medium was confirmed to be free of pollution. After 7-10 days of culture, the HAT culture medium was replaced by HT culture medium, and the culture continued for 3-4 days. The supernatant of each well was taken for ELISA detection.

[0065] The ELISA screening steps were as follows:

[0066] (1) Antigen coating: after the pure antigen human CLL1 ECD-His (human CLL1 extracellular domain with His tag) at a concentration of 50 ng/mL was diluted with the coating solution, 100 μL was added to each well of the polystyrene enzyme-linked detection plate followed by incubating at 4° C. overnight;

[0067] (2) Blocking: the plate was equilibrated to room temperature the next day, washed with PBS three times, and 100 μL blocking solution was added to each well, incubated at room temperature for one hour, washed with PBS three times, and patted dry;

[0068] (3) Addition sample to be tested: the fusion cell culture supernatant was taken under aseptic conditions, and added 35˜50 μL/well to the sealed ELISA plate, at the same time, negative control wells (no cell growth) and positive control wells (adding positive serum) were set up, incubated at room temperature for 1 hour, washed with PBST (0.5% Tween) three times, and washed with PBS twice;

[0069] (4) Addition secondary antibody: the diluted enzyme-labeled secondary antibody was added in an amount of 50 μL/well, incubated at 37° C. for 30 min, washed 3 times with PBS, and patted dry;

[0070] (5) Color development: two-component TMB color-developing solution stop solution (Solarbio, Cat#PR1210) was added 50 μL/well after homogeneous mixing, and color development was performed at 37° C. for 15-30 min, and then 50 μL/well stop solution was added to stop the reaction;

[0071] (6) Reading: the OD value of each well was measured with a single wavelength of 450 nm, and the multiple clones with high readings were selected for the next step of functional screening according with the order of OD values from high to low.

[0072] The positive wells initially screened by ELISA were selected, and the cells in the positive wells with high confluence rate were passed to a 24-well plate, and further performed functional experiments to determine the clone number for subcloning; the limiting dilution method was used to carry out cell subcloning, while the seeds were cryopreserved. The details were as follows: the cell clones were diluted and spread on a 96-well plate, and each clone was spread on a plate separately, cultured in HT medium for 7-10 days, and observed under the microscope on the 7th day. The supernatant of the monoclonal cells was selected and the positive clones were further screened by ELISA;

[0073] The clones were sequenced and the amino acid sequences of clones 23D7, 19C1, and 27H4 were obtained, wherein, the heavy chain variable region of 23D7 is shown in SEQ ID NO: 18, and the light chain variable region of 23D7 is shown in SEQ ID NO: 19; the heavy chain variable region of 19C1 is shown in SEQ ID NO: 20, and the light chain variable region of 19C1 is shown in SEQ ID NO: 21; the heavy chain variable region of 27H4 is shown in SEQ ID NO: 22, and the light chain variable region of 27H4 is shown in SEQ ID NO: 23.

Example 2 Expression and Purification of Antibodies

[0074] In the present example, specific primers were designed according to the results of monoclonal identification and sequencing, and the genes of antibodies 23D7, 19C1, and 27H4 were obtained by PCR, and the genes were cloned to the upstream of the coding gene of the Fc segment of the constant region of human IgG1 heavy chain to construct a recombinant eukaryotic expression vector to obtain human-mouse chimeric antibody expression plasmid;

[0075] The human-mouse chimeric antibody expression plasmid was transiently transfected into 293F cells, and the chimeric antibodies ch23D7, ch19C1, and ch27H4 with 23D7, 19C1, and 27H4 as parents, respectively, were obtained by transient expression and affinity purification.

Example 3 Affinity Test of Antibody

[0076] In the present example, the ForteBio affinity measurement method (P. Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning MAbs, 2013. 5(2): 270-278.) was used to detect the affinity of the chimeric antibodies ch23D7, ch19C1, and ch27H4, and the VL and VL chains (SEQ ID NO: 29) of the anti-human CLL-1 antibody 1075.7 (U.S. Pat. No. 8,536,310B2) were selected as the control antibody (Anti-CLL1-Ref).

[0077] In short, the antibody was loaded on the anti-human IgG capture (AHC) biosensor, the sensor was equilibrated in the assay buffer for 30 min offline, and monitored online for 60 s to establish the baseline; the antibody-loaded sensor was incubated with the 100 nM antigen human CLL1 ECD-His for 5 min, then transferred to the assay buffer, and the dissociation rate was measured after 5 min; the kinetic analysis was carried out using a 1:1 combination model.

[0078] The results are shown in Table 1 and FIG. 1. The binding affinity of ch23D7, ch27H4 and ch19C1 to the antigen CLL1 were 2.19 nM, 3.83 nM and 10.9 nM, respectively, and the binding affinity of the control antibody 1075.7 to the antigen CLL1 was 1.08 nM.

TABLE-US-00009 TABLE 1 Concentration Sample Number (nM) Response KD(M) Kon(1/Ms) Kdis(1/s) RMax ch23D7 100 0.6536 2.19E−09 1.30E+05 2.84E−04 0.6693 ch27H4 100 0.6341 3.83E−09 2.65E+05 1.01E−03 0.6237 ch19C1 100 0.6194 1.09E−08 1.80E+05 1.97E−03 0.6574 ch1075.7 100 0.2361 4.14E−10 1.08E+05 4.48E−05 0.2384

TABLE-US-00010 SEQ ID NO: 29: ENVLTQSPAIMSASPGEKVTMTCRASSNVISSYVHWYQQRSGASPKLWI YSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLT FGAGTKLELGGGGSGGGGSGGGGSDIQLQESGPGLVKPSQSLSLTCSVT GYSITSAYYWNWIRQFPGNKLEWMGYISYDGRNNYNPSLKNRISITRDT SKNQFFLKLNSVTTEDTATYYCAKEGDYDVGNYYAMDYWGQGTSVTVSS.

Example 4 Binding of Antibody to CLL1 on HEK293 Cells

[0079] In the present example, flow cytometry was used to detect the binding of antibodies ch23D7, ch27H4, and ch19C1 to CLL1 on HEK293 cells. The steps are as follows:

[0080] 5×10.sup.5 HEK293 cells overexpressing CLL1 were resuspended in PBS+5% BSA and incubated at 4° C. for 30 min; antibodies of different concentrations (1 μg/mL˜0.01 μg/mL, 10-fold dilutions) were added and incubated at 4° C. for 60 min; after centrifugation and washing, a PBS+5% BSA solution containing FITC-labeled goat anti-human IgG-Fc secondary antibody (1:200, sigma, F9512) was added, and incubated on ice for 30 min in the dark; the cells were washed 3 times and then analyzed by flow cytometry;

[0081] The control group CLL1-RefAb (ch1075.7), Cell+Secondary antibody and Blank were set up.

[0082] The results are shown in FIG. 2. The chimeric antibodies ch23D7, ch27H4, ch19C1, and ch1075.7 can effectively bind to HEK293-CLL1 cells, and as the antibody concentration increased, the average fluorescence intensity (MFI) also increased.

Example 5 Humanization of Antibodies

[0083] In the present example, 27H4 was humanized. In short, the gene sequence of the 27H4 antibody was compared with the human antibody Germline database to find out the sequence with high homology, while avoiding the rarely used or subclass Germline; after selecting a humanized template, rearranged the frequency of the amino acid of the antibody at a specific FR site, and performed CDR grafting to avoid the introduction of glycosylation and other protein modification sites and sites that are prone to chemical degradation; if the affinity of the sequence decreased after CDR transplantation, reverse mutation was performed. The mutation principle was to perform stepwise single point mutations in the FR region; if there were modification sites or chemical degradation sites in the CDR region, and it had an impact on protein control, single-point mutations were performed on the sites step by step; Affinity (KD/Kon/Koff) test was performed after each round of mutation; the humanized sequence finally obtained had the best performance in terms of affinity (within the difference of 3 times compared with the parent), stability, and protein quality (PA>90% in one step).

[0084] According to the above principles, one heavy chain variable region hz27H4H1 (SEQ ID NO: 24) and three light chain variable regions hz27H4L1 (SEQ ID NO: 25), hz27H4L2 (SEQ ID NO: 26) and hz27H4L3 (SEQ ID NO: 27) of the humanized hz27H4 antibody were obtained.

Example 6 ForteBIo Analysis of the Difference in Affinity Between Antibody and CLL1 Before and After Humanization

[0085] In short, 4 μg/mL antibody was loaded on the anti-human IgG capture (AHC) biosensor, the sensor was equilibrated offline for 30 min in the assay buffer, and the online monitoring was used for 60 s to establish the baseline; the antibody-loaded sensor was incubated with the 60 nM antigen human CLL1 ECD-His for 3 min, then transferred to the assay buffer, and the dissociation rate was measured after 3 min; the kinetic analysis was performed using a 1:1 binding model.

[0086] The results are shown in Table 2. Compared with the parent antibody (ch27H4), the three humanized antibodies (hz27H4H1L1, hz27H4H1L2, and hz27H4H1L3) all retained higher affinity.

TABLE-US-00011 TABLE 2 ForteBIo analysis of the difference in affinity between antibody and CLL1 before and after humanization Sample Number Concentration (nM) Response KD (M) Kon (1/Ms) Kdis (1/s) hz27H4H1L1 60 0.6123 2.29E−09 3.77E+05 8.62E−04 hz27H4H1L2 60 0.7255 2.21E−09 4.41E+05 9.76E−04 hz27H4H1L3 60 0.7224 2.49E−09 4.38E+05 1.09E−03 ch27H4 60 0.5972 3.88E−09 3.20E+05 1.24E−03 Anti-CLL1 Ref Ab 60 0.2189 6.76E−09 1.48E+05 9.97E−04

Example 7 Flow Cytometric Detection of the Binding of Humanized Antibody to CLL1

[0087] In the present example, flow cytometry was used to detect the binding of antibodies hz27H4H1L1, hz27H4H1L2 and CLL1 on HEK293 cells. The steps are as follows:

[0088] 2×10.sup.5 HEK293 cells overexpressing CLL1 were resuspended in PBS+5% BSA and incubated at 4° C. for 30 min; different concentrations of antibodies (5 μg/mL˜0.002286 μg/mL, 3-fold gradient dilution) were added, and incubated at 4° C. for 60 min; after centrifugation and washing, a PBS+5% BSA solution containing FITC-labeled goat anti-human IgG-Fc secondary antibody (1:200, sigma, F9512) was added, and incubated on ice for 30 min in the dark; and the cells were washed 3 times and then analyzed by flow cytometry; Graphpad software calculated EC50.

[0089] The control group ch27H4, CLL1-RefAb (ch1075.7) and NC-huIgG1 were set up.

[0090] The results are shown in FIG. 3, FIG. 4 and Table 3. ch27H4 combined with CLL1 in a dose-dependent manner, with an EC50 (n=1) of 0.3583 μg/mL; hz27H4H1L1 combined with CLL1 in a dose-dependent manner, with an EC50 (n=1) of 0.2681 μg/mL; hz27H4H1L2 combined with CLL1 in a dose-dependent manner, with an EC50 (n=1) of 0.3214 μg/mL; CLL1-refAb combined with CLL1 in a dose-dependent manner, with an EC50 (n=1) of 0.264 μg/mL; ch27H4, hz27H4H1L1 and hz27H4H1L2 combined with human CLL1 with EC50 similar to CLL1-refAb.

TABLE-US-00012 TABLE 3 Flow cytometric detection of the binding of humanized antibodies to HEK293-CLL1 cells (EC50) — ch27H4 hz27H4H1L1 hz27H4H1L2 CLL1-refAb EC50 (μg/mL) 0.3583 0.2618 0.3214 0.2446

Example 8 Membrane Proteome Array Evaluation of Antibody Specificity

[0091] In the present example, a Membrane Proteome Array (MPA) was used to verify the non-target binding interaction of antibodies. Membrane Proteome Array (MPA) is a platform for analyzing human membrane proteins targeted by specific antibodies and other ligands, and can be used to determine the specificity of antibody targets.

[0092] Plasmids containing about 6000 membrane protein clones (accounting for more than 94% of the human membrane proteome) were transfected into HEK-293T cells (ATCC, CRL-3216) or QT6 cells (ATCC, CRL-1708), and seeded into 384-well cell culture plates (Corning, 3764) at a density of 18000 cells/well; after 36 hours of incubation, the test antibody was added to the membrane proteome array matrix plate at a predetermined concentration, and flow cytometry was used to directly detect the binding of antibody scFv to about 6000 membrane protein expressing cells. All target proteins had natural conformations and appropriated post-translational modifications. The single-chain antibody (scFv) expressed in mammals had a VL-(G4S).sub.3-VH structure, and the C-terminal fusion expressed human hIgG1-Fc. See Table 4 for specific information.

[0093] The test results are shown in FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D. 23D7-scFv-hFc, 27H4-scFv-hFc, 19C1-scFv-hFc, Hz27H4-scFv-hFc can specifically bind to the CLL1 target antigen, among which FCGR1A, FCGR2B, and FCGR3B were IgG Fc receptors.

TABLE-US-00013 TABLE 4 Information on antibodies used Target Antibody Basic Information Antigen Uniprot 23D7-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9 27H4-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9 19C1-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9 Hz27H4-scFv-hFc Anti-CLL1 scFv-hIgG1 CLL1 Q5QGZ9

Example 9 Determination of Antibody Affinity by Surface Plasmon Resonance Method

[0094] In the present example, Surface Plasmon Resonance (SPR) technology was used to detect and compare the affinities between two CLL-1 antigens (recombinant human CLL-1, Acro, article number: CLA-H5245, batch number: 3413a-9B8F1-SQ; recombinant cynomolgus monkey CLL-1, Acro, article number: CLA-H5263, batch number: 3765-2079F1-SS) and 6 antibodies. The single-chain antibody (scFv) has a VL-(G4S).sub.3-VH structure, and the C-terminal fusion expresses human hIgG1-Fc. The sample information table is shown in Table 5.

TABLE-US-00014 TABLE 5 Sample information table Molecular Concentration Storage Name Weight (mg/mL) Conditions Recombinant 35 KDa 0.25 After Human CLL-1 dissolving - Human CLL-1 20° C.  Protein (his) Recombinant 44 KDa 0.25 After Cynomolgus Monkey dissolving - CLL-1 Cynomolgus 20° C.  CLL-1 Protein (his) 27H4-scFv-hFc 106 KDa 1.6 4° C. 19C1-scFv-hFc 106 KDa 1.3 4° C. 23D7-scFv-hFc 106 KDa 1.2 4° C. Hz27H4-scFv-hFc 106 KDa 2.6 4° C. h27H4H1L1 145 KDa 1 4° C. (complete antibody) h27H4H1L2 145 KDa 1.5 4° C. (complete antibody)

[0095] (1) Sample Preparation

[0096] Antibody diluent (ligand): the antibody was diluted to 5 μg/mL with 1×HBS-EP+ running buffer;

[0097] Recombinant human CLL-1 diluent (Analyte 1): recombinant human CLL-1 (250 μg/mL) was diluted to 50 nM with running buffer, and a 2-fold serial dilution was used to obtain recombinant human CLL-1 dilutions of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM and 0 nM;

[0098] Recombinant cynomolgus monkey CLL-1 diluent (analyte 2): recombinant cynomolgus monkey CLL-1 (250 μg/mL) was diluted to 50 nM with running buffer, and a 2-fold serial dilution was used to obtain recombinant cynomolgus monkey CLL-1 dilutions of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM and 0 nM;

[0099] (2) Analysis of Recombinant Human CLL-1 Antigen

[0100] The Protein A chip was used for testing. 5 μg/mL antibody dilution was passed through the experimental flow path (Fc2, Fc4) at a flow rate of 10 μL/min, and the capture volume was about 454 RU after 20 s capture. And then, the flow rate was adjusted to 30 μL/min, and different concentrations of recombinant human CLL-1 dilutions (0, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) were sequentially added, and passed through the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) at the same time. The binding time was 85 s and the dissociation time was 70 s. Finally, glycine solution (Glycine, pH=1.5) was added and lasted for 60 s to regenerate the chip and allow the chip to enter the next cycle.

[0101] (3) Analysis of Recombinant Cynomolgus Monkey CLL-1 Antigen

[0102] The Protein A chip was used for testing. 5 μg/mL antibody dilution was passed through the experimental flow path (Fc2, Fc4) at a flow rate of 10 μL/min, and the capture volume was about 454 RU after 20 s capture. And then, the flow rate was adjusted to 30 μL/min, and different concentrations of recombinant cynomolgus monkey CLL-1 dilutions (0, 3.125 Nm, 6.25 nM, 12.5 nM, 25 nM, 50 nM) were sequentially added, and passed through the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) at the same time. The binding time was 85 s and the dissociation time was 70 s. Finally, glycine solution (Glycine, pH=1.5) was added and lasted for 60 s to regenerate the chip and allow the chip to enter the next cycle.

[0103] (4) Data Analysis

[0104] The data analysis software Evaluation Software 3.1 was used to analyze the test results. The sensor signal collected from the sample test flow path was used for deduction of both the reference flow path and sample blank, and the kinetic “1:1” model was used for fitting, and the kinetic parameters (Ka: association rate; kd: dissociation rate; kD: association and dissociation equilibrium constant) of each batch of samples with shTNF-α were obtained. The kinetic fitting results of the binding of 6 antibodies to recombinant human CLL-1 are shown in Table 6 and FIG. 6A. The kinetic fitting results of binding of antibodies to recombinant cynomolgus monkey CLL-1 are shown in FIG. 6B.

TABLE-US-00015 TABLE 6 Statistics table of the kinetic fitting results of antibody binding to recombinant human CLL-1 Sample ka(1/Ms) kd(1/s) KD(M) Rmax(RU) Chi.sup.2(RU.sup.2) 27H4-scFv-hFc 4.11E+06 1.26E−02 3.06E−09 249.8 0.839 19C1-scFv-hFc 8.86E+05 5.09E−03 5.75E−09 273.8 0.11 23D7-scFv-hFc 2.93E+05 3.22E−02 1.10E−07 272.1 0.232 Hz27H4-scFv-hFc 4.12E+06 6.25E−03 1.52E−09 273.9 0.638 h27H4H1L1 4.91E+06 5.49E−03 1.12E−09 256.3 0.67 (complete antibody) h27H4H1L2 5.04E+06 5.44E−03 1.08E−09 207.1 0.342 (complete antibody)

[0105] The results showed that, except for the low binding affinity of 23D7-scFv-hFc to recombinant human CLL-1, the other 5 antibodies have binding affinity to recombinant human CLL-1 between 1 nM and 6 nM; and none of the 6 antibodies bound to recombinant cynomolgus monkey CLL-1 (FIG. 6B).

[0106] In summary, the anti-CLL1 antibodies 23D7, 27H4, and 19C1 of the present application have significant binding ability to CLL1. After humanization, the affinity of the antibody to CLL1 is further improved. It has important application prospects in the clinical diagnosis and/or treatment of tumors.

[0107] The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.