An injection formulation of an anti-siglec-15 monoclonal antibody

Abstract

The present application relates to the field of biological medicine, and specifically provides an injection preparation of anti-Siglec-15 monoclonal antibody, the injection preparation comprising: anti-Siglec-15 monoclonal antibody; buffer salt; protein protective agent; surfactant; the pH value of the injection preparation is 5.5-7.5.The anti-Siglec-15 monoclonal antibody disclosed in the present application can be specifically combined with Siglec-15, effectively inhibit tumor growth, and be used for treating cancer or immunological diseases; secondly, the injection preparation provided in the present application provides a good storage microenvironment for anti-Siglec-15 monoclonal antibody through the synergistic cooperation of buffer solution, protein protective agent, osmotic pressure regulator, surfactant and the like in injection solution, effectively reduces the generation rate of aggregates or sub-visible particles, improves the physical stability of the antibody, ensures the biological activity of anti-Siglec-15 monoclonal antibody, and reduces potential safety risks.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an injection preparation of anti-Siglec-15 monoclonal antibody. BACKGROUND

[0002] With the continuous development of biological medicine technology, antibodies as targeted therapeutic drugs have been proven to be a reliable and effective choice in the treatment of tumors and autoimmune diseases. Based on the unique therapeutic advantages and low side effects of antibody drugs, the development of antibody drugs has attracted great attention from pharmaceutical companies worldwide.

[0003] Sialic acid-binding Ig-like lectin 15 (Siglec-15) is a member of the SIGLEC gene family and a DAP12-associated immune receptor, belonging to the immunoglobulin superfamily and the sialic acid-binding Ig-like lectin family. Studies have found that Siglec-15 has the function of inhibiting T cell activity, and Siglec-15 expressed by macrophages can directly inhibit the proliferation and activity of human and mouse T cells and inhibit the secretion of IFN-γ and TNF-α; gene knockout and antibody blocking of Siglec-15 in mice can enhance tumor immunity in the microenvironment and inhibit tumor growth in some mouse models. Therefore, Siglec-15 is a novel immunosuppressive molecule widely present in various tumors, which has potential clinical relevance. Therefore, the development of anti-Siglec-15 monoclonal antibody drugs has important clinical significance.

[0004] Anti-Siglec-15 monoclonal antibody protein has a complex higher structure, which often aggregates under the influence of various factors during production, transportation and use, and undergoes various chemical and physical denaturation or degradation. Compared with traditional small molecule drugs, protein molecules have complex structures, including primary, secondary, tertiary and higher structures, and the structure of the protein, especially the higher structure, is prone to change, such as denaturation, aggregation or precipitation. The structure of the product is variable, which has a great impact on the safety of biological pharmaceuticals, especially some protein aggregates can stimulate the immune response of the human body, which can reduce the efficacy of biological drugs, and even cause death of patients. Antibody drugs not only need to obtain high-purity products during production, but also need to maintain structural stability during transportation, storage and use. Therefore, in order to ensure the long-term stability of the anti-Siglec-15 monoclonal antibody provided by the present application, the present application provides an injection preparation of anti-Siglec-15 monoclonal antibody. SUMMARY

[0005] In order to ensure the biological activity of the anti-Siglec-15 monoclonal antibody during storage, transportation and use, and ensure the stability of the anti-Siglec-15 monoclonal antibody protein, the application discloses an injection preparation of an anti-Siglec-15 monoclonal antibody.

[0006] The specific technical scheme of the application is as follows:

[0007] The application provides an injection preparation of an anti-Siglec-15 monoclonal antibody, which comprises the following components:

[0008] (i) an anti-Siglec-15 monoclonal antibody;

[0009] (ii) a buffer salt;

[0010] (iii) a protein protective agent;

[0011] (iv) a surfactant;

[0012] The pH value of the injection preparation is 5.5-7.5;

[0013] The anti-Siglec-15 monoclonal antibody comprises three heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3 respectively and three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No: 6.

[0014] The application provides an injection preparation of an anti-Siglec-15 monoclonal antibody through the synergistic cooperation of a buffer salt, a protein protective agent and a surfactant, the injection preparation can effectively improve the available concentration of the anti-Siglec-15 monoclonal antibody, ensure the stability, solubility, body fluid balance (pH) and blood isotonicity of the protein, the injection preparation is administered through an intravenous injection route, and the combination of the components in the preparation effectively improves the appearance and particle performance of the high-concentration preparation.

[0015] The beneficial effects of the present application are as follows: first, the anti-Siglec-15 monoclonal antibody disclosed in the present application can specifically bind to Siglec-15, block the binding of Siglec-15 to cell surface receptors, inhibit the conduction of intracellular signal pathways, and achieve the effect of inhibiting tumor growth, and is used for treating cancer or immunological diseases, the cancer includes but is not limited to brain glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma or leukemia, etc., and the immunological diseases include but are not limited to psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis and autoimmune hepatitis, etc.; second, the injection preparation provided by the present application provides a good storage microenvironment for the anti-Siglec-15 monoclonal antibody through the synergistic cooperation of buffer solutions, protein protectants, osmotic pressure regulators, surfactants and the like in the injection liquid, so that the generation rate of aggregates or sub-visible particles can be effectively reduced during storage and transportation, the physical stability of the antibody is improved, the biological activity of the anti-Siglec-15 monoclonal antibody is ensured, and the potential safety risk is reduced; the anti-Siglec-15 monoclonal antibody preparation provided by the present application has the characteristics of simple preparation process, low cost, higher concentration and better stability. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The plasmid map of the pScFv-Disb-HS vector in Example 2 of the present application is shown in Figure 1.

[0017] Figure 2 The comparison chart of the affinity of the gradient dilution ELISA anti-Siglec-15 phage monoclonal antibody in Example 3 of the present application is shown in Figure 2.

[0018] Figure 3 The map of the vector pTSE in Example 5 of the present application is shown in Figure 5.

[0019] Figure 4 The denaturing polyacrylamide gel electrophoresis chart of the murine antibody molecule in Example 5 of the present application is shown in Figure 6.

[0020] Figure 5 The comparison chart of the binding ability of the murine antibody molecule and Siglec-15 in Example 6 of the present application is shown in Figure 7.

[0021] Figure 6 The comparison chart of the ability of the murine antibody to inhibit the binding of Siglec-15 to the Jurkat cell surface receptor in Example 7 of the present application is shown in Figure 8.

[0022] Figure 7 The comparison chart of the ability of the murine antibody molecule to inhibit the binding of Siglec-15 to the CHOSLV-LRRC4C cell surface receptor in Example 8 of the present application is shown in Figure 9.

[0023] Figure 8 Figure 8 is a graph showing the promotion of human TNF-a cytokine release by the murine antibody molecule in Example 9 of the present application;

[0024] Figure 9 Figure 9 is a graph showing the promotion of human IFN-γ cytokine release by the murine antibody molecule in Example 9 of the present application;

[0025] Figure 10 Figure 10 is a graph showing the promotion of T cell activation and proliferation by the murine antibody molecule in Example 9 of the present application;

[0026] Figure 11 Figure 11 is a graph showing the biological activity detection of the murine antibody molecule in Example 10 of the present application;

[0027] Figure 12 Figure 12 is a denaturing polyacrylamide gel electrophoresis graph of the humanized antibody molecule in Example 15 of the present application;

[0028] Figure 13 Figure 13 is a graph showing the comparison of the binding ability of the humanized antibody molecule in Example 16 of the present application to Siglec-15;

[0029] Figure 14 Figure 14 is a graph showing the cross-binding experiment of the humanized antibody to different species of Siglec-15 in Example 17 of the present application;

[0030] Figure 15 Figure 15 is a graph showing the binding ability of the humanized antibody molecule in Example 18 of the present application to inhibit the binding of Siglec-15 to the surface receptor of Jurkat cells;

[0031] Figure 16 Figure 16 is a graph showing the biological activity detection of the humanized antibody molecule in Example 19 of the present application;

[0032] Figure 17 Figure 17 is a graph showing the tumor volume growth curve of the anti-Siglec-15 monoclonal antibody HA-I in the MC38-Siglec-15 colorectal cancer model in mice in Example 20 of the present application;

[0033] Figure 18 Figure 18 is a graph showing the thermal stability evaluation of the anti-Siglec-15 monoclonal antibody HA-I in Example 21 of the present application. DETAILED DESCRIPTION

[0034] In order that the application can be more readily understood, certain technical and scientific terms are defined below, immediately prior to the detailed description. The terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting.

[0035] The term "antibody" as used herein includes whole antibodies and any antigen binding fragments thereof, the antibody includes murine antibody, humanized antibody, bispecific antibody or chimeric antibody, the antibody can also be Fab, F(ab)2, Fv or ScFv (single chain antibody), the antibody can be naturally occurring antibody or antibody altered by changing (such as mutation, deletion, substitution, etc.).

[0036] The terms "variable region" and "constant region" as used herein, the sequence region close to N segment of antibody heavy chain and light chain is variable region (V region), the remaining amino acid sequence close to C segment is relatively stable, which is constant region (C region), the variable region includes 3 complementarity determining regions (CDRs) and 4 framework regions (FRs), each light chain variable region and heavy chain variable region are composed of 3 CDR regions and 4 FR regions, the 3 CDR regions of heavy chain are represented by HCDR1, HCDR2 and HCDR3 respectively, and the 3 CDR regions of light chain are represented by LCDR1, LCDR2 and LCDR3 respectively.

[0037] The term "murine antibody molecule" as used herein is the antibody obtained after immunizing mice with Siglec-15 antigen.

[0038] The term "chimeric antibody molecule" as used herein is the antibody obtained by fusing the variable region of murine antibody with the constant region of humanized antibody, which can reduce the immune response reaction induced by murine antibody in human body. The chimeric antibody is obtained by using DNA recombination technology to insert the light and heavy chain variable region genes of murine monoclonal antibody into an expression vector containing human antibody constant region, so that the light and heavy chain variable regions of the antibody molecule expressed are of murine origin, and the constant region is of human origin, and the whole antibody molecule is about 2 / 3 of human origin. The antibody produced in this way reduces the immunogenicity of murine antibody while retaining the ability of the parent antibody to specifically bind to antigen.

[0039] The term "humanized antibody molecule" as used herein is the CDR of murine monoclonal antibody transplanted to the variable region of human antibody, replacing the CDR of human antibody, so that the human antibody acquires the antigen binding specificity of murine monoclonal antibody while reducing its heterogeneity.

[0040] The application will be further described in detail below in combination with the following examples.

[0041] Example 1

[0042] An injection preparation of anti-Siglec-15 monoclonal antibody, the injection preparation includes the following components:

[0043] (i) anti-Siglec-15 monoclonal antibody;

[0044] (ii) buffer salt;

[0045] (iii) a protein protective agent;

[0046] (iv) a surfactant;

[0047] The pH value of the injection preparation is 5.5-7.5.

[0048] wherein the anti-Siglec-15 monoclonal antibody comprises 3 heavy chain complementarity determining regions denoted as HCDR1, HCDR2 and HCDR3 respectively and 3 light chain complementarity determining regions denoted as LCDR1, LCDR2 and LCDR3 respectively, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No: 6.

[0049] Heavy chain complementarity determining region Amino acid sequence Light chain complementarity determining region Amino acid sequence HCDR1 (SEQ ID No: 1) DYNMF LCDR1 (SEQ ID No: 4) RASQDISNYLN HCDR2 (SEQ ID No: 2) YIYPDNGGTGYNQNFKS LCDR2 (SEQ ID No: 5) YTSRLHS HCDR3 (SEQ ID No: 3) SEYDYFDY LCDR3 (SEQ ID No: 6) QQGNTLPLT

[0050] Example 2: Screening of mouse-derived antibody molecules

[0051] The present application immunizes mice with Siglec-15 antigen (Siglec-15 protein extracellular segment, the Siglec-15 antigen and protein used in subsequent experiments are Siglec-15 extracellular segment), optimizes the immunization method, creates a phage display library and establishes an antigen site screening method. The construction and screening identification of the phage display library are as follows:

[0052] Step one: immunizing mice with Siglec-15 antigen

[0053] 1. Experimental animals:

[0054] Species strain: BALB / c, female, mouse;

[0055] Body weight: 18-20g;

[0056] Supplier of experimental animals: Beijing Huafukang Biotechnology Co., Ltd.

[0057] 2. Immunization: immunize mice, and the immunization antigen is human Siglec-15 (synthetic gene by Nanjing Kingsriver Biotechnology Co., Ltd., and the vector is constructed and expressed and purified by the company).

[0058] Step two: construction of phage antibody library

[0059] The mouse spleen cells with higher titer were taken, and the total RNA in the mouse spleen cells was extracted by using Trizol reagent (purchased from Ambion, item number: 15596026), and the cDNA was obtained by RT-PCR, and the degenerate primers (the used degenerate primers refer to the reference: Journal of Immunological Methods 233 (2000) 167-177) were used for PCR amplification, so as to obtain the immune mouse antibody heavy chain variable region gene library (VH) and light chain variable region gene library (VL), and the light and heavy chains were double-digested respectively, and then were connected to the vector which was also treated by enzyme digestion in the same step, so as to construct the pScFv-Disb-HS-VH-VL gene library, and the pScFv-Disb-HS vector was modified by using a series of gene cloning methods on the vector pComb3 vector (purchased from the Chinese plasmid vector strain cell strain gene preservation center), so as to be used for the construction and expression of the phage single-chain antibody library. The modified vector is named as pScFv-Disb-HS vector, and its plasmid map is shown in Figure 1 , and the mouse immune phage antibody library is constructed based on the vector.

[0060] Step three: the Siglec-15 is used as an antigen to coat the immunotube, the antigen coating amount is 5 μg / 500 μL / tube, and the immunotube is coated at 4°C overnight, and then 4% skimmed milk / PBST is used for blocking the immunotube and the immune phage antibody library respectively, and the blocking is carried out at room temperature for 1 h. After the blocking, the immune phage antibody library is added into the immunotube for antigen-antibody combination, and the phage input amount is about 10 9 ~ 10 12 The phage is reacted at room temperature for 1 h, and then the unbound phage is washed away by using PBST-PBS, and then the phage is eluted by using 0.1M pH 2.2 Glycine-HCl, and finally the eluted phage antibody solution is neutralized to about pH 7.0 by using 1.5M pH 8.8 Tris-HCl.

[0061] Step four: the neutralized phage is used to infect 10 ml of TG1 bacterial liquid which is grown to the logarithmic phase, and the bacterial liquid is placed in a 37°C incubator for 30 min, and then part of the bacterial liquid is taken out for gradient dilution, and is coated on a 2YTAG plate for calculating the phage output. The remaining bacterial liquid is centrifuged, and the supernatant is discarded, and then the bacterial body is resuspended in a small amount of culture medium, and is sucked out and coated on a 2YTAG large plate for the next round of screening.

[0062] Step five: the bacterial body coated on the large plate is scraped off from the large plate, and is inoculated into 2YTAG liquid medium, and is shaken to the logarithmic phase, and then M13KO7 helper phage superinfection is added, and the phage is prepared by culturing at 28°C under the condition of 220 rpm overnight, and the phage is purified by PEG / NaCl sedimentation for the next round of screening, and a round of phage library enrichment screening is carried out.

[0063] Step six: Screening of Siglec-15 phage single-chain antibody positive clones: After one round of screening, well-separated single colonies were picked and inoculated into 96-well deep plates with 2YTAG liquid medium, and cultured at 37°C and 220 rpm until the logarithmic growth phase, about 10 10 μL helper phage M13KO7 was added, and the bacteria were infected at 37°C for 30 min. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the bacteria were resuspended and precipitated with 2YTAK. The bacteria were cultured overnight at 28°C and 220 rpm. After centrifugation at 4000 rpm and 4°C for 15 min, the amplified phage supernatant was aspirated for ELISA identification. Finally, four mouse antibody molecules with high affinity to Siglec-15 were screened, and were named MA-I, MA-II, MA-III, and MA-IV, respectively. The above obtained monoclonal antibodies were subjected to gene sequencing to determine the correct antibody sequence. After sequencing, the sequences of the four monoclonal antibodies screened above were as follows.

[0064]

[0065]

[0066] Example 3 Gradient dilution ELISA for comparing the affinity of anti-Siglec-15 phage monoclonal antibodies

[0067] The four mouse antibody molecules (MA-I, MA-II, MA-III, and MA-IV) obtained in Example 2 were subjected to monoclonal phage display and purification, and then subjected to phage gradient dilution ELISA to identify the affinity. The control antibody was a monoclonal antibody against SIGLEC-15 (also known as 5G12, patent application number 201780067999.3, and patent name Antibodies Against SIGLEC-15 and Methods of Use Thereof) from Nuscreen Corporation. The specific method was as follows:

[0068] The Siglec-15 antigen was coated with a carbonate buffer at pH 9.6, 100 ng / well / 100 μL, and coated overnight at 4°C, washed three times with PBST, and the four phage monoclonal antibodies screened in Example 2 were each diluted with PBST in a five-fold gradient, 100 μL of the diluted sample was added to each well, and incubated at room temperature for 1 hour. The ELISA plate was washed with PBST, and the HRP-anti-M13 (purchased from Bio-viewshine, item number: GE27-9421-01) monoclonal antibody diluted with PBST was added to the ELISA plate, and incubated at room temperature for 1 hour. The TMB color developing kit was used for color development, and color development was performed at room temperature for 10 minutes. After termination with 2M H2SO4, the microplate reader was read at 450 nm / 630 nm, and the corresponding EC50 value was calculated. The specific data are as follows:

[0069] Clone MA-I MA-II MA-III MA-IV 5G12 EC50 (ng / mL) 0.5995 7.805 21.9 23.96 1.629

[0070] As shown by the above data and as shown in Figure 2 , the four different murine antibody molecules screened in Example 2 can all bind to Siglec-15, and the monoclonal antibodies provided by the present application all have high affinity for Siglec-15.

[0071] Example 4

[0072] In Example 4 of the present application, the murine antibody molecule is further limited to include a heavy chain constant region and a light chain constant region. The heavy chain constant region is one of the constant regions of murine IgG1, IgG2a, IgG2b or IgG3, and the light chain constant region is a constant region of murine C k . The amino acid sequence of the constant region of IgG1 is shown in SEQ ID No: 16, the amino acid sequence of the constant region of IgG2a is shown in SEQ ID No: 17, the amino acid sequence of the constant region of IgG2b is shown in SEQ ID No: 18, and the amino acid sequence of the constant region of IgG3 is shown in SEQ ID No: 19.

[0073] Example 5 Preparation of Anti-Siglec-15 Monoclonal Antibody Murine Antibody Molecule

[0074] In Example 5 of the present application, the murine antibody molecule is preferably limited to include a heavy chain constant region of murine IgG1 (the amino acid sequence of which is shown in SEQ ID No: 16) and a light chain constant region of murine C k . The antibody preparation method is as follows:

[0075] 1. The coding genes for the heavy chain VH and light chain VL of the four monoclonal antibodies screened in Example 2 were cloned into the vector pTSE (e.g., ...) containing the heavy chain and light chain constant region genes, respectively. Figure 3 As shown in SEQ ID No: 16), the preferred heavy chain constant region is the mouse IgG1 type constant region (amino acid sequence shown in SEQ ID No: 16), and the light chain constant region is mouse C k Chain (amino acid sequence as shown in SEQ ID No:15), pTSE vector structure as shown Figure 3 As shown (for the preparation process of the pTSE vector, please refer to paragraph

[0019] on page 3 of the instruction manual CN103525868A).

[0076] 2. HEK293E cells were transiently transfected (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression. Four monoclonal antibodies were purified using an AKTA instrument via a protein A affinity column. Protein concentration was determined using a BCA kit (purchased from Beijing Huitian Oriental Technology Co., Ltd., catalog number BCA0020). Protein size was then identified by SDS-PAGE. The results are shown below. Figure 4 As shown, from left to right, the images represent non-reduced MA-I, MA-II, MA-III, and MA-IV, protein molecular weight Marker 1, protein molecular weight Marker 2, and reduced MA-I, MA-II, MA-III, and MA-IV murine anti-Siglec-15 monoclonal antibodies. The molecular weight of each band is consistent with the theoretical values.

[0077] Example 6: Binding experiment of mouse antibody with Siglec-15

[0078] Siglec-15 antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST for blocking at 37°C for 1 h, added different dilution concentrations of MA-I, MA-II, MA-III and MA-IV mouse antibody molecules, the starting highest concentration of the four antibody molecules was 5 μg / ml, respectively, 5-fold gradient dilution, each antibody was diluted for 8 gradients, incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, then added Goat Anti-Mouse IgG-HRP (purchased from solarbio, item number: SE131) diluted 1:2000 with 1% BSA-PBST, incubated at 37°C for 1 h. TMB color developing kit color development, 100 μL / well, color development at room temperature for 8 min, then color development was terminated with 2M H2SO4. The microplate reader was read at 450 nm / 630 nm, and the corresponding EC50 value was calculated, and the specific data were as follows:

[0079] Clone MA-I MA-II MA-III MA-IV 5G12 EC50 (ng / mL) 13.67 21.43 40.94 60.94 20.72

[0080] According to the above data and as shown in Figure 5 , the four different mouse antibody molecules screened can bind to Siglec-15.

[0081] Example 7: Mouse antibody inhibits the binding of Siglec-15 to Jurkat cell surface receptor

[0082] First, four mouse antibodies (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were respectively prepared into protein solutions with a concentration of 600 μg / mL, and 25 μL of each solution was added to a 96-well plate. Second, the Siglec-15 ligand protein was prepared into a solution with a concentration of 200 μg / mL, and 25 μL of the solution was added to the 96-well plate. Third, the Jurkat cell strain was counted, centrifuged, resuspended with PBS buffer, and adjusted to a cell density of 2E+6 cells / mL, and 50 μL of the cell solution was added to the 96-well plate. All samples and protein dilutions were performed using PBS buffer. Finally, the 96-well plate was placed in a 4°C incubator for 1 h. After removal, 100 μL of PBS buffer was added to each well, and the cells were centrifuged at 3000 rpm for one time to clean the cells, and the cell precipitate was collected. The AF488-anti human IgG-Fc antibody (purchased from SouthernBiotech, item number 2048-30) diluent prepared in advance was added to the cell precipitate, and the mixture was incubated at 4°C for 1 h. After removal, the mixture was centrifuged at 3000 rpm for one time, resuspended with 200 μL of PBS, and then detected by flow cytometry to collect the fluorescence signal in the FL1-A channel.

[0083] The results are shown in Table 2. Figure 6 As shown in Table 2, the four mouse antibodies screened in Example 2 can all inhibit the binding of Siglec-15 to the receptor on the surface of Jurkat cells, and are equivalent to the control antibody 5G12 at the same concentration.

[0084] Example 8: Inhibition of the binding of Siglec-15 to the receptor on the surface of CHOSLV-LRRC4C cells by mouse antibodies

[0085] Gradient dilution of four mouse-derived antibody molecules (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were prepared at a concentration of 200 pg / mL, 3x gradient dilution, a total of 8 gradients, 25 pL per well was added to the corresponding position of the 96-well plate. Dilute Siglec-15-Fc ligand protein, prepare a concentration of 40 pg / mL, 25 pL per well was added to the corresponding position of the 96-well plate. Count the CHOSLV-LRRC4C cell strain, take a certain number of cells, centrifuge and resuspend with PBS buffer, adjust the cell density to 2E+6 cells / mL, 50 pL per well was added to the 96-well plate. All sample and protein dilutions were performed using PBS buffer. The 96-well plate was placed at 4°C after the sample was added, and incubated for 1 h. After taking out, 100 pL of PBS buffer was added per well, and the cells were washed once by centrifugation at 3000 rpm, and the cell pellet was collected. Add AF488-anti human IgG-Fc antibody (purchased from SouthernBiotech, catalog number 2048-30) prepared in advance to the cell pellet, incubate at 4°C for 1 h, then wash once at 3000 rpm, resuspend in 200 pL of PBS, and then detect by flow cytometry, collect the fluorescence signal in the FL1-A channel. Draw the dose-effect curve, and calculate the inhibition of Siglec-15 ligand protein binding to cell surface LRRC4C receptor by the candidate molecule.

[0086] Candidate molecule 5G12 MA-I MA-II MA-III MA-IV IC50 (pg / mL) 0.5919 0.3645 0.6946 0.4400 3.918

[0087] Conclusion: Through the above data and Figure 7 It can be seen that the four mouse-derived candidate molecules (MA-I, MA-II, MA-III, MA-IV) can block the binding of Siglec-15 to its receptor.

[0088] Example 9 Mouse-derived antibodies promote T cell activation and proliferation

[0089] PBMC cells were collected after centrifugation, resuspended with RPMI1640 complete medium and counted, and the cell density was adjusted to 2E+6 cells / mL, 50 μL per well was added to a 96-well plate. The Siglec-15 ligand protein was prepared at a concentration of 20 μg / mL, 50 μL per well was added to the corresponding position of the 96-well plate. The final concentration of the anti-CD3 antibody was 0.5 μg / well, and the preparation concentration was 10 μg / mL, 50 μL per well was added to the corresponding position of the 96-well plate. Four mouse molecules (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were prepared at an initial concentration of 100 μg / mL, 3x gradient dilution, a total of 8 gradients. 50 μL per well was added to the corresponding position of the 96-well plate. The dilution of proteins and antibodies was carried out using RPMI1640 complete medium, the 96-well plate was mixed, and incubated at 37°C in the dark for 3 days. The cell culture supernatant was diluted 10 times and used for cytokine detection. The activation of natural T cells by anti-Siglec-15 mouse antibody molecules was evaluated from the following three aspects: human TNF-α cytokine release, human IFN-γ cytokine release, and T cell proliferation, which were performed as follows:

[0090] Human IFN-γ detection kit (purchased from Yikosai Biotechnology Co., Ltd., item number H008-96): Diluted supernatant and standard were added to sample wells, 100 μL per well, covered with sealing film, and incubated at room temperature for 1.5 h. After incubation, the plate was washed 3 times. Biotinylated antibody diluent in the human IFN-γ detection kit was added, 100 μL per well, covered with sealing film, and incubated at room temperature for 1 h. After incubation, the plate was washed 3 times. Streptavidin-HRP working solution was added, 100 μL per well, covered with sealing film, and incubated at room temperature for 30 min. After incubation, the plate was washed 3 times. TMB color developing solution was added, 100 μL per well, and incubated at room temperature for about 15 minutes. 100 μL of stop solution was added to each well to terminate the reaction. After reading the OD value by the enzyme-labeled instrument, the dose-effect curve was drawn.

[0091] Human TNF-a detection kit (purchased from Eko-Ca Biotech Co., Ltd., item number EM008-96): Diluted supernatant and standard were added to sample wells, 100 μL per well, covered with sealing film, and incubated at room temperature for 1.5 h. After incubation, the plate was washed 3 times. Biotinylated antibody diluent was added, 100 μL per well, covered with sealing film, and incubated at room temperature for 1 h. After incubation, the plate was washed 3 times. Streptavidin-HRP working solution in the human IFN-γ detection kit was added, 100 μL per well, covered with sealing film, and incubated at room temperature for 30 min. After incubation, the plate was washed 3 times. TMB color developing solution was added, 100 μL per well, and incubated at room temperature for about 15 min. 100 μL of stop solution was added to each well to terminate the reaction. After reading the OD value by a microplate reader, a dose-effect curve was drawn.

[0092] The cell pellet was collected, and the cells were stained with CD3e Monoclonal Antibody (purchased from Thermo Fisher Scientific, item number MA1-10177), and incubated at room temperature in the dark for 15 min. After washing the plate once, the cells were resuspended in 100 μL of PBS buffer, and absolute counting was performed by flow cytometry. A dose-effect curve was drawn.

[0093]

[0094] From the above data and Figure 8-10 It can be seen that the four mouse antibody molecules (MA-I, MA-II, MA-III, and MA-IV) can all block the binding of Siglec-15 ligand protein to the natural T cell surface receptor by binding to Siglec-15, block the intracellular inhibitory signal pathway, activate T cells, and promote T cell proliferation and activation (release of TNF-a and IFN-γ).

[0095] Example 10: Detection of biological activity of mouse antibody molecules (reporter gene method)

[0096] Jurkat-NFAT-Luc engineered cell line was counted, and the cell density was adjusted to 2E+6 cells / mL using sample diluent (the components of which include 90% RPMI1640, 10% FBS, 0.5 μg / ml Puromycin). After gentle mixing, 50 μL / well of cell solution was added to a 96-well plate. Four mouse-derived molecules (MA-I, MA-II, MA-III, and MA-IV) were diluted to an initial concentration of 800 μg / mL using sample diluent, 5-fold gradient dilution, a total of 8 gradients, 50 μL / well, and added to the corresponding positions of the 96-well plate. Each sample concentration was set up in duplicate wells. Siglec-15 antigen was prepared, and 50 μL / well was added to the 96-well plate to make the final concentration 16 μg / mL. Human CD3 antibody (purchased from Yewu Shenzhou Biotechnology Co., Ltd., item number 10977-H001) was prepared, and 50 μL / well was added to the 96-well plate to make the final concentration 1 μg / mL. The cell culture plate was gently mixed and incubated in a 37°C CO2 incubator for 6 h. The supernatant was discarded by centrifugation, and 10 μL of lysis solution was added to each well of a 384-well plate. An equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., item number E2610) was added, and the reaction was carried out at room temperature for 5 min. The fluorescence value was read on a microplate reader, and the corresponding IC50 value was calculated. The specific data are as follows:

[0097] Candidate molecule 5G12 MA-I MA-II MA-III MA-IV EC50 (pg / mL) 0.3059 0.1138 0.1448 0.3244 0.4852

[0098] As shown by the above data and Figure 11 , the four different mouse-derived antibodies and the control antibody selected can bind to Siglec-15 and inhibit the binding of Siglec-15 to the surface receptor of Jurkat cells, block the intracellular inhibitory signal pathway, and reactivate T cells. The construction of the engineered cell line Jurkat-NFAT-Luc can simulate T cells. Siglec-15 inhibits and down-regulates the intracellular activation signal pathway (NFAT-Luc) by binding to the surface receptor of T cells. The four mouse-derived antibody molecules can effectively block the binding of Siglec-15 to the cell surface receptor and reactivate the intracellular signal pathway of T cells.

[0099] Example 11

[0100] The anti-Siglec-15 monoclonal antibody or antigen binding fragment thereof in the embodiment 11 of the present application is further defined as a chimeric antibody molecule, which includes the heavy chain variable region of the murine antibody molecule in the embodiment 2, the light chain variable region of the murine antibody molecule, and a humanized antibody constant region. The humanized antibody constant region includes a humanized antibody heavy chain constant region and a humanized antibody light chain constant region. The humanized antibody heavy chain constant region is one of the constant regions of human IgG1, IgG2 or IgG4, the amino acid sequence of the constant region of IgG1 is shown in SEQ ID No: 20, the amino acid sequence of the constant region of IgG2 is shown in SEQ ID No: 21, and the amino acid sequence of the constant region of IgG4 is shown in SEQ ID No: 22. The humanized antibody light chain constant region is a constant region of human C k .

[0101] Preparation of the chimeric antibody molecule in the embodiment 12

[0102] The humanized antibody constant region in the embodiment 12 of the present application is further defined based on the embodiment 7, and includes a heavy chain constant region of human IgG1 (the amino acid sequence of which is shown in SEQ ID No: 20) and a light chain constant region of human C k .

[0103] Specific preparation method

[0104] The heavy chain variable region VH (SEQ ID No: 7) and the light chain variable region VL gene (SEQ ID No: 8) of the antibody molecule MA-I obtained by screening the phage antibody library in the embodiment 2 are kept as the murine sequences, and are respectively cloned into the vector pTSE (as shown in SEQ ID No: 24) loaded with the heavy chain constant region and the light chain constant region genes. The heavy chain constant region is human IgG1 (the amino acid sequence of which is shown in SEQ ID No: 20), and the light chain constant region is human C k . Figure 3 The HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, with the item number GNHu43) are transiently transfected to express the antibody, and the chimeric antibody CA-I is obtained.

[0105] Humanization of the murine antibody molecule in the embodiment 13

[0106] First, the sequence of the murine antibody molecule MA-1 from Example 2 was compared with the human antibody lineage database (v-base) to identify human antibody light and heavy chain lineages with high homology as candidate sequences. Then, the CDR sequence of the murine antibody molecule MA-I was transplanted onto the human candidate sequences for homology modeling. Next, three-dimensional structural simulations were used to calculate key framework amino acid residues that might play an important role in maintaining the CDR ring structure, thereby designing reversion mutations for humanized antibodies. The light and heavy chain variable region sequences of the designed humanized antibodies containing reversion mutations were optimized and synthesized by Nanjing Genscript Biotech Co., Ltd., and then ligated into a transient expression vector. Analysis of the humanized light and heavy chain combinations yielded the following humanized antibody molecules: HA-I and HA-II. The two monoclonal antibody sequences screened above are as follows.

[0107]

[0108] Example 14

[0109] Example 14 of the present invention further specifies, based on Example 13, that the humanized antibody molecule also includes a humanized antibody constant region; the humanized antibody constant region includes a heavy chain constant region selected from human IgG1, IgG2 or IgG4 and human C k The amino acid sequences of the light chain constant region of type IgG1, the heavy chain constant region of type IgG2 are shown in SEQ ID No:21, and the heavy chain constant region of type IgG4 are shown in SEQ ID No:22. Human C k The amino acid sequence of the light chain constant region of the type is shown in SEQ ID No:23.

[0110] The specific sequence of the constant region of the above-mentioned humanized antibody is the same as that in Example 11.

[0111] Example 15 Preparation of humanized antibody molecules

[0112] Example 15 of the present invention further defines the humanized antibody constant region based on Example 10, including the heavy chain constant region of human IgG1 type (whose amino acid sequence is shown in SEQ ID No: 20) and human C k The light chain constant region of type (its amino acid sequence is shown in SEQ ID No:23).

[0113] The coding genes for the heavy chain VH and light chain VL of the two humanized antibody molecules obtained in Example 13 were cloned into the vector pTSE (e.g., ...) containing the heavy chain constant region and light chain constant region genes, respectively. Figure 3The heavy chain constant region is human IgGl type (amino acid sequence as shown in SEQ ID NO: 20), and the light chain constant region is C k The heavy chain constant region is human IgGl type (amino acid sequence as shown in SEQ ID NO: 20), and the light chain constant region is C

[0114] The humanized antibody molecules HA-I and HA-II were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number GNHu43) for antibody expression. The monoclonal antibodies were purified by protein A affinity column using AKTA instrument, and the protein concentration was determined using BCA kit (purchased from Beijing Huitian Dongfang Science and Technology Co., Ltd., item number BCA0020). The protein size was identified by SDS-PAGE, and the results are shown in Figure 12. From left to right, the non-reduced protein molecular weights of HA-I, HA-II, chimeric antibody CA-I prepared in Example 12, non-reduced protein molecular weight Marker 1 and reduced protein molecular weight Marker 2, HA-I, HA-II, and chimeric antibody CA-I are shown. The molecular weight of each band is consistent with the theoretical value. Figure 12

[0115] Example 16 Binding experiment of humanized antibody molecules with Siglec-15

[0116] Siglec-15 antigen was coated with carbonate buffer at pH 9.6, 200 ng / well / 100 μL, and coated overnight at 4°C. Washed five times with 300 μL / well PBST, then added 1% BSA-PBST for blocking at 37°C for 1 h. Added humanized antibodies HA-I and HA-II and chimeric antibody CA-I prepared in Example 12 at different dilution concentrations, with the highest starting concentration of 50 μg / mL for each of the three antibodies. After 3-fold dilution, 10 gradients were prepared for each antibody. Incubated at 37°C for 1 h. Washed five times with 300 μL / well PBST, then added Goat Anti-Human IgG-HRP diluted 1:5000 with 1% BSA-PBST (purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., item number ZB-2304) and incubated at 37°C for 1 h. Color development with TMB color development kit, 100 μL / well, color development at room temperature for 5 min, then color development was terminated with 2M H2SO4. Readings were taken at 450 nm / 630 nm using a microplate reader, and the corresponding EC50 values were calculated. The specific data are as follows:

[0117]

[0118] The above data and experimental results are shown in Figure 13. Figure 13 ​As shown, both different humanized antibody molecules can bind to Siglec-15, and the EC50 values ​​of both humanized antibody molecules are close to those of the chimeric antibody CA-I, indicating that the humanized antibody molecules retain the high binding ability of the mouse parent antibody MA-I to Siglec-15.

[0119] Example 17 Cross-binding experiment of humanized antibody with Siglec-15 from different species

[0120] Human Siglec-15, mouse Siglec-15-His (purchased from Nearshore Protein Technology Co., Ltd., catalog number: CW71), and cynomolgus monkey Siglec-15-His (purchased from Nearshore Protein Technology Co., Ltd., catalog number: CW70) were coated with 100 ng / well / 100 μL of carbonate buffer at pH 9.6 and incubated overnight at 4°C. The cells were washed five times with 300 μL / well of PBST, then blocked with 1% BSA-PBST at 37°C for 1 h. Different dilutions of humanized antibodies HA-I and HA-II were added. The initial maximum concentration of both humanized antibodies was 25 μg / mL. Eight gradients were performed for each antibody after 5-fold dilution, and the cells were incubated at 37°C for 1 h. The cells were washed five times with 300 μL / well of PBST, then Goat Anti Human IgG-HRP diluted 1:5000 with 1% BSA-PBST was added, and the cells were incubated at 37°C for 1 h. The TMB chromogenic kit was used for color development. 100 μL / well was incubated at room temperature for 5 min, then the color development was stopped with 2 M H₂SO₄. Readings were taken at 450 nm and 630 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Specific data are as follows:

[0121]

[0122] Based on the above data and as follows Figure 14 As shown, the two different humanized antibody molecules screened can both bind to human Siglec-15, cynomolgus monkey Siglec-15, and mouse Siglec-15.

[0123] Example 18: Humanized antibody molecules inhibit the binding of Siglec-15 to the Jurkat cell surface receptor.

[0124] Gradient dilution of two humanized antibody molecules (HA-I, HA-II) and control antibody 5G12, concentration of 200 μg / mL, 5 gradient dilutions, a total of 8 gradients, 25 μL per well added to the corresponding position of the 96-well plate. The Siglec-15 protein was labeled with a FITC fluorescent labeling kit (purchased from Thermo Fisher Scientific, catalog number F6434) to prepare Siglec-15-FITC protein. The concentration of Siglec-15-FITC protein was adjusted with PBS buffer, and a concentration of 40 μg / mL was prepared, 25 μL per well added to the corresponding position of the 96-well plate. The Jurkat cell line was counted, a certain amount of cells were centrifuged and resuspended with PBS buffer, the cell density was adjusted to 2E+6 cells / mL, 50 μL per well added to the 96-well plate. All sample and protein dilutions were performed using PBS buffer. The 96-well plate after sample addition was placed at 4°C for incubation for 1 h. After removal, 100 μL of PBS buffer was added per well, and the cells were washed once by centrifugation at 3000 rpm, the cell precipitate was collected, resuspended with 200 μL of PBS, and then detected by flow cytometry, and the fluorescence signal in the FL1-A channel was collected. The dose-effect curve was drawn, and the binding of the candidate molecule to the Siglec-15 ligand protein and the Jurkat cell surface receptor was calculated.

[0125]

[0126] From the above data and Figure 15 It can be seen that both humanized candidate molecules (HA-I, HA-II) can block the binding of Siglec-15 to the receptor on the surface of Jurkat cells.

[0127] Example 19 Biological activity detection of humanized antibody molecules (reporter gene)

[0128] Jurkat-NFAT-Luc engineered cell line was counted, and the cell density was adjusted to 2E+6 cells / mL using sample diluent (the components of which include 90% RPMI1640, 10% FBS, 0.5 μg / ml Puromycin). After gentle mixing, 50 μL / well of cell solution was added to a 96-well plate. Two humanized antibody molecules (HA-I, HA-II) were diluted to an initial concentration of 800 μg / ml using sample diluent, 5-fold gradient dilution, a total of 8 gradients, 50 μL / well, added to the corresponding positions of the 96-well plate, and two replicate wells were set up for each sample concentration. Siglec-15 antigen was prepared, 50 μL per well was added to the 96-well plate, and the final concentration was adjusted to 16 μg / mL. Anti-CD3 antibody (purchased from Yewu Shenzhou Biotechnology Co., Ltd., item number 10977-H001) was prepared, 50 μL per well was added to the 96-well plate, and the concentration was adjusted to 1 μg / mL. The cell culture plate was gently mixed and incubated in a 37°C CO2 incubator for 6 h. The supernatant was discarded by centrifugation, 10 μL of lysis solution was added per well to a 384-well plate, an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., item number E2610) was added, and the reaction was carried out at room temperature for 5 min. The fluorescence value was read on a microplate reader, and the corresponding IC50 value was calculated. The specific data are as follows:

[0129]

[0130] As shown by the above data and Figure 16 , the two humanized antibody molecules screened can bind to Siglec-15 and inhibit the binding of Siglec-15 to the receptor on the surface of Jurkat cells, block the inhibitory signal pathway in the cell, and reactivate T cells.

[0131] Example 20 Inhibition of MC38-Siglec-15 colorectal cancer in mice by anti-Siglec-15 monoclonal antibody HA-I

[0132] 1. Experimental animals: species and strain: C57BL / 6JGpt mice; age: 6-8 weeks;

[0133] Supplier of experimental animals: Jiangsu Jizhuangkang Biotechnology Co., Ltd.

[0134] 2. Cell culture: MC38 tumor cells (YK-CL-256-02) (purchased from Biovector NTCC Inc., catalog number: NTCC-MC38) were used as the original cells to construct the MC38-Siglec-15 tumor cell line. Tumor cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum (ExCell Bio, catalog number: FND500), 100 U / mL penicillin, 100 μg / mL streptomycin, 250 μg / mL Hygromycin B (purchased from Gibco, catalog number: 10687010), and 2 mM glutamine at 37°C in a 5% CO2 incubator. After the cells reached confluence every 3 to 4 days, they were passaged into individual flasks. Tumor cells in the logarithmic growth phase were used for in vivo tumor inoculation.

[0135] Bone marrow-derived macrophages (BMDM) were isolated from C57BL / 6 mice. After culturing in RPMI 1640 medium (Thermo Fisher Scientific (China) Co., Ltd. (Gibco), catalog number A10491-01) containing inactivated 10% fetal bovine serum (ExCell Bio, catalog number: FND500), 100 U / mL penicillin, 100 μg / mL streptomycin, 20 ng / mL mouse M-CSF (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 51112-MNAH), and 20 ng / mL mouse IL-10 (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 50245-MNAE) for 4 days in an incubator at 37°C and 5% CO2, they were ready for use in in vivo tumor models.

[0136] 3. Inoculation and grouping of tumor cells: MC38-Siglec-15 tumor cells resuspended in PBS at a cell density of 1.0 × 10⁻⁶ cells / year. 6 / mL, mixed evenly with a certain amount of BMDMs cell suspension, and inoculated subcutaneously into the right flank of experimental animals, 100μL / animal. Inoculation continued until the tumor grew to 43mm. 3 Two groups of five animals were administered the drug at approximately 10:00-10:00 AM. The control group consisted of a vehicle (intra-pharmacy, 1 tw x 3 w) and a HA-1 (10 mg / kg, intra-pharmacy, 1 tw x 3 w).

[0137] 4. Detection index: The tumor volume was measured twice a week using a vernier caliper, and the long diameter and short diameter of the tumor were measured, and the volume calculation formula was: volume = 0.5 x long diameter x short diameter 2 ; record the change of tumor volume

[0138] The experimental results are shown in Figure 17 .

[0139] The Figure 17 data show that the anti-Siglec-15 monoclonal antibody HA-1 can inhibit the growth of tumors, and shows a dose-dependent response.

[0140] Example 21 Evaluation of the thermal stability of anti-Siglec-15 monoclonal antibody HA-I

[0141] The thermal stability of anti-Siglec-15 monoclonal antibody HA-I was evaluated using a multi-functional protein thermal stability analysis system (purchased from Unchained Labs). Changes in protein conformation were detected by monitoring changes in intrinsic fluorescence of the protein with temperature (starting from 25°C, increasing to 95°C at a temperature increasing rate of 0.3°C / min), so as to determine the protein melting temperature Tm and evaluate the stability of the protein conformation. When the sample aggregates, it will cause interference of scattered light waves, and the scattered light signal will increase, and the colloidal stability of the protein is determined by static light scattering (characterized by Tagg), and the results are shown in the following table and Figure 18 .

[0142]

[0143] The temperature of anti-Siglec-15 monoclonal antibody HA-I was 72.9°C, and the average Tagg was 72.0°C, showing good conformational stability and colloidal stability.

[0144] Example 22

[0145] The anti-Siglec-15 monoclonal antibody of the embodiment 22 of the present application is further limited to a protein content of 40-100 mg / ml based on the embodiment 1.

[0146] Example 23

[0147] The concentration of the buffer salt in the embodiment 23 of the present application is further limited to 5-50 mM based on the embodiment 1.

[0148] Preferably, the buffer salt is one or more combinations of phosphate buffer, citrate buffer, and acetate buffer.

[0149] Example 24

[0150] The embodiment 24 of the present application is further limited on the basis of the embodiment 1 that the concentration of the protein protective agent is 10-200 mM.

[0151] Preferably, the protein protective agent is one or more combinations of sucrose, mannitol, trehalose, arginine hydrochloride, glycine, proline.

[0152] Embodiment 25

[0153] The embodiment 25 of the present application is further limited on the basis of the embodiment 1 that the injection preparation further comprises an isotonicity adjusting agent with a concentration of 0-100 mM.

[0154] Preferably, the isotonicity adjusting agent is a sodium chloride solution.

[0155] Embodiment 26

[0156] The embodiment 26 of the present application is further limited on the basis of the embodiment 1 that the content of the surfactant is 0.005%-0.02% (w / v).

[0157] Preferably, the surfactant is one or more combinations of polysorbate 20, polysorbate 80, poloxamer.

[0158] Embodiment 27

[0159] The embodiment 27 of the present application is further limited on the basis of the embodiment 1 that the pH value of the injection preparation is 5.5-6.5; the protein content of the anti-Siglec-15 monoclonal antibody is 70-90 mg / ml; the concentration of the buffer salt is 10-20 mM; the protein protective agent is one or more combinations of mannitol, sorbitol, trehalose, sucrose, maltose, glycine, proline, methionine, lysine hydrochloride, arginine hydrochloride, preferably trehalose, especially alpha-trehalose; the injection preparation further comprises an isotonicity adjusting agent with a concentration of 20-50 mM, and the isotonicity adjusting agent is preferably a sodium chloride solution; and the surfactant is preferably polysorbate 20.

[0160] Selection of buffer and pH value range in the preparation of embodiment 28

[0161] The present application provides a preparation method of the injection preparation of the anti-Siglec-15 monoclonal antibody: the anti-Siglec-15 monoclonal antibody HA-1 is ultrafiltrated and exchanged into a buffer with different pH values, sterilized and filtered through a 0.22 μm filter, and then divided and packaged into 2 ml vials, 1 ml / vial.

[0162] (1) Selection of pH value range

[0163] After the sub-packaging is completed, the protein thermal stability parameters under different buffer conditions are detected, then the sample is placed in a 40±2℃ stability test box, and the stability of the protein concentration, purity (SEC-HPLC), charge isomer and particle size distribution is investigated, and the relatively stable pH range is screened.

[0164] Analysis and detection method: the thermal stability is detected by using a multifunctional protein stability analysis system (UNcle) to detect the melting temperature (Tm) and the aggregation temperature (Tagg); the protein concentration is detected by using an ultraviolet spectrophotometry; the purity is detected by using a molecular exclusion high performance liquid chromatography (SEC-HPLC); the charge isomer is detected by using a weak cation exchange chromatography (CEX-HPLC); the appearance is visually detected by using a clarity meter; the sub-visible microparticles are detected by using a microflow imaging particle method; and the particle size distribution is detected by using a dynamic light scattering module of the multifunctional protein stability analysis system (UNcle).

[0165] The experimental group is designed as follows:

[0166]

[0167] The thermal stability detection results are as follows:

[0168]

[0169] The 40±2℃ accelerated stability investigation results are as follows:

[0170]

[0171] The protein is treated at a higher protein concentration of 100 mg / ml, and it is found that, during the sample treatment process, the anti-Siglec-15 monoclonal antibody HA-1 has inconsistent liquid exchange rates under different buffer conditions at different pH values, and after the accelerated stability is placed, the protein appears opalescence, and some samples also appear protein particulate matter, but the thermal stability and solubility of the protein are good, and the protein has the main conditions of preparation. According to the analysis of the thermal stability and the 40±2℃ accelerated stability investigation results, the pH value of the injection preparation is in the range of 5.5-7.5, and the experimental examples 2-4 can ensure the stability of the anti-Siglec-15 monoclonal antibody HA-1 protein, more preferably, the pH value of the injection preparation provided in the experimental examples 2 and 3 is in the range of pH 5.5-6.5, which can more effectively maintain the stability of the anti-Siglec-15 monoclonal antibody HA-1 protein, therefore, the pH value range of the buffer of the injection preparation of the anti-Siglec-15 monoclonal antibody provided in the application is preferably 5.5-6.5.

[0172] (ii) Buffer type screening

[0173] The screening of buffer system and pH is carried out in a buffer with a buffer capacity of about 5.5-6.5, and the screening of the type of preparation buffer is carried out by indexes such as Tm, Tagg, particle size, protein concentration, purity (SEC-HPLC), charge isomer (WCX-HPLC), sub-visible particles, viscosity and CE-SDS (NR), and the protein concentration is set to 80 mg / ml, and the buffer design composition is as follows:

[0174]

[0175] The thermal stability detection results are as follows:

[0176]

[0177] The accelerated stability investigation results (80 mg / ml concentration) at 40±2 ℃ are shown in the following table:

[0178] The results show that after 2 weeks of acceleration at 40±2 ℃, the proportion of aggregates of experimental examples 11-13 increases more obviously, the purity of the main peak of charge isomers of experimental example 12 decreases the most, the CE-SDS (NR) purity of the samples of each experimental example decreases in different degrees, and the decrease proportion of experimental example 12 is the most, which is 5.9%, and the monomer purity of experimental examples 11-13 decreases relatively more, which decreases by about 3%, therefore, the stability of the buffer salt in experimental examples 6-10 and 14-15, which is one of acetate buffer, citrate buffer or phosphate buffer, is better than that of the stability of the hydrochloric acid histidine buffer of experimental examples 11-13, so the preferred buffer salt of the present application is acetate buffer, citrate buffer or phosphate buffer.

[0179] In addition, among the SEC-HPLC purity (proportion of aggregate growth), the purity of the main peak of charge isomers, the CE-SDS (NR) purity of the samples of each experimental example and the monomer purity, experimental example 7 is obviously better than experimental example 6, experimental example 9 is obviously better than experimental examples 8 and 10, and experimental example 14 is obviously better than 15, so the preferred pH value of the buffer salt of the present application is 6.0.

[0180] The B 22 and k D The suitable buffer system is further screened through B 22 and k DThe determination of B and k further compared the stability of the protein in these buffers. The anti-Siglec-15 monoclonal antibody HA-1 protein concentration exchange was performed again using the three buffers, and the buffer composition and samples are as follows:

[0181]

[0182] Then the samples after exchange were diluted to 10 mg / ml, 8 mg / ml, 6 mg / ml, 4 mg / ml, 2 mg / ml in five series of concentrations using the corresponding formulation buffer, respectively, and three repeated holes were set for each concentration sample (i.e. one sample was repeated three times), and the blank formulation buffer was single-hole sampled, and B 22 and k D detection, and the results are shown in the following table:

[0183]

[0184] B 22 and k D The results of the investigation show that the B 22 of anti-Siglec-15 monoclonal antibody HA-1 in three buffer conditions is positive, and the k D is negative, and when the positive and negative are inconsistent, the B 22 is given priority. When the pH value is 6, the B 22 value of the protein sample of experimental example 16 is significantly greater than that of experimental examples 17 and 18, indicating that the protein sample of experimental example 16 has a relatively better repulsion in 20 mM acetate buffer. Based on the results of the thermal stability, the accelerated stability investigation at 40±2℃ (using protein concentration, purity, charge isomer, CE-SDS(NR), viscosity, and sub-visible particles as indicators) and B 22 , k D analysis, the preferred buffer for anti-Siglec-15 monoclonal antibody injection formulation is 20 mM acetate buffer, pH 6.0.

[0185] Example 29: Screening of other excipient types

[0186] Based on the buffer selected by Example 28, which is 20 mM acetate buffer with pH 6.0, this Example 29 further screens other excipient types. The detection method and screening method of the formulation refer to Example 28.

[0187] (I) Screening of protein protectants and surfactants

[0188]

[0189] The samples with the label were placed in the stability test box, and the stability investigation items and investigation time point data are as follows:

[0190]

[0191] The results of the accelerated stability test at 40±2℃ are as follows:

[0192]

[0193] Note: In the appearance test, A represents "colorless and clear liquid, no visible foreign matter", and B represents "slight opalescence".

[0194] From the above data, it can be concluded that, compared with other experimental examples, experimental example 21 in which the protein protective agent is trehalose is better in terms of purity (SEC-HPLC), charge isomer, sub-visible particles, CE-SDS (NR), etc., so it can be concluded that the preferred protein protective agent of the present application is trehalose.

[0195] In addition, surfactants are added in experimental examples 29 to 31, and the sub-visible particles perform better, and the sample in experimental example 30 in which the surfactant is polysorbate 20 is relatively better; therefore, the preferred surfactant of the present application is preferably polysorbate 20.

[0196] (ii) Screening of isotonicity regulators and surfactants

[0197] Through the above screening of protein protective agents and surfactants, the preferred protein protective agent is trehalose and the preferred surfactant is polysorbate 20, and through the thermal stability and 40±2℃ accelerated stability, the content of polysorbate 20 and the isotonicity regulator are further screened, and the specific components are as follows.

[0198]

[0199] The sample with the label is placed in the stability test box, and the stability test items and test time points are as follows:

[0200]

[0201] After being placed at 40±2℃ for 4 weeks, the stability test results of each sample are shown in the following table:

[0202]

[0203] Note: In the appearance test, A represents "colorless and clear liquid, no visible foreign matter", and B represents "slight opalescence".

[0204] From the above data, first, the results of the appearance (opalescence), thermal stability, charge isomer, purity (SEC-HPLC), CE-SDS (NR), and sub-visible particles show that the selection of sodium chloride as the isotonicity adjusting agent can enhance the opalescence and sub-visible particles of the anti-Siglec-15 monoclonal antibody protein, and compared with other experimental examples, considering the principle of as few as possible under the condition of meeting the requirements of the formulation components, the combination of trehalose and sodium chloride in experimental example 36 can reduce the amount of sugar, thereby saving costs; second, too much or too little surfactant is not conducive to maintaining the stability of the anti-Siglec-15 monoclonal antibody protein, and the present application preferably uses 0.02% (w / v) polysorbate 20 as the surfactant.

[0205] In summary, through the above screening of excipients, the present application preferably provides an injection preparation of anti-Siglec-15 monoclonal antibody, which comprises the following components:

[0206] (i) anti-Siglec-15 monoclonal antibody HA-1;

[0207] (ii) 5-50 mM acetate buffer;

[0208] (iii) 10-200 mM trehalose;

[0209] (iv) 0-100 mM sodium chloride solution;

[0210] (v) 0.02% (w / v) polysorbate 20;

[0211] The pH value of the injection preparation is 6.0.

[0212] Example 30 prescription confirmation

[0213] Three batches of samples with different concentrations were prepared by the above preferred injection preparation formula, and long-term, accelerated and influence factor stability tests were carried out on the formulation prescription. The stability of the formulation prescription during production, storage and use was simulated, and the sample composition was as follows:

[0214] Experimental group Buffer composition Protein stabilizer Isotonicity adjusting agent Surfactant pH Protein concentration (mg / ml) Experimental Example 40 20 mM acetate buffer 120 mM trehalose 50 mM sodium chloride 0.02% (w / v) polysorbate 20 6 40 mg / ml of anti-Siglec-15 monoclonal antibody HA-1 Experimental Example 41 20 mM acetate buffer 120 mM trehalose 50 mM sodium chloride 0.02% (w / v) polysorbate 20 6 90 mg / ml of anti-Siglec-15 monoclonal antibody HA-1 Experimental Example 42 20 mM acetate buffer 120 mM trehalose 50 mM sodium chloride 0.02% (w / v) polysorbate 20 6 100 mg / ml of anti-Siglec-15 monoclonal antibody HA-1

[0215] The test plan is as follows:

[0216]

[0217] The stability test results after 12 months at 2-8°C and the accelerated stability test results at 25±2°C are shown in the following table:

[0218]

[0219] The stability investigation results under the shaking condition simulating transportation and the light stability investigation results are shown in the following table:

[0220]

[0221] In summary, the injection preparations of different concentrations of anti-Siglec-15 monoclonal antibodies provided by the present application can effectively improve the appearance (opalescence) and sub-visible particle performance of anti-Siglec-15 monoclonal antibody proteins, and each index meets the requirements of pharmaceutical properties, and can maintain the tolerance of the preparation to various influencing factors during production, storage, transportation and use.

[0222] The present application is not limited to the above best mode, and anyone can derive other various forms of products under the inspiration of the present application, but regardless of any changes in shape or structure, any technical solution with the same or similar to the present application falls within the scope of the present application.

Claims

1. An injection preparation of an anti-Siglec-15 monoclonal antibody, characterized by, The injectable formulation comprises the following components: (i) Anti-Siglec-15 monoclonal antibody; (ii) Buffer salts; (iii) Protein protectants; (iv) Surfactants; The pH value of the injectable formulation is 5.5-7.5; The anti-Siglec-15 monoclonal antibody comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3), respectively. The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:2, the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:3, the amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:

6.

2. The injection preparation of the anti-Siglec-15 monoclonal antibody according to claim 1, characterized by, The anti-Siglec-15 monoclonal antibody is a murine antibody molecule, which includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

8.

3. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 2, characterized in that, The murine antibody molecule further comprises a heavy chain constant region which is one of a murine IgGl, IgG2a, IgG2b or IgG3 type constant region, and a light chain constant region which is a murine C k type constant region having an amino acid sequence as shown in SEQ ID No: 15, the IgGl type constant region having an amino acid sequence as shown in SEQ ID No: 16, the IgG2a type constant region having an amino acid sequence as shown in SEQ ID No: 17, the IgG2b type constant region having an amino acid sequence as shown in SEQ ID No: 18, and the IgG3 type constant region having an amino acid sequence as shown in SEQ ID No:

19.

4. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 1, characterized in that, The anti-Siglec-15 monoclonal antibody is a humanized antibody molecule, which includes a heavy chain variable region and a light chain variable region, and is selected from any one of the following: HA-Ⅰ: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:24, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:25; HA-Ⅱ: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No:24, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:

26.

5. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 4, characterized in that, The humanized antibody molecule further comprises a humanized antibody constant region, the humanized antibody constant region comprising a humanized antibody heavy chain constant region and a humanized antibody light chain constant region, the humanized antibody heavy chain constant region being one of a constant region of human IgG1 type, IgG2 type or IgG4 type, the amino acid sequence of the constant region of IgG1 type being as shown in SEQ ID No: 20, the amino acid sequence of the constant region of IgG2 type being as shown in SEQ ID No: 21, the amino acid sequence of the constant region of IgG4 type being as shown in SEQ ID No: 22, the humanized antibody light chain constant region being a constant region of human C k type with the amino acid sequence as shown in SEQ ID No:

23.

6. The injectable formulation of the anti-Siglec-15 monoclonal antibody according to any one of claims 1-5, characterized in that, The protein content of the anti-Siglec-15 monoclonal antibody is 40-100 mg / ml.

7. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 6, characterized in that, The protein content of the anti-Siglec-15 monoclonal antibody is 70-90 mg / ml.

8. The injectable formulation of the anti-Siglec-15 monoclonal antibody according to any one of claims 1-5, characterized in that, The concentration of the buffer salt is 5-50 mM.

9. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 8, characterized in that, The buffer salt is one or more combinations of phosphate buffer, citrate buffer, and acetate buffer.

10. The injectable formulation of the anti-Siglec-15 monoclonal antibody according to any one of claims 1-5, characterized in that, The concentration of the protein protectant is 10-200 mM.

11. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 10, characterized in that, The protein protectant is one or more combinations of mannitol, sorbitol, trehalose, sucrose, maltose, glycine, proline, methionine, lysine hydrochloride, and arginine hydrochloride.

12. The injectable formulation of the anti-Siglec-15 monoclonal antibody according to any one of claims 1-5, characterized in that, The injectable formulation also includes an isotonic adjuster with a concentration of 50-100 mM.

13. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 12, characterized in that, The isotonicity regulator is a sodium chloride solution.

14. The injectable formulation of the anti-Siglec-15 monoclonal antibody according to any one of claims 1-5, characterized in that, The surfactant content is 0.005%-0.02% (w / v).

15. The injectable formulation of the anti-Siglec-15 monoclonal antibody as described in claim 14, characterized in that, The surfactant is one or more of polysorbate 20, polysorbate 80, and poloxamer.

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