Bispecific antibody and application thereof

Abstract

The invention discloses a bispecific antibody which comprises a CD22 antibody and an antibody of a cerebrovascular system endothelial cell high expression target. The bispecific antibody disclosed by the invention not only can inhibit CD22 in a targeting manner, but also can pass through a blood brain barrier by being combined with a high-expression target of endothelial cells of a cerebrovascular system, so that the function of CD22 in microglial cells is successfully blocked, cicatrization after spinal cord injury is inhibited, and functional recovery of fine movement of hind limbs of a mouse 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 a bispecific antibody and its application. 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-based 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 erythrocytes. 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 its 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, and 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 preparing drugs for treating spinal cord injury-related diseases, and the drugs are substances that inhibit the expression of the CD22 gene, selected from one or more 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 use of CD22 as a target for preparing drugs 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] A bispecific antibody, which is an antibody containing two specific antigen-binding sites, and the two specific antigen-binding site antibodies are a CD22 antibody and an antibody against a target highly expressed on the endothelial cells of the cerebrovascular system.

[0007] Preferably, the targets highly expressed by the endothelial cells of the cerebrovascular system are selected from transferrin receptor, insulin receptor, members of the low-density lipoprotein receptor family, melanotransferrin, or CD98.

[0008] The antibodies with two specific antigen-binding sites in the present invention can be polyclonal antibodies or monoclonal antibodies.

[0009] 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.

[0010] 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.

[0011] The bispecific antibody in the present invention is preferably a Knob-into-hole type bispecific antibody KIH-BsAb.

[0012] Preferably, the CD22 antibody and the antibody against the target highly expressed by the endothelial cells of the cerebrovascular system in the present invention are in the form of Fab-scfv and are paired through the KIH (knob-into-hole) structure at the FC end. KIH structure: Based on human IgG1 FC, the Knob end has S354C and T366W mutations, and the Hole end has T366S, L368A, Y407V, and Y349C mutations. Reference: Carter, J Immunol Methods 248, 7-15 (2001).

[0013] Preferably, the CD22 antibody is selected from HB22.7 (WO2003072036), Epratuzumab, m971, Inotuzumab, Moxetumomab, Pinatuzumab, Suciraslimab, Bectumomab or Rezetamig. The antibody against the target highly expressed on the endothelial cells of the cerebrovascular system is the transferrin receptor antibody TFR1, which is selected from 8D3, OX26, RI7217, CX-2029 or TFR1 ScFv. For example, 8D3 is a murine antibody and is widely used in the study of mouse blood-brain barrier penetration; OX26 is often used in brain-targeted delivery experiments, especially for drug delivery to the central nervous system; RI7217 is a murine TFR antibody and is used to study the transport mechanism of drugs through the blood-brain barrier; TFR ScFv (single-chain antibody): is used in the study of blood-brain barrier permeability and can be linked to different drugs or therapeutic molecules to assist drug delivery. The present invention selects the TFR antibody as TFR ScFv (CN104520329A), which is combined with the ScFv and Fab fragments of HB22.7 for constructing a complete KIH-BsAb antibody.

[0014] A specific example is KIH-BsAb (HB22.7×TFR, ScFv-Fab).

[0015] In a specific example of the present invention, the bispecific antibody is characterized in that 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; the transferrin receptor 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, 50-66 and 99-107 of SEQ ID NO:3; the light chain variable region has complementarity-determining regions shown by amino acid residues at positions 24-34, 50-56 and 89-97 of SEQ ID NO:4. 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; the amino acid sequence of the heavy chain variable region of the transferrin receptor antibody is as shown in SEQ ID NO:3; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:4.

[0016] In a specific example of the present invention, the amino acid sequence information of the bispecific antibody is shown as follows.

[0017] The amino acid sequence of mTfR-VH-CH is shown in SEQ ID NO:5; the amino acid sequence of mTfR-VL-CL is shown in SEQ ID NO:7; the amino acid sequence of CD22-scFv-Fc is shown in SEQ ID NO:6.

[0018] Another object of the present invention is to provide the use of the bispecific 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.

[0019] In a specific example of the present invention, the drug is an injection preparation. Treatment can be carried out by intravenous injection.

[0020] Advantages of the present invention:

[0021] (1) The research results of the present invention show that using HB22.7 (prepared by Shanghai Genechem Co., Ltd.) to target and block the binding of CD22 to its ligand in humanized CD22 mice blocks the function of CD22 in microglia. Behavioral irregular horizontal ladder test, BMS score, catwalk experiment and Tse fine motor system analysis show that using CD22 antibody to block the binding of CD22 ligand and receptor significantly promotes the behavioral recovery of mice after spinal cord injury; spinal electrophysiological detection finds that inhibiting the binding of CD22 ligand and receptor promotes nerve conduction ability; mechanical pain test shows that blocking the binding of CD22 ligand and receptor significantly promotes the sensitivity of the hind limb on the injured side; immunohistochemical staining shows that blocking the function of CD22 ligand and receptor significantly promotes the repair after spinal cord injury. (2) The research results of the present invention show that using the bispecific antibody KIH-BsAb targeting humanized CD22 and humanized TFR1 (prepared by Shanghai Genechem Co., Ltd.) can promote the antibody to cross the blood-brain barrier of humanized CD22 / humanized TFR1 mice to target and inhibit the function of CD22, enabling the antibody to better act on the spinal cord injury site to reduce inflammatory response and promote motor coordination. Brief Description of the Drawings

[0022] Figure 1 Injecting HB22.7 increases the BMS score and horizontal ladder score of humanized CD22 mice after spinal cord injury.

[0023] A. Administration time point after spinal cord injury; B. Statistical error rate (%) of irregular horizontal ladder; C. Statistical BMS score. n = 6, data are expressed as Mean±SD, Student's t-test is used, *p<0.05, **p<0.01, ***p<0.001 indicate significant differences.

[0024] Figure 2 Injecting HB22.7 promotes the recovery of fine motor function of the hind limbs of humanized CD22 mice after spinal cord injury.

[0025] A. TSE test of the movement map of the right hind limb; B. Statistical chart of the knee - right ankle - right hind foot angle. n = 6, data are expressed as Mean ± SEM, one - way ANOVA is used, and Tukey's HSD multiple comparisons are performed. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0026] Figure 3 Injection of HB22.7 promotes electrophysiological recovery after spinal cord injury in humanized CD22 mice. 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, one - way ANOVA and Tukey's HSD multiple comparisons are used. *p < 0.05, **p < 0.01,

[0027] ***p < 0.001 indicate significant differences.

[0028] Figure 4 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 feet at about 8W after spinal cord injury in mice, one - way ANOVA and Tukey's HSD multiple comparisons are used. n = 6, *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0029] Figure 5 Injection of HB22.7 promotes the recovery of fine motor function of the hind limbs after spinal cord injury in humanized CD22 mice.

[0030] A. Mouse gait footprint; B. Coordination degree statistics of mice at 4W, 6W, and 8W after spinal cord injury; C. Statistical chart of the footprint area of the right foot of mice (mm 2 ). n = 6, data are expressed as Mean ± SEM, Student’s t - test is used, *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0031] Figure 6Injecting HB22.7 promotes the regeneration axons of humanized CD22 mice to penetrate the scar area after spinal cord injury. A. Co-labeling results of Tuj1, CD31, and DAPI in spinal cord tissues of BALB / c mice and humanized CD22 mice at 8 weeks after injury. 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 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 positive controls. n = 6, data are presented as Mean ± SEM, and Student's t-test was used, with *p < 0.05, **p < 0.01, ***p < 0.001 indicating significant differences.

[0032] Figure 7 Injecting HB22.7 inhibits the formation of scars by microglia in humanized CD22 mice after spinal cord injury. A. Co-labeling of spinal cord tissues of BALB / c mice and humanized CD22 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; B. Statistical analysis of the proportion of activated microglia. n = 6, data are presented as Mean ± SEM, and Student's t-test was used, with *p < 0.05, **p < 0.01, ***p < 0.001 indicating significant differences.

[0033] Figure 8 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 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 positive controls. n = 6, data are presented as Mean ± SEM, and Student's t-test was used, with *p < 0.05, **p < 0.01, ***p < 0.001 indicating significant differences.

[0034] Figure 9 Schematic diagram of the structure of the bispecific antibody KIH-BsAb described in the present invention.

[0035] Figure 10Injection of KIH-BsAb improved the BMS score and horizontal ladder score in humanized hCD22 / hTFR1 mice after spinal cord injury. A. Time points of drug administration after spinal cord injury; B. Statistics of the error rate (%) of the irregular horizontal ladder, with the error rate (%) on the Y-axis and the time points on the X-axis; C. Statistics of the BMS score, with the BMS score on the Y-axis and the time points on the X-axis; n = 6, data are expressed as Mean±SD. BALB / c mice injected with mouse IgG were used as the positive control. Student's t-test was used, *p<0.05, **p<0.01, ***p<0.001 indicated significant differences.

[0036] Figure 11 Injection of KIH-BsAb promoted the recovery of fine motor function of the hindlimbs in humanized hCD22 / hTFR1 mice after spinal cord injury. A. Gait footprint diagrams of mice; B. Coordination degree at 2W and 4W after spinal cord injury in mice, with the coordination degree (%) on the Y-axis; C. Statistical chart of the right foot print area at 2W and 4W after spinal cord injury in mice, with the footprint area (mm 2 ) on the Y-axis. n = 6, data are expressed as Mean±SEM. BALB / c mice injected with mouse IgG were used as the positive control. Student's t-test was used, *p<0.05, **p<0.01,

[0037] ***p<0.001 indicated significant differences.

[0038] Figure 12 Injection of KIH-BsAb promoted the regeneration axons to penetrate the glial scar area in humanized hCD22 / hTFR1 mice after spinal cord injury. A. Co-labeling of spinal cord tissues of BALB / c mice and hCD22 + / - / hTFR1 + / - mice at 8 weeks after injury with Tuj1, CD31, and DAPI. Tuj1 (green) represents the regenerated axons in the scar area, CD31 (red) represents the blood vessels in the scar area, and DAPI shows the cell nuclei. Bar = 100μm; B. Statistics of the number of Tuj1-positive axons penetrating the scar area; C. Statistics of the number of CD31-positive blood vessels in the scar area. n = 6, data are expressed as Mean±SEM. BALB / c mice injected with mouse IgG were used as the positive control. Student's t-test was used, *p<0.05, **p<0.01, ***p<0.001 indicated significant differences.

[0039] Figure 13 Injection of KIH-BsAb inhibited the formation of scars by microglia in humanized hCD22 / hTFR1 mice after spinal cord injury. A. Co-labeling of spinal cord tissues of BALB / c mice and hCD22 + / - / hTFR1 + / -Mouse spinal cord tissue, Iba1 (green) indicates microglia in the scar area, and CD68 (red) indicates activated microglia.

[0040] Bar = 100 μm; B. Statistical analysis of the proportion of activated microglia. n = 6, data are expressed as Mean ± SEM. BALB / c mice injected with mouse IgG were used as positive controls. Student's t-test was used, and *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0041] Figure 14 Injection of KIH-BsAb inhibits the formation of fibrous scar after spinal cord injury in humanized hCD22 / hTFR1 mice.

[0042] A. Results of immunofluorescence staining of spinal cord tissue. P4HB (red) indicates fibroblasts in the scar area, GFAP (green) indicates astrocytes, and DAPI (blue) shows cell nuclei. Bar = 100 μm; B. Measurement results of the fibrous scar area. C. Average fluorescence intensity of P4HB. n = 6, data are expressed as Mean ± SEM. BALB / c mice injected with mouse IgG were used as positive controls. Student's t-test was used, and *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences. Detailed implementation manners

[0043] The above content of the present invention will be further described in detail below through specific implementation manners in the form of examples. 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.

[0044] Example 1 Behavioral horizontal ladder test and BMS score after injection of HB22.7 in humanized CD22 mice with spinal cord injury

[0045] I. Adult mouse spinal cord T10 right hemisection model

[0046] 6 eight-week-old female humanized CD22 mice (Jiangsu Jicui Yakang Biotechnology Co., Ltd., product number:

[0047] T54197), and 6 eight-week-old female BALB / c mice (Jiangsu Huachuang Xinnuo Medical Technology Co., Ltd.). After anesthesia, the skin was incised along the midline of the thoracic vertebrae, a T10 laminectomy was performed, and 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 spinal cord. The muscle layer was sutured, and then the skin was fixed with wound clips. The animals 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 Provincial Laboratory Animal Management Committee. The animal experiment license was SYXK (Su) 2022-0046. II. Injection of monoclonal antibody HB22.7

[0048] Since the blood-brain barrier (BBB) is damaged at the initial stage of model establishment, the antibodies injected via the tail vein are more likely to reach the spinal cord injury site through the BBB. Subsequently, as the barrier gradually recovers, it becomes difficult for the antibodies injected via the tail vein to cross the BBB again. Intrathecal injection can bypass the BBB and directly deliver the antibodies to the spinal cord, ensuring an effective concentration at the specific treatment site. By directly acting on the spinal cord region through intrathecal injection, the drug efficacy can be ensured to be long-lasting and systemic side effects can be reduced. Therefore, in this experiment, the drug was administered by combining tail vein injection and intrathecal injection ( Figure 1 A). Half an hour after model establishment, humanized CD22 mice were injected with monoclonal antibody HB22.7 (prepared by Shanghai Genechem Co., Ltd. under commission) via the tail vein. The mice were fixed with a venous-visible mouse tail injection fixator, the mouse tails 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 model establishment. The positions of the midlines of the bilateral iliac bones were felt by hand, and a syringe (30G) was used to inject the diluted antibody into the L5-L6 intervertebral space.

[0049] The amino acid sequence information of HB22.7 is as follows:

[0050] HB22.7 VH (mouse IgG2a)

[0051] EVQLQESGPGLVAPSQSLSITCTVSGFSLSDYGVNWVRQIPGKGLEWLGIIWGDGRTDYNSA LKSRLNISKDNSKSQVFLKMNSLKADDTARYYCARAPGNRAMEYWGQGTSVTVSS (SEQ ID NO:1).

[0052] HB22.7 CH (mouse IgG2a)

[0053] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLY

[0054] TLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFP

[0055] PKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA

[0056] LPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLT

[0057] CMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0058] HB22.7 VL (mouse Kappa)

[0059] IVMTQTPKFLLVSAGDRITLTCKASQSVTNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFT GSGYGTDFTFTISTVQAEDLAVYFCQQDYRSPWTFGGGTKLEIK (SEQ ID NO:2)。

[0060] HB22.7 CL (mouse Kappa)

[0061] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0062] III. Irregular horizontal ladder test

[0063] Three days before model establishment, each group of experimental mice underwent horizontal ladder training, with each mouse undergoing training three times per day. A right hemisection model of the spinal cord at T10 was established in adult mice. After model establishment, irregular horizontal ladder testing was performed weekly. The ladders were randomly spaced at each testing time point, and video was recorded three times per mouse. The total experimental duration was 8 weeks. Two researchers, unaware of the experimental conditions, calculated the error rate of the right hind foot in each group. Figure 1 B) Statistics for the error rate (%) of irregular horizontal ladders. The Y-axis represents the error rate (%), and the X-axis represents the time point. Data are expressed as Mean ± SD. Student's t-test was used. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences. The results showed that injection of the monoclonal antibody HB22.7 reduced the error rate of the horizontal ladder after spinal cord injury.

[0064] 4. Motor Function Evaluation Experiment after Spinal Cord Injury - Open Field Test BMS Score

[0065] The day before modeling, normal mice were placed in an open field to familiarize themselves with the environment and a right hemisection model of spinal cord T10 was established in adult mice. After modeling, BMS scores were performed weekly and recorded with a camera. Scoring was performed according to the scoring rules of the BMS primary and secondary scoring systems. The total experimental duration was 8 weeks. Figure 1 C) shows the BMS score. Data are expressed as mean ± SD using the Student's t-test. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences. The results showed that HB22.7 injection improved the BMS score after spinal cord injury.

[0066] Example 2: TSE MotoRater System for evaluating hind limb fine motor recovery in humanized CD22 mice injected with HB22.7 after spinal cord injury

[0067] 1. Referring to Example 1, a right hemisection model of the spinal cord T10 in adult mice was constructed.

[0068] 2. Refer to Example 1 to administer the drug to adult mice with spinal cord injury.

[0069] 3. TSE testing and statistics

[0070] Eight weeks after modeling, TSE tests were performed on both groups of mice. TSE tests were also performed on uninjured BALB / c mice of the same age (16 weeks). The right side of the mice was shaved and luminous patches were applied to the right pelvis, right hip, knee, right ankle, and right hind foot of the mice. TSE Motion test was used to analyze the footstep trajectory ( Figure 2 A) and knee-right ankle-right hindfoot angle statistics ( Figure 2B). One-way ANOVA was used, followed by Tukey's HSD multiple comparisons. Data are presented as Mean ± SEM, with *p < 0.05, **p < 0.01, and ***p < 0.001 indicating significant differences. The results showed that the step frequency, step length, and knee-right ankle bone-right hind foot angle of the mice injected with HB22.7 were closer to those of uninjured normal mice.

[0071] Example 3 Electrophysiological Test of Humanized CD22 Mice after Spinal Cord Injury with Injection of HB22.7

[0072] I. Referring to Example 1, a right T10 hemisection model of the spinal cord in adult mice was constructed.

[0073] II. Referring to Example 1, the adult mice after spinal cord injury were administered drugs.

[0074] III. Electrophysiological Test and Statistics

[0075] Eight weeks after modeling, 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 T9 lamina was removed to expose the upper segment of the hemisected spinal cord, and a channel full-function electromyogram evoked potential instrument was used to test and analyze the CAMP amplitude ( Figure 3 A). It was found that the CAMP amplitude of the mice injected with the monoclonal antibody HB22.7 was higher than that of the BALB / c mice injected with mouse IgG ( Figure 3 B). One-way ANOVA was used, followed by Tukey's HSD multiple comparisons. Data are presented as Mean ± SEM, with *p < 0.05, **p < 0.01, and ***p < 0.001 indicating significant differences.

[0076] Example 4 Hindlimb Mechanical Pain Test of Humanized CD22 Mice after Spinal Cord Injury with Injection of HB22.7

[0077] I. Referring to Example 1, a right T10 hemisection model of the spinal cord in adult mice was constructed.

[0078] II. Referring to Example 1, the adult mice after spinal cord injury were administered drugs.

[0079] III. Mechanical Pain Test and Statistics

[0080] Eight weeks after modeling, a hindlimb mechanical pain test was performed on the two groups of mice. A von Frey fiber was used to apply a mechanical force 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, and 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 for triggering the response was obtained. The results are as Figure 4As shown. The results showed that the right foot of the mice injected with the 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.

[0081] Example 5 Catwalk test of the hind limbs of humanized CD22 mice after spinal cord injury following injection of HB22.7

[0082] I. Refer to Example 1 to construct a right hemisection model of the T10 spinal cord in adult mice.

[0083] II. Refer to Example 1 to administer drugs to the adult mice after spinal cord injury.

[0084] III. Catwalk test and statistics

[0085] At 4 weeks, 6 weeks, and 8 weeks after modeling, the two groups of mice were subjected to the catwalk test. Using a gait analyzer for testing, 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 5 A). The results showed that the mice injected with HB22.7 had higher gait coordination ( Figure 5 B) and a larger right foot landing area ( Figure 5 C). 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

[0086] Example 6 Axon and blood vessel regeneration in humanized CD22 mice after spinal cord injury following injection of HB22.7

[0087] I. Refer to Example 1 to construct a right hemisection model of the T10 spinal cord in adult mice.

[0088] II. Refer to Example 1 to administer drugs to the adult mice after spinal cord injury.

[0089] III. Immunofluorescence staining of spinal cord tissue and measurement of scar formation by astrocytes

[0090] After 8 weeks, perfusion with 4% paraformaldehyde. Then post-fixed with 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, wash three times with PBS, 10 minutes each time. Dissect the laminae to expose the spinal cord, and try to keep the spinal cord intact. For the spinal cord tissue taken out, 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 thickness of frozen sections is 10 μm. Add immunohistochemical blocking solution and block at room temperature for 2 hours. Dilute the primary antibodies Tuj1 (BioLegend, B249869, Mouse, 1:1000), CD31 (R&D, AF3628, goat, 1:100) 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 minutes each time. 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 Secondary Antibody, AlexaFluor TM 647 (invitrogen, A31571, 1:500) with immunohistochemical secondary antibody diluent. After adding the secondary antibodies, incubate for 2 hours at room temperature in the dark. Discard the secondary antibodies and wash 3 times with PBS, 5 minutes 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 6 shown.( Figure 6 (A) Co-labeling of spinal cord tissues of BALB / c mice and humanized CD22 mice at 8 weeks after injury with 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 6 (B) Counting the number of Tuj1-positive axons in the scar area;( Figure 6 (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. 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.

[0091] Example 7 Formation of scars after injection of HB22.7 microglia in spinal cord injury of humanized CD22 mice

[0092] 1. Construct an adult mouse spinal cord T10 right hemisection model with reference to Example 1.

[0093] 2. Administer drugs to the adult mice after spinal cord injury with reference to Example 1.

[0094] 3. Immunofluorescence staining of spinal cord tissue and measurement of inflammatory cells in the scar area

[0095] 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 minutes each time. Dissect the vertebral lamina to expose the spinal cord, and try to keep the integrity of the spinal cord. 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 frozen sections 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 minutes each time. 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 minutes each time. Add an appropriate amount of fluorescent mounting medium and observe and take pictures under a ZEISS upright fluorescence microscope. [[ID=I7]]

[0096] 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 7 shown, ( Figure 7 A) Co-label the spinal cord tissues of humanized CD22 mice and BALB / c mice 8 weeks after injury with Iba1 and CD68. Iba1 (green) represents microglia in the scar area, and CD68 (red) represents activated microglia. Bar = 100 μm. ( Figure 7B) Statistics of the proportion of activated microglia. 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. The results showed 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.

[0097] Example 8 Fibrous Scar Formation after Injection of HB22.7 in Humanized CD22 Mice with Spinal Cord Injury

[0098] I. Refer to Example 1 to construct a right-sided hemisection model of the T10 spinal cord in adult mice.

[0099] II. Refer to Example 1 to administer drugs to adult mice after spinal cord injury.

[0100] III. Immunofluorescence staining of spinal cord tissue and measurement of fibrous scar area

[0101] 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 minutes each time. Dissect the lamina to expose the spinal cord, and try to keep the spinal cord intact. For the removed 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 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 minutes 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). 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 minutes each time. Add an appropriate amount of fluorescent mounting medium, observe under a ZEISS upright fluorescence microscope, and take pictures. Observe the fibroblast distribution at the injury site, take pictures and count the size of the fibrous scar area in each group. The results are as Figure 8 shown. ( Figure 8A) 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) Results of measurement of fibrous scar area. Average fluorescence intensity of P4HB; ( Figure 8 C) Statistics of fluorescence density of positive astrocytes; Mice injected with mouse IgG after spinal cord injury in BALB / c mice were used as positive controls. 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. The results showed that the number of fibroblasts in the spinal cord scar area of mice in the group injected with humanized CD22 HB22.7 decreased, and the width of the gap in the scar center formed by GFAP-positive astrocytes significantly narrowed.

[0102] Example 9 hCD22 + / - / hTFR1 + / - Behavioral horizontal ladder test and BMS score of mice injected with KIH-BsAb after spinal cord injury

[0103] I. T10 right hemisection model of adult mouse spinal cord

[0104] Humanized CD22 mice (Jiangsu Genscript Biogene Co., Ltd., catalog number: T54197) were mated with humanized TFR1 (Jiangsu Genscript Biogene Co., Ltd., catalog number: T055118) mice to obtain 6 8-week-old female hCD22 + / - / hTFR1 + / - mice, and 6 8-week-old female BALB / c mice (Jiangsu Huachuang Xinnuo Medical Technology Co., Ltd.). After anesthesia, the skin was incised along the midline of the thoracic vertebrae, a T10 laminectomy was performed, and the tip of an iris knife (BVI Beaver, Oakville, Canada) was carefully inserted into the posterior median sulcus of the spinal cord to completely cut the right spinal cord. The muscle layer was sutured, and then the skin was fixed with wound clips. They were placed at 37 °C for resuscitation until fully awake, and a pain relief gel was given for pain relief. All animal experiments were conducted in accordance with animal care guidelines and were ethically approved by the Jiangsu Provincial Laboratory Animal Management Committee. The animal experiment license is SYXK (Su) 2022-0046.

[0105] II. Injection of KIH-BsAb

[0106] In this experiment, the drug was administered by tail vein injection ( Figure 10 A), and the mice were injected with bispecific antibody KIH-BsAb via the tail vein half an hour after modeling. The structure is as Figure 9As shown (prepared by Shanghai Genechem Co., Ltd.). Fix the mouse using a tail vein visualization mouse tail injection fixator. Warm the mouse's tail with warm water and wipe the mouse's tail with an alcohol cotton ball. After the blood vessels dilate, use a 1 ml syringe (30G) to aspirate the diluted antibody and inject it into the mouse. And inject the same dose of the drug 2 weeks after modeling.

[0107] The amino acid sequence information of the bispecific antibody KIH-BsAb is as follows:

[0108] mTfR-VH-knob(VH-CH)(Human IgG1 S354C T366W):

[0109] EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSSKMNY

[0110] ADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVTVSSA

[0111] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL

[0112] YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF

[0113] LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

[0114] VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKN

[0115] QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:5).

[0116] CD22-hole(scFv-Fc)(Human IgG1 Mutation)

[0117] EVQLQESGPGLVAPSQSLSITCTVSGFSLSDYGVNWVRQIPGKGLEWLGIIWGDGRTDYNS

[0118] ALKSRLNISKDNSKSQVFLKMNSLKADDTARYYCARAPGNRAMEYWGQGTSVTVSSGGG

[0119] GSGGGGSGGGGSSIVMTQTPKFLLVSAGDRITLTCKASQSVTNDVAWYQQKPGQSPKLLIY

[0120] YASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYRSPWTFGGGTKLEIKGGG

[0121] GSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0122] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0123] KTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:6)。

[0124] mTfR-VL-CL(Human Kappa)

[0125] DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGVPSRF

[0126] SGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQL

[0127] KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:7)。

[0128] III. Irregular horizontal ladder test

[0129] Three days before modeling, horizontal ladder training was carried out on each group of experimental mice three times a day for each mouse. An adult mouse spinal cord right T10 hemisection model was constructed. After modeling, the irregular horizontal ladder test was carried out weekly. The spacing of the ladder was randomly arranged at each test time point, and the video was recorded by a camera. Each mouse was photographed 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 foot in each group.( Figure 10 B) shows the statistical results of the irregular horizontal ladder error rate (%). The error rate (%) is on the Y-axis, and the time point is on the X-axis. 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 injection of bispecific antibody KIH-BsAb reduced the error rate of the horizontal ladder after spinal cord injury.

[0130] IV. Motor function evaluation experiment after spinal cord injury - Open field test BMS score

[0131] One day before modeling, normal mice were placed in the open field to familiarize with the environment. An adult mouse spinal cord right T10 hemisection model was constructed. After modeling, the BMS score was carried out weekly, and the video was recorded by a camera. Scoring was carried out according to the scoring rules of the BMS main and sub-scoring systems. The total experimental duration was 8 weeks.( Figure 10 C) shows the BMS score results. 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 injection of KIH-BsAb improved the BMS score after spinal cord injury.

[0132] Example 10 hCD22 + / - / hTFR1 + / - Catwalk test of the hind limbs of mice after injection of KIH-BsAb after spinal cord injury

[0133] I. Refer to Example 9 to construct an adult mouse spinal cord right T10 hemisection model.

[0134] II. Refer to Example 9 to administer drugs to adult mice after spinal cord injury.

[0135] III. Catwalk test and statistics

[0136] At 2 weeks and 4 weeks after modeling, the catwalk test was carried out on the two groups of mice. The gait analyzer was used to test each mouse three times and analyze the motor coordination and the footprint area of the right hind foot of the mouse. Figure 11A). It was found that the mice injected with KIH-BsAb antibody had higher gait coordination ( Figure 11 B) and a larger right foot contact area ( Figure 11 C). Data are presented as Mean ± SEM, and Student's t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences.

[0137] Example 11 hCD22 + / - / hTFR1 + / - Axonal and vascular regeneration after injection of KIH-BsAb in mice with spinal cord injury

[0138] I. Refer to Example 9 to construct a right T10 hemisection model of the spinal cord in adult mice.

[0139] II. Refer to Example 9 to administer drugs to adult mice after spinal cord injury.

[0140] III. Immunofluorescence staining of spinal cord tissue and measurement of scar formation by astrocytes

[0141] After 8 weeks, perfusion with 4% paraformaldehyde. Then post-fix with 4% paraformaldehyde for 12 hours. After discarding 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 frozen section thickness is 10 μm. Add immunohistochemical blocking solution and block at room temperature for 2 h. Dilute the primary antibodies Tuj1 (BioLegend, B249869, Mouse, 1:1000), CD31 (R&D, AF3628, goat, 1:100) 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-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, AlexaFluor 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-labeled 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) Count the number of Tuj1-positive axons in the scar area;( Figure 12 C) Count 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 in humanized CD22 + / - / humanized TFR1 + / - mice, the blood vessels in the spinal cord scar area increased significantly after injecting KIH-BsAb after spinal cord injury.

[0142] Example 12 h CD22 + / - / hTFR1 + / - The situation of microglia forming scars after injecting KIH-BsAb in mice with spinal cord injury

[0143] I. Refer to Example 9 to construct a right T10 hemisection model of the adult mouse spinal cord.

[0144] II. Refer to Example 9 to administer drugs to adult mice after spinal cord injury.

[0145] III. Immunofluorescence staining of spinal cord tissues and measurement of inflammatory cells in the scar area

[0146] After 8 weeks, perfusion with 4% paraformaldehyde. Then post-fixed with 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, wash three times with PBS, 10 minutes each time. Dissect the laminae to expose the spinal cord, and try to maintain the integrity of the spinal cord. For the taken 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 thickness of the frozen sections is 10 μm. Add immunohistochemical blocking solution and block at room temperature for 1 hour. Dilute the primary antibodies Iba1 (invitrogen ZG4398731, Goat 1:1000) and CD68 (BIO-RAD 160940, Mouse 1:500) with the 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 minutes each time. 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 hours at room temperature in the dark. Discard the secondary antibodies and wash 3 times with PBS, 5 minutes each time. Add an appropriate amount of fluorescent mounting medium and observe and take pictures under a ZEISS upright fluorescence microscope.

[0147] 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 analysis 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 activated CD68 + / - / humanized TFR1 + / - microglia in the spinal cord scar area of humanized CD22 + mice significantly decreased after injection with KIH-BsAb.

[0148] Example 13 Humanized CD22 + / - / Humanized TFR1 + / - Fibrous scar formation after injection of KIH-BsAb in mice with spinal cord injury

[0149] I. Refer to Example 9 to construct a right-sided T10 hemisection model of the spinal cord in adult mice.

[0150] II. Refer to Example 9 to administer drugs to mice after adult spinal cord injury.

[0151] III. Immunofluorescence staining of spinal cord tissue and measurement of fibrous scar area

[0152] After 8 weeks, perfuse with 4% paraformaldehyde. Then post-fix with 4% paraformaldehyde for 12 hours. After discarding paraformaldehyde, wash three times with PBS, 10 minutes each time. Dissect the lamina to expose the spinal cord, and try to keep the spinal cord intact. For the spinal cord tissue taken out, 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 thickness of frozen sections 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 minutes 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 �mM sodium azide (invitrogen 2752568, 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 minutes 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 count 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 14B) Results of fiber scar area measurement. Mean fluorescence intensity of P4HB; ( Figure 14 C) Statistical analysis of the fluorescence density of positive astrocytes; Mice injected with mouse IgG after spinal cord injury in BALB / c mice were used as positive controls. 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 hCD22 + / - / hTFR1 + / - After spinal cord injury in mice, the number of fibroblasts in the spinal cord scar area of the mice injected with KIH-BsAb decreased, and the width of the gap in the scar center formed by GFAP-positive astrocytes became narrower.

Claims

1. A bispecific antibody, an antibody comprising two specific antigen-binding sites, characterized in that The antibodies with the two specific antigen-binding sites are CD22 antibodies and antibodies against targets highly expressed on endothelial cells of the cerebrovascular system.

2. The bispecific antibody according to claim 1, wherein The targets highly expressed on endothelial cells of the cerebrovascular system are selected from transferrin receptor, insulin receptor, members of the low-density lipoprotein receptor family, melanotransferrin or CD98.

3. The bispecific antibody according to claim 1, wherein The antibodies with the two specific antigen-binding sites are polyclonal antibodies or monoclonal antibodies.

4. The bispecific antibody according to claim 3, wherein The monoclonal antibodies are non-human monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies or fully human monoclonal antibodies.

5. The bispecific antibody according to claim 3, wherein The monoclonal antibodies are genetically engineered antibodies, including single-chain antibodies, bispecific antibodies, Fc fusion proteins, antibody fragments.

6. The bispecific antibody according to claim 5, wherein The antibody fragments are VH single-domain antibodies, Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain variable fragments scFv or disulfide-stabilized variable fragments dsFv.

7. The bispecific antibody according to claim 1, characterized in that The CD22 antibodies are selected from HB22.7, Epratuzumab, m971, Inotuzumab, Moxetumomab, Pinatuzumab, Suciraslimab, Bectumomab or Rezetamig, and the antibody against the target highly expressed on endothelial cells of the cerebrovascular system is the transferrin receptor antibody TFR1, selected from 8D3, OX26, RI7217, CX-2029 or TFR ScFv.

8. The bispecific antibody according to any one of claims 1-7, characterized in that The bispecific antibody is a Knob-into-hole type bispecific antibody.

9. The bispecific antibody according to claim 8, wherein The Knob-into-hole type bispecific antibody has mutations of S354C and T366W at the Knob end and mutations of T366S, L368A, Y407V and Y349C at the Hole end based on human IgG1 FC.

10. The bispecific antibody according to claim 8, wherein The bispecific antibody is formed by pairing TFR1-Fab and CD22-ScFv through the knob-into-hole structure at the FC end.

11. The bispecific antibody according to claim 10, wherein The CD22 antibody includes 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; The transferrin receptor antibody includes 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, 50-66 and 99-107 of SEQ ID NO:3; the light chain variable region has complementarity-determining regions shown by amino acid residues at positions 24-34, 50-56 and 89-97 of SEQ ID NO:

4.

12. The bispecific antibody according to claim 11, wherein The amino acid sequence of the heavy chain variable region of the CD22 antibody is shown as SEQ ID NO: 1; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the transferrin receptor antibody is shown as SEQ ID NO: 3; the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

4.

13. The bispecific antibody according to claim 12, wherein The amino acid sequence information of the bispecific antibody is as follows: The amino acid sequence of mTfR-VH-CH is shown as SEQ ID NO: 5; the amino acid sequence of mTfR-VL-CL is shown as SEQ ID NO: 7; The amino acid sequence of CD22-scFv-Fc is shown as SEQ ID NO:

6.

14. Use of the bispecific antibody according to any one of claims 1-13 in the preparation of a medicament for treating spinal cord injury-related diseases.

15. The application according to claim 13, characterized in that The medicament reduces the formation of glial scars after spinal cord injury.

Images