Application of CD22 antibody in preparation of medicine for treating spinal cord injury related diseases

Abstract

The invention discloses application of an antibody of a cell surface adhesion molecule CD22 in preparation of drugs for treating spinal cord injury related diseases. The CD22 antibody is used for successfully blocking the function of CD22 in microglial cells, cicatrization after spinal cord injury is inhibited, and functional recovery of fine movement of hind limbs of mice after spinal cord injury is promoted. The invention provides a new possibility for development of drugs for spinal cord injury and treatment of spinal cord injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a CD22 antibody in the preparation of a drug for treating spinal cord injury-related diseases. Background Art

[0002] CD22 is a type I transmembrane protein initially discovered on the surface of B cells, which participates in regulating B cell receptor (BCR) signaling to inhibit excessive immune responses. CD22 has multiple ligands, and its extracellular domain can specifically bind to α2-6 sialic acid residues present on various cell surface glycoproteins, and inhibit B cell receptor (BCR) signaling through its immunoreceptor tyrosine inhibitory motif, playing a role in maintaining the homeostasis of humoral immunity, and is commonly present in normal B cells and B cell malignancies. CD22 can interact with sialic acid-containing cells, including T cells, B cells, neutrophils, monocytes, and red blood cells. Recent studies have found that CD22 expression has also been observed in murine intestinal eosinophils, indicating that CD22 may have eosinophil regulatory functions. Clinically, CD22 antibodies have been used to treat various B cell malignancies, including non-Hodgkin lymphoma and acute leukemia. For example, HB22.7 is a monoclonal antibody specifically targeting CD22, and studies have shown that by blocking the binding of CD22 to ligands, the activation and inflammatory responses of B cells can be significantly reduced. In tumor immunotherapy, HB22.7 helps reduce B cell-mediated dangerous responses and the progression of autoimmune diseases by regulating B cell functions.

[0003] Previous studies of the present invention first disclosed that CD22 is specifically upregulated in the glial scar area. Inhibiting CD22 can significantly reduce the formation of glial scars after spinal cord injury and promote the recovery of fine motor function of the hind limbs of mice after spinal cord injury. (Chinese Patent Application No. 202410215286.9). The CD22 gene can be used as a target for the preparation of a drug for treating spinal cord injury-related diseases, and the drug is a substance that inhibits the expression of the CD22 gene, selected from one or several of compounds, proteins, polypeptides, polysaccharides, glycoproteins, glycopeptides, and nucleic acids. Summary of the Invention

[0004] Based on previous studies, the present invention further studies the application of CD22 as a target for the preparation of a drug for treating spinal cord injury-related diseases, and finds that blocking the function of CD22 in microglia with a CD22 antibody can inhibit the formation of scars after spinal cord injury and promote the recovery of fine motor function of the hind limbs of mice after spinal cord injury.

[0005] The specific technical solution of the present invention is as follows:

[0006] The application of a CD22 antibody in the preparation of a drug for treating spinal cord injury-related diseases. The drug can reduce the formation of glial scars after spinal cord injury.

[0007] The CD22 antibody described in the present invention can be a polyclonal antibody or a monoclonal antibody.

[0008] In a specific example of the present invention, the antibody is a monoclonal antibody. The monoclonal antibody can be a non-human monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody or a fully human monoclonal antibody.

[0009] Furthermore, the monoclonal antibody can be a genetically engineered antibody, including single-chain antibody, bispecific antibody, Fc fusion protein, antibody fragment. Preferably, the antibody fragment is a VH single-domain antibody, Fab fragment, Fab' fragment, F(ab)'2 fragment, single-chain variable fragment scFv or disulfide-stabilized variable fragment dsFv.

[0010] For example, the CD22 antibody can be selected from HB22.7 (WO2003072036), Epratuzumab, m971, Inotuzumab, Moxetumomab, Pinatuzumab, Suciraslimab, Bectumomab or Rezetamig.

[0011] In a specific example of the present invention, the CD22 antibody is a monoclonal antibody targeting CD22 of C57BL / 6J mice (BioXCell, BE0011) and a monoclonal antibody HB22.7 targeting humanized CD22 mice (WO2003072036, prepared by Shanghai Genechem Co., Ltd.).

[0012] In a specific example of the present invention, the CD22 antibody comprises a heavy-chain variable region and a light-chain variable region. The heavy-chain variable region has complementarity-determining regions shown by amino acid residues at positions 31-35, 50-65 and 98-106 of SEQ ID NO:1; the light-chain variable region has complementarity-determining regions shown by amino acid residues at positions 23-33, 49-55 and 88-96 of SEQ ID NO:2. Further preferably, the amino acid sequence of the heavy-chain variable region of the CD22 antibody is as shown in SEQ ID NO:1; the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:2.

[0013] The amino acid sequence of HB22.7 is shown as follows.

[0014] HB22.7 VH(mouse IgG2a)

[0015]

[0016] HB22.7 CH(mouse IgG2a)

[0017]

[0018] HB22.7 VL (mouse Kappa)

[0019]

[0020] HB22.7 CL (mouse Kappa)

[0021]

[0022] In a specific example of the present invention, the drug is an injection preparation. Treatment can be carried out by intrathecal injection. The drug takes the CD22 antibody as the main active ingredient.

[0023] In a preferred embodiment of the present invention, the drug has a blood-brain barrier drug delivery system. Specifically,

[0024] (1) The drug delivery system can target highly expressed targets in endothelial cells of the cerebrovascular system, such as transferrin receptor (TfR), insulin receptor, members of the low-density lipoprotein receptor (LDLR) family, melanotransferrin (MTf), and CD98 heavy chain (CD98hc, also known as SLC3A2).

[0025] (2) The drug delivery system uses adeno-associated virus (AAV) or lentivirus as a drug carrier to enter immune cells to achieve cross-blood-brain barrier (BBB), direct cross-BBB endocytosis, or deliver drugs by bypassing the BBB through peripheral or sensory nerves.

[0026] (3) The drug delivery system is a nanoparticle system containing various carriers, such as liposomes, polymer nanoparticles, or solid lipid nanoparticles (SLNP).

[0027] (4) The drug delivery system is exosomes (modified and unmodified), cell-mediated brain drug delivery, cell-penetrating peptides, and receptor-mediated BBB-opening drug delivery methods.

[0028] Advantages of the present invention:

[0029] The research results of the present invention show that the use of CD22 antibody (BioXCell, BE0011) can specifically recognize and bind to CD22 molecules in 8WC57BL / 6J mice, block their functions, and block the functions of CD22 in microglia. HB22.7 (prepared by Shanghai Genechem Co., Ltd. on commission) was used to target and block the binding of CD22 to its ligand in humanized CD22 mice, blocking the functions of CD22 in microglia. Behavioral irregular horizontal ladder tests, BMS scores, catwalk experiments, and Tse fine motor system analyses showed that blocking the binding of CD22 ligand and receptor with CD22 antibody significantly promoted the behavioral recovery of mice after spinal cord injury; spinal cord electrophysiological detection found that inhibiting the binding of CD22 ligand and receptor promoted nerve conduction ability; mechanical pain tests showed that blocking the binding of CD22 ligand and receptor significantly promoted the sensitivity of the hind limb on the injured side; immunohistochemical staining showed that blocking the functions of CD22 ligand and receptor significantly promoted the repair after spinal cord injury. Brief Description of the Drawings

[0030] Figure 1 Injecting CD22 antibody improved the BMS score and horizontal ladder score of C57BL / 6J mice after spinal cord injury.

[0031] A. Time points for injecting CD22 antibody after spinal cord injury; B. Statistical chart of error rates (%) of irregular horizontal ladders; C. Statistical chart of BMS scores. n = 6, data are expressed as Mean±SD, Student's t-test was used, *p<0.05, **p<0.01, ***p<0.001 indicate significant differences.

[0032] Figure 2 Injecting CD22 antibody promoted the recovery of fine motor function of the hind limbs of C57BL / 6J mice after spinal cord injury.

[0033] A. Movement map of the right hind limb in TSE test; B. Statistical chart of the angle of knee - right ankle - right hind foot. n = 6, data are expressed as Mean±SD, one-way ANOVA was used, and Tukey's HSD multiple comparisons were performed, *p<0.05, **p<0.01, ***p<0.001 indicate significant differences.

[0034] Figure 3 Injecting CD22 antibody promoted the electrophysiological recovery of C57BL / 6J mice after spinal cord injury.

[0035] A. Electrophysiological nerve conduction CAMP amplitude map 8 weeks after spinal cord injury in mice; B. CAMP amplitude statistical chart. n = 6, data are expressed as Mean ± SD, and one-way ANOVA and Tukey's HSD multiple comparisons are used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0036] Figure 4 Injecting CD22 antibody promotes the recovery of hindlimb motor function in C57BL / 6J mice after spinal cord injury.

[0037] A. Mouse gait footprint; B. Coordination degree statistics of mice at 4W, 6W, and 8W after spinal cord injury; C. Statistical of the footprint area of the right foot of the mouse (mm 2 ). n = 6, data are expressed as Mean ± SD, and Student’s t-test is used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0038] Figure 5 Injecting CD22 antibody inhibits the formation of scars by astrocytes in C57BL / 6J mice after spinal cord injury.

[0039] A. Mouse spinal cord tissue at 8 weeks after injury is labeled with GFAP, and GFAP (green) indicates astrocytes. Bar = 100μm; B. Statistical of the gap width in the scar center area formed by GFAP-positive astrocytes. n = 6, data are expressed as Mean ± SD, and Student’s t-test is used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0040] Figure 6 Injecting CD22 antibody inhibits the formation of fibrous scars in the spinal cord injury area of C57BL / 6J mice.

[0041] A. Results of immunofluorescence staining of spinal cord tissue, P4HB (red) indicates fibroblasts in the scar area. DAPI (blue) shows the cell nucleus. Bar = 100μm; B. Measurement results of the fibrous scar area. The average fluorescence intensity of P4HB, the mouse IgG group is used as a positive control, n = 6, data are expressed as Mean ± SD, and Student’s t-test is used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0042] Figure 7 Injecting HB22.7 improves the BMS score and horizontal ladder score in humanized CD22 mice after spinal cord injury.

[0043] A. Time points of drug administration after spinal cord injury; B. Error rate statistics (%) of the irregular horizontal ladder; C) BMS score statistics. n = 6, data are expressed as Mean ± SD, and Student's t-test is used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0044] Figure 8 Injection of HB22.7 promotes the recovery of fine motor function of the hindlimbs in humanized CD22 mice after spinal cord injury.

[0045] A. Movement map of the right hindlimb in the TSE test; B. Statistical chart of the knee-right ankle bone-right hindfoot angle. n = 6, data are expressed as Mean ± SEM, and one-way ANOVA is used, followed by Tukey's HSD multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0046] Figure 9 Injection of HB22.7 promotes electrophysiological recovery in humanized CD22 mice after spinal cord injury. A. Electrophysiological nerve conduction CAMP amplitude map at 8 weeks after spinal cord injury in mice; B. Statistical chart of CAMP amplitude. n = 6, data are expressed as Mean ± SEM, and one-way ANOVA and Tukey's HSD multiple comparison are used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0047] Figure 10 Injection of HB22.7 enhances the sensitivity of mechanical pain in the injured lower limb after spinal cord injury in humanized CD22 mice. Statistical chart of mechanical pain in the left and right hindlimbs of mice at about 8 weeks after spinal cord injury, using one-way ANOVA and Tukey's HSD multiple comparison. n = 6, *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0048] Figure 11 Injection of HB22.7 promotes the recovery of fine motor function of the hindlimbs in humanized CD22 mice after spinal cord injury.

[0049] A. Mouse gait footprint; B. Coordination statistics of mice at 4W, 6W, and 8W after spinal cord injury; C. Statistical chart of the footprint area of the right hindlimb of mice (mm 2 )). n = 6, data are expressed as Mean ± SEM, and Student's t-test is used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0050] Figure 12Injecting HB22.7 promotes the regeneration axons of humanized CD22 mice to penetrate the scar area after spinal cord injury. A. The co-labeling results of Tuj1, CD31 and DAPI in the spinal cord tissues of BALB / c mice and humanized CD22 mice at 8 weeks after injury are shown. Tuj1 (green) represents the regenerated axons in the scar area, CD31 (red) represents the blood vessels in the scar area, and DAPI is used to label the cell nuclei. Scale bar: Bar = 100 μm. B. Statistical analysis of the number of Tuj1-positive axons penetrating the scar area. C. Statistical analysis of the number of CD31-positive blood vessels in the scar area. BALB / c mice injected with mouse IgG were used as the positive control. n = 6, the data are expressed as Mean±SEM, and Student's t-test was used, *p<0.05, **p<0.01, ***p<0.001 indicate significant differences.

[0051] Figure 13 Injecting HB22.7 inhibits the formation of scars by microglia in humanized CD22 mice after spinal cord injury. A. The spinal cord tissues of BALB / c mice and humanized CD22 mice at 8 weeks after injury were co-labeled with Iba1 and CD68. Iba1 (green) represents the microglia in the scar area, and CD68 (red) represents the activated microglia. Bar = 100 μm; B. Statistical analysis of the proportion of activated microglia. n = 6, the data are expressed as Mean±SEM, and Student's t-test was used, *p<0.05, **p<0.01, ***p<0.001 indicate significant differences.

[0052] Figure 14 Injecting HB22.7 inhibits the formation of fibrous scars in humanized CD22 mice after spinal cord injury. A. Results of immunofluorescence staining of spinal cord tissues. P4HB (red) represents fibroblasts in the scar area, GFAP (green) represents astrocytes, and DAPI is used to label the cell nuclei. Scale bar: Bar = 100 μm. B. Measurement results of the fibrous scar area. C. Average fluorescence intensity of P4HB. BALB / c mice injected with mouse IgG were used as the positive control. n = 6, the data are expressed as Mean±SEM, and Student's t-test was used, *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. Detailed implementation manners

[0053] The above content of the present invention will be further described in detail through the specific implementation manners in the form of examples below. However, the examples should not be construed as any limitation to the present invention. The protection scope of the present invention is subject to the claims. Unless otherwise specified, conventional existing technologies are used in the following examples.

[0054] Example 1: Behavioral ladder test and BMS score of mice injected with CD22 antibody after spinal cord injury in C57BL / 6J mice. I. Right hemisection model of the spinal cord at T10 in adult mice

[0055] Twelve 8-week-old female C57BL / 6J mice (Jiangsu Huachuang Xinnuo Medical Technology Co., Ltd.) were anesthetized, and the skin was incised along the midline of the thoracic vertebrae. A laminectomy of T10 was performed. The tip of an iris knife (BVI Beaver, Oakville, Canada) was carefully inserted into the posterior median sulcus of the spinal cord to completely transect the right side of the spinal cord. The muscle layer was sutured, and then the skin was fixed with wound clips. The mice were placed at 37°C for recovery until fully awake, and pain was relieved with pain-relieving injections. All animal experiments were conducted in accordance with the animal care guidelines and were ethically approved by the Jiangsu Provincial Laboratory Animal Management Committee. The animal experiment license was SYXK (Su) 2022-0046.

[0056] II. Injection of CD22 antibody

[0057] In this experiment, CD22 antibody (BioXCell, BE0011) was injected into the tail vein of mice half an hour after modeling. The mice were fixed with a venous visible mouse tail injection fixator. The mouse tail was warmed with warm water and wiped with an alcohol cotton ball. After the blood vessels dilated, a 1 ml syringe (30G) was used to aspirate the diluted antibody and inject it into the mouse body. Intrathecal injections were performed at 3W, 4W, 5W, 6W, and 7W after modeling ( Figure 1 A), and the positions of the midlines of the bilateral iliac bones were felt by hand. A 1 ml syringe (30G) was used to inject the diluted antibody into the L5-L6 intervertebral space.

[0058] III. Irregular horizontal ladder test

[0059] Mice in each experimental group were trained for horizontal ladder climbing in mice three days before modeling, with each mouse trained three times a day. A right hemisection model of the spinal cord at T10 in adult mice was constructed. After modeling, an irregular horizontal ladder test was performed weekly. The spacing of the ladder was randomly arranged at each test time point, and the mice were videotaped. Each mouse was filmed 3 times. The total experimental duration was 8 weeks. The error rate of the right hind foot of each group was counted by two persons who were unaware of the experimental situation. ( Figure 1 B) is the statistical chart of the irregular horizontal ladder error rate (%), with the error rate (%) on the Y-axis and the time point on the X-axis. The data are expressed as Mean±SD. Student's t-test was used. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results showed that the injection of CD22 antibody reduced the error rate of the horizontal ladder after spinal cord injury.

[0060] IV. Kinematic evaluation experiment after spinal cord injury - Open field experiment BMS score

[0061] One day before modeling, normal mice were placed in the open field to familiarize them with the environment, and a right hemisection model of the spinal cord at T10 in adult mice was constructed. After modeling, the BMS score was measured weekly and the mice were videotaped. Scoring was performed according to the scoring criteria of the BMS primary and secondary scoring systems. The total experimental duration was 8 weeks. Figure 1 C) shows the BMS score results. Data are expressed as Mean±SD, and Student's t-test was used. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results showed that injection of CD22 antibody increased the BMS score after spinal cord injury.

[0062] Example 2 Evaluation of the recovery of fine motor function of the hindlimbs in mice injected with CD22 antibody after spinal cord injury using the TSE MotoRater System

[0063] I. Refer to Example 1 to construct a right hemisection model of the spinal cord at T10 in adult mice.

[0064] II. Refer to Example 1 to administer drugs to adult mice after spinal cord injury.

[0065] III. TSE testing and statistics

[0066] Eight weeks after modeling, TSE testing was performed on the two groups of mice, and TSE testing was also performed on uninjured C57BL / 6J mice of the same age (16 weeks). The hair on the right side of the mice was shaved, and a luminescent sticker was used and attached to the right pelvis, right hip, knee, right ankle bone, and right hind foot of the mice. The TSE Motion was used to test and analyze the foot trajectory and the angle of the knee-right ankle bone-right hind foot ( Figure 2 A). One-way ANOVA was used, and Tukey's HSD multiple comparisons were performed. Data are expressed as Mean±SD, and *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results showed that the step frequency and the angle of the knee-right ankle bone-right hind foot in the mice injected with CD22 antibody were closer to those of uninjured normal mice.

[0067] Example 3 Electrophysiological testing of mice injected with CD22 antibody after spinal cord injury in C57BL / 6J mice

[0068] I. Refer to Example 1 to construct a right hemisection model of the spinal cord at T10 in adult mice.

[0069] II. Refer to Example 1 to administer drugs to adult mice after spinal cord injury.

[0070] III. Electrophysiological testing and statistics

[0071] After 8 weeks of model establishment, electrophysiological tests were performed on the two groups of mice, and electrophysiological tests were also performed on uninjured C57BL / 6J mice of the same age (16 weeks). The lamina of the T9 region was removed to expose the upper segment of the spinal cord after hemisection. The CAMP amplitude was measured and analyzed using a channel full-functional electromyogram evoked potential instrument ( Figure 3 A), and the statistics of the CAMP amplitude were performed ( Figure 3 B). One-way ANOVA was used, and Tukey's HSD multiple comparisons were carried out. The data were expressed as Mean±SD, and *p<0.05, **p<0.01, ***p<0.001 indicated significant differences. The results showed that the CAMP amplitude of the mice injected with CD22 antibody was closer to that of the uninjured normal mice.

[0072] Example 4 Catwalk test of the hind limbs of C57BL / 6J mice after spinal cord injury and injection of CD22 antibody

[0073] I. Refer to Example 1 to construct a right-sided T10 hemisection model of the spinal cord in adult mice.

[0074] II. Refer to Example 1 to administer drugs to the adult mice after spinal cord injury.

[0075] III. Catwalk test and statistics

[0076] At 4 weeks, 6 weeks, and 8 weeks after model establishment, catwalk tests were performed on the two groups of mice. The test was carried out using a gait analyzer. The mice were placed on the test platform 30 minutes before the test to adapt to the environment, and the gait test was performed after they calmed down. Each mouse was tested three times, and the motor coordination and the footprint area of the right hind foot of the mouse were analyzed ( Figure 4 A). The data were expressed as Mean±SD, and Student's t-test was used. *p<0.05, **p<0.01, ***p<0.001 indicated significant differences. The results showed that the mice injected with CD22 antibody had higher gait coordination ( Figure 4 B) and a larger right foot contact area ( Figure 4 C).

[0077] Example 5 Scar formation of astrocytes in the area injected with CD22 antibody after spinal cord injury in C57BL / 6J mice

[0078] I. Refer to Example 1 to construct a right-sided T10 hemisection model of the spinal cord in adult mice.

[0079] II. Refer to Example 2 to administer drugs to the adult mice after spinal cord injury.

[0080] III. Immunofluorescence staining of spinal cord tissue and measurement of scar formation of astrocytes

[0081] After 8 weeks, perfusion with 4% paraformaldehyde was performed. Then, post-fixation with 4% paraformaldehyde was carried out for 12 hours. After discarding the paraformaldehyde, the sample was washed three times with PBS for 10 minutes each time. The lamina was dissected to expose the spinal cord, and the integrity of the spinal cord was maintained as much as possible. For the removed spinal cord tissue, the spinal cord with a 3-mm distance before and after the injury site was collected. After dehydration with 30% sucrose, it was embedded in OCT, and the frozen sections were 10 μm thick. Immunohistochemical blocking solution was added and blocked at room temperature for 2 hours. The primary antibody GFAP (abcam AB4674, Chicken 1:500) was diluted with the immunohistochemical primary antibody diluent. After adding the primary antibody, it was incubated overnight at 4°C. The primary antibody was discarded, and the sample was washed 3 times with PBS for 5 minutes each time. The fluorescent secondary antibody Alexa Fluor TM 488 Goat anti-Chicken IgY(H+L)(invitrogen, A11039, 1:500) was diluted with the immunohistochemical secondary antibody diluent. After adding the secondary antibody, it was incubated for 2 hours at room temperature in the dark. The secondary antibody was discarded, and the sample was washed 3 times with PBS for 5 minutes each time. An appropriate amount of fluorescent mounting medium was added, and it was observed and photographed under a ZEISS upright fluorescence microscope.

[0082] Observe the morphology of astrocytes at the injury site, and count the number and boundaries of astrocytes in each group. The results are as Figure 5 shown, ( Figure 5 A) shows the spinal cord tissues of mice injected with CD22 antibody and PBS 8 weeks after injury labeled with GFAP. GFAP (green) indicates astrocytes. Bar = 100 μm. ( Figure 5 B) shows the statistical results of the fluorescence density of GFAP-positive astrocytes. Mean ± SD, Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001, which is statistically significant. The results show that the width of the gap in the scar center formed by GFAP-positive astrocytes significantly narrows 8 weeks after injury, and some GFAP-positive astrocytes are interconnected.

[0083] Example 6 Fibrous Scar Formation in the Region of the Spinal Cord Injured in C57BL / 6J Mice after Injection of CD22 Antibody

[0084] I. Refer to Example 1 to construct a right-sided T10 hemisection model of the spinal cord in adult mice.

[0085] II. Refer to Example 1 to administer drugs to adult mice after spinal cord injury.

[0086] III. Immunofluorescence staining of spinal cord tissue and measurement of fibrous scar area

[0087] After 8 weeks, perfuse with 4% paraformaldehyde. Fix with 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, wash with PBS three times, 10 minutes each time. Peel off the vertebral lamina, expose the spinal cord, and try to maintain the integrity of the spinal cord. The spinal cord tissue was removed and the spinal cord 3 mm before and after the injury site was collected. After dehydration with 30% sucrose, it was embedded in OCT and the thickness of the frozen section was 10 μm. Add immunohistochemistry blocking solution and block at room temperature for 2 hours. Dilute the primary antibody P4HB (abcam ab137110, Rabbit 1:1000) with immunohistochemistry primary antibody diluent. After adding the primary antibody, incubate at 4°C overnight. Discard the primary antibody and wash with PBS 3 times, 5 minutes each time. Dilute the fluorescent secondary antibody Alexa with immunohistochemistry secondary antibody diluent. 647AffiniPure Sheep Anti-mouse IgG (invitrogen A31571, 1:500), after adding the secondary antibody, incubate in the dark at room temperature for 2 hours. Discard the secondary antibody and wash 3 times with PBS, 5 minutes each time. Add appropriate amount of fluorescent mounting medium, observe under ZEISS upright fluorescence microscope, and take pictures. Observe the distribution of fibroblasts at the injury site, take pictures, and count the size of the fibrous scar area in each group. The results are as follows Figure 6 As shown. ( Figure 6 A) Immunofluorescence staining results of spinal cord tissue. P4HB (red) indicates fibroblasts in the scar area, and DAPI (blue) shows cell nuclei. Bar = 100 μm; ( Figure 6 B) Fibroblast scar area measurement. P4HB mean fluorescence intensity. C57BL / 6J mice injected with PBS after spinal cord injury served as a positive control. Mean ± SD, Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant. The results show that the number of fibroblasts in the spinal cord scar area of mice injected with CD22 antibodies is reduced.

[0088] Example 7 Behavioral level ladder test and BMS score in humanized CD22 mice injected with HB22.7 after spinal cord injury

[0089] 1. Adult Mouse Spinal Cord T10 Right Hemisection Model

[0090] Six 8-week-old female humanized CD22 mice (Jiangsu GICC BioTech Co., Ltd., product number: T54197) and six 8-week-old female BALB / c mice (Jiangsu Huachuang Xinnuo Medical Technology Co., Ltd.) were anesthetized. The skin was incised along the midline of the thoracic vertebrae, and a T10 laminectomy was performed. The tip of an iris knife (BVI Beaver, Oakville, Canada) was carefully inserted into the posterior median sulcus of the spinal cord, and the right spinal cord was completely transected. The muscle layer was sutured, and then the skin was fixed with wound clips. The mice were placed at 37°C for recovery until fully awake, and pain was relieved with pain-relieving injections. All animal experiments were conducted in accordance with animal care guidelines and were ethically approved by the Jiangsu Laboratory Animal Management Committee. The animal experiment license number is SYXK (Su) 2022-0046.

[0091] II. Injection of monoclonal antibody HB22.7

[0092] In this experiment, the drug was administered by combining tail vein injection and intrathecal injection ( Figure 7 A). Half an hour after modeling, humanized CD22 mice were injected with monoclonal antibody HB22.7 (prepared by commissioning Shanghai Genechem Co., Ltd.) via the tail vein. The mice were fixed with a tail vein visual mouse injection fixator. The tails of the mice were warmed with warm water and wiped with alcohol cotton balls. After the blood vessels dilated, a 1 ml syringe (30G) was used to aspirate the diluted antibody and inject it into the mice. Intrathecal injections were performed at 3W, 4W, 5W, 6W, and 7W after modeling. The positions of the midlines of the bilateral iliac bones were felt by hand, and the diluted antibody was injected into the L5-L6 intervertebral space using a syringe (30G).

[0093] III. Irregular horizontal ladder test

[0094] Three days before modeling, each experimental mouse was trained in horizontal ladder climbing three times a day. A model of right-sided T10 hemisection of the spinal cord in adult mice was constructed. After modeling, an irregular horizontal ladder test was performed weekly. The spacing of the ladders was randomly arranged at each test time point, and the test was recorded with a camera. Each mouse was filmed 3 times. The total experimental duration was 8 weeks. Two personnel who were unaware of the experimental situation counted the error rate of the right hind paw in each group. ( Figure 7 B) shows the statistical results of the irregular horizontal ladder error rate (%). The error rate (%) is on the Y-axis, and the time points are on the X-axis. The data are expressed as Mean±SD. Student's t-test was used. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results showed that the injection of monoclonal antibody HB22.7 reduced the error rate of the horizontal ladder after spinal cord injury.

[0095] IV. Kinematic evaluation experiment after spinal cord injury - Open field experiment BMS score

[0096] One day before modeling, normal mice were placed in an open field to familiarize them with the environment, and a right hemisection model of the T10 spinal cord of adult mice was constructed. After modeling, the BMS score was measured weekly, and the mice were videotaped. Scoring was performed according to the scoring rules of the main and sub BMS scoring systems. The total experimental duration was 8 weeks.( Figure 7 C) shows the BMS score results. Data are expressed as Mean±SD, and Student's t-test was used. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results showed that injection of HB22.7 improved the BMS score after spinal cord injury.

[0097] Example 8 Evaluation of the recovery of fine motor function of the hind limbs after injection of HB22.7 in humanized CD22 mice with spinal cord injury using a fine motor dynamic assessment system (TSE MotoRater System)

[0098] I. Referring to Example 7, a right hemisection model of the T10 spinal cord of adult mice was constructed.

[0099] II. Referring to Example 7, adult mice with spinal cord injury were administered drugs.

[0100] III. TSE testing and statistics

[0101] Eight weeks after modeling, TSE tests were performed on two groups of mice, and TSE tests were also performed on uninjured BALB / c mice of the same age (16 weeks). The hair on the right side of the mice was shaved, and a luminescent sticker was used and attached to the right pelvis, right hip, knee, right ankle bone, and right hind foot of the mice. The TSE Motion was used to test and analyze the foot trajectories( Figure 8 A) and the statistics of the knee - right ankle bone - right hind foot angle( Figure 8 B). One-way ANOVA was used, and Tukey's HSD multiple comparisons were performed. Data are expressed as Mean±SEM, and *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results showed that the step frequency, step spacing, and knee - right ankle bone - right hind foot angle of the mice injected with HB22.7 were closer to those of uninjured normal mice.

[0102] Example 9 Electrophysiological testing of humanized CD22 mice with spinal cord injury after injection of HB22.7

[0103] I. Referring to Example 7, a right hemisection model of the T10 spinal cord of adult mice was constructed.

[0104] II. Referring to Example 7, adult mice with spinal cord injury were administered drugs.

[0105] III. Electrophysiological testing and statistics

[0106] After 8 weeks of model establishment, electrophysiological tests were performed on the two groups of mice, and electrophysiological tests were also performed on uninjured BALB / c mice of the same age (16 weeks). The lamina of the T9 region was removed to expose the upper segment of the spinal cord after hemisection. The CAMP amplitude was measured and analyzed using a channel full-functional electromyogram evoked potential instrument ( Figure 9 A). The results showed that the CAMP amplitude of the mice injected with monoclonal antibody HB22.7 was higher than that of the BALB / c mice injected with mouse IgG ( Figure 9 B). One-way ANOVA was used, followed by Tukey's HSD multiple comparisons. The data were expressed as Mean±SEM, and *p<0.05, **p<0.01, ***p<0.001 indicated significant differences.

[0107] Example 10 Mechanical pain test of the hind limbs of humanized CD22 mice after spinal cord injury with injection of HB22.7

[0108] I. Refer to Example 7 to construct a right hemisection model of the spinal cord of adult mice at T10.

[0109] II. Refer to Example 7 to administer drugs to the adult mice after spinal cord injury.

[0110] III. Mechanical pain test and statistics

[0111] After 8 weeks of model establishment, the hind limb mechanical pain test was performed on the two groups of mice. Using von Frey fibers, mechanical force was applied to the center of the sole of the foot starting from 0.16 g. If there was no response, the force was gradually increased until 2.0 g. The responses of the mice such as lifting the foot, licking the paw, or sudden twitching were recorded. Then, according to the test results and the mouse mechanical pain reference table, the threshold value that caused the response was obtained. The results were as Figure 10 , the right foot of the mice injected with monoclonal antibody HB22.7 was more sensitive than that of the BALB / c mice injected with mouse IgG, indicating that the injection of HB22.7 promoted the recovery of the plantar nerve. The data were expressed as Mean±SEM, and Student’s t-test was used. *p<0.05, **p<0.01, ***p<0.001 indicated significant differences.

[0112] Example 11 Catwalk test of the hind limbs of humanized CD22 mice after spinal cord injury with injection of HB22.7

[0113] I. Refer to Example 7 to construct a right hemisection model of the spinal cord of adult mice at T10.

[0114] II. Refer to Example 7 to administer drugs to the adult mice after spinal cord injury.

[0115] III. Catwalk test and statistics

[0116] At 4 weeks, 6 weeks, and 8 weeks after model establishment, catwalk tests were performed on the two groups of mice. Using a gait analyzer, the mice were placed on the test platform 30 minutes before the test to adapt to the environment, and the gait test was performed after they calmed down. Each mouse was tested three times, and the motor coordination and the footprint area of the right hind foot of the mice were analyzed ( Figure 11 A). The results showed that the mice injected with HB22.7 had higher gait coordination ( Figure 11 B) and a larger right foot contact area ( Figure 11 C). The data are expressed as Mean±SEM, and Student's t-test was used. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences

[0117] Example 12 Axonal and vascular regeneration after injection of HB22.7 in humanized CD22 mice with spinal cord injury

[0118] I. Refer to Example 7 to construct a right hemisection model of the T10 spinal cord in adult mice.

[0119] II. Refer to Example 7 to administer drugs to adult mice after spinal cord injury.

[0120] III. Immunofluorescence staining of spinal cord tissue and measurement of scar formation by astrocytes

[0121] After 8 weeks, perfusion was performed with 4% paraformaldehyde. Then, it was post-fixed with 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, it was washed three times with PBS, 10 minutes each time. The vertebral lamina was stripped to expose the spinal cord, and the integrity of the spinal cord was maintained as much as possible. The removed spinal cord tissue was collected from the spinal cord at a distance of 3 mm before and after the injury site. After dehydration with 30% sucrose, it was embedded in OCT, and the thickness of the frozen section was 10 μm. Immunohistochemical blocking solution was added and blocked at room temperature for 2 h. The primary antibodies Tuj1 (BioLegend, B249869, Mouse, 1:1000) and CD31 (R&D, AF3628, goat, 1:100) were diluted with immunohistochemical primary antibody diluent. After adding the primary antibody, it was incubated overnight at 4°C. The primary antibody was discarded, and it was washed 3 times with PBS, 5 minutes each time. The fluorescent secondary antibodies Donkey anti-Goat IgG(H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM Plus 488 (invitrogen, YA363610, 1:500), Donkey anti-Mouse IgG(H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TMAfter adding 647 (Invitrogen, A31571, 1:500) as the secondary antibody, incubate in the dark at room temperature for 2 h. Discard the secondary antibody and wash 3 times with PBS for 5 min each time. Add an appropriate amount of fluorescent mounting medium and observe and take pictures under a ZEISS upright fluorescence microscope. Observe the distribution of blood vessels and axons at the injury site, take pictures and count the number of axons and blood vessels in the scar area of each group. The results are as Figure 12 shown. ([[]] Figure 12 A) Co-labeling of spinal cord tissues of BALB / c mice and humanized CD22 mice at 8 weeks after injury using CD31, Tuj1, and DAPI. Tuj1 (green) represents axons in the scar area, CD31 (red) represents blood vessels in the scar area, and DAPI shows cell nuclei. Bar = 100 μm; ([[]] Figure 12 B) Counting the number of Tuj1-positive axons in the scar area; ([[]] Figure 12 C) Counting the number of CD31-positive blood vessels in the scar area. BALB / c is used as a positive control. The data are expressed as Mean ± SEM, and Student's t-test is used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. The results show that the regeneration of blood vessels and axons in the spinal cord scar area of mice injected with HB22.7 is better than that of the group injected with mouse IgG.

[0122] Example 13 Formation of scars after injection of HB22.7 microglia in spinal cord injured humanized CD22 mice

[0123] I. Refer to Example 7 to construct a right T10 hemisection model of the spinal cord in adult mice.

[0124] II. Refer to Example 7 to administer drugs to adult mice after spinal cord injury.

[0125] III. Immunofluorescence staining of spinal cord tissues and measurement of inflammatory cells in the scar area

[0126] After 8 weeks, perfusion with 4% paraformaldehyde. Then post-fix in 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, wash three times with PBS, 10 min for each wash. Dissect the laminae to expose the spinal cord, and try to maintain the integrity of the spinal cord. For the excised spinal cord tissue, collect the spinal cord at a distance of 3 mm before and after the injury site. After dehydration with 30% sucrose, embed in OCT, and the frozen section thickness is 10 μm. Add immunohistochemical blocking solution and block at room temperature for 1 h. Dilute the primary antibodies Iba1 (invitrogen ZG4398731, Goat 1:1000) and CD68 (BIO-RAD 160940, Mouse 1:500) with immunohistochemical primary antibody diluent. After adding the primary antibodies, incubate overnight at 4°C. Discard the primary antibodies and wash 3 times with PBS, 5 min for each wash. Dilute the fluorescent secondary antibodies Donkey anti-Goat IgG(H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM Plus 488 (invitrogen, YA363610, 1:500), Donkey anti-Mouse IgG(H+L) Highly Cross-Adsorbed SecondaryAntibody, Alexa Fluor TM 647 (invitrogen, A31571, 1:500). After adding the secondary antibodies, incubate for 2 h at room temperature in the dark. Discard the secondary antibodies and wash 3 times with PBS, 5 min for each wash. Add an appropriate amount of fluorescent mounting medium and observe and take pictures under a ZEISS upright fluorescence microscope.

[0127] Observe the morphology of activated microglia and astrocytes at the injury site, and count the number and boundaries of activated microglia and astrocytes in each group. The results are as Figure 13 shown, ( Figure 13 A) Co-labeling of spinal cord tissues of humanized CD22 mice and BALB / c mice at 8 weeks after injury using Iba1 and CD68. Iba1 (green) represents microglia in the scar area, and CD68 (red) represents activated microglia. Bar = 100 μm. ( Figure 13 B) Statistical chart of the proportion of activated microglia. Data are expressed as Mean±SEM, and Student's t-test is used. *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results show that at 8 weeks after injury, the number of CD68-positive activated microglia in the spinal cord scar area of the humanized CD22 injection HB22.7 group was significantly reduced.

[0128] Example 14 Fibrous scar formation in the spinal cord of humanized CD22 mice after spinal cord injury

[0129] 1. Construct a right-sided hemisection model of the adult mouse spinal cord at T10 with reference to Example 7.

[0130] 2. Administer drugs to the adult mice after spinal cord injury with reference to Example 7.

[0131] 3. Immunofluorescence staining of spinal cord tissue and measurement of the area of fibrous scar

[0132] After 8 weeks, perfuse with 4% paraformaldehyde. Then post-fix with 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, wash three times with PBS, 10 min each time. Dissect the vertebral lamina to expose the spinal cord, and try to keep the spinal cord intact. Take out the spinal cord tissue, collect the spinal cord at a distance of 3 mm before and after the injury site, dehydrate with 30% sucrose, embed in OCT, and the thickness of the frozen section is 10 μm. Add immunohistochemical blocking solution and block at room temperature for 2 h. Dilute the primary antibodies P4HB (abcam AB137110, Mouse 1:1000) and GFAP (abcam AB4674, Chicken 1:1000) with immunohistochemical primary antibody diluent. After adding the primary antibodies, incubate overnight at 4°C. Discard the primary antibodies and wash 3 times with PBS, 5 min each time. Dilute the fluorescent secondary antibodies Donkey anti-Mouse IgG(H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 647 (invitrogen, A31571, 1:500), Alexa Fluor TM 488 donkey anti-chicken IgY(H+L)*2mg / mL**contains 5mM sodium azide (invitrogen 2752568, 1:500) with immunohistochemical secondary antibody diluent. After adding the secondary antibodies, incubate for 2 h at room temperature in the dark. Discard the secondary antibodies and wash 3 times with PBS, 5 min each time. Add an appropriate amount of fluorescent mounting medium, observe and take pictures under a ZEISS upright fluorescence microscope. Observe the distribution of fibroblasts at the injury site, take pictures and statistically analyze the size of the fibrous scar area in each group. The results are as Figure 14 shown. ( Figure 14 A) shows the results of immunofluorescence staining of spinal cord tissue. P4HB (red) represents fibroblasts in the scar area, GFAP (green) represents astrocytes, and DAPI (blue) shows cell nuclei. Bar = 100 μm; ( Figure 14 B) shows the results of measurement of the fibrous scar area. Average fluorescence intensity of P4HB; ( Figure 14C) Fluorescence density statistics of positive astrocytes; Mice injected with mouse IgG after spinal cord injury in BALB / c mice were used as positive controls. The data are expressed as Mean±SEM. Student's t-test was used, and *p<0.05, **p<0.01, ***p<0.001 indicate significant differences. The results showed that the number of fibroblasts in the spinal cord scar area of mice in the humanized CD22 injection HB22.7 group decreased, and the gap width in the scar center area formed by GFAP-positive astrocytes was significantly narrowed.

Claims

1. Use of a CD22 antibody in the preparation of a medicament for treating spinal cord injury-related diseases.

2. The application according to claim 1, characterized in that The medicament reduces the formation of glial scars after spinal cord injury.

3. The application according to claim 1, wherein The CD22 antibody is a polyclonal antibody or a monoclonal antibody.

4. The application according to claim 3, characterized in that The monoclonal antibody is a non-human monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody or a fully human monoclonal antibody.

5. The application according to claim 3, characterized in that The monoclonal antibody is a genetically engineered antibody, including a single-chain antibody, a bispecific antibody, an Fc fusion protein, an antibody fragment.

6. The application according to claim 1, wherein The antibody fragment is a VH single-domain antibody, a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single-chain variable fragment scFv or a disulfide-stabilized variable fragment dsFv.

7. The application according to claim 1, wherein The CD22 antibody is selected from HB22.7, Epratuzumab, m971, Inotuzumab, Moxetumomab, Pinatuzumab, Suciraslimab, Bectumomab or Rezetamig.

8. The application according to claim 1, wherein The medicament is an injection.

9. The application according to claim 8, wherein The medicament has a blood-brain barrier drug delivery system.

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