Use of an anti-CXCL16 antibody in the preparation of a medicament for treating spinal cord injury

Abstract

The present invention discloses the use of an anti-CXCL16 antibody as the sole active ingredient in the preparation of a medicament for treating spinal cord injury. The inventors' research found that injecting the CXCL16 antibody can effectively neutralize CXCL16 in the spinal cord of mice, significantly reduce the number of CD8+ T cells and the number of GZMB+ CD8+ T cells in the spinal cord of mice, and can significantly improve the hind limb movement of mice after spinal cord injury, increase the maximum height of the iliac crest and the maximum height of the toes during the movement of mice. Therefore, it is inferred that the anti-CXCL16 antibody can be used as a therapeutic drug for spinal cord injury, providing a new way for the treatment of spinal cord injury. In addition, compared with the current therapeutic drugs for spinal cord injury, the CXCL16 antibody has low toxicity and side effects, and the CXCL16 antibody has a wider treatment time window and is more convenient to use.

Description

Technical Field

[0001] The present invention relates to the use of an anti-CXCL16 antibody in the preparation of a medicament for treating spinal cord injury, and belongs to the field of biomedical technology. Background Art

[0002] Spinal cord injury is a severe traumatic disease of the central nervous system, often leading to irreversible motor, sensory, urinary and fecal, and multi-system dysfunction below the injured segment, which not only causes great harm to the physical and mental health of patients, but also is a heavy burden on the entire family and society. Although many research teams at home and abroad are currently committed to the research on spinal cord injury, due to its manifestation as a complex pathophysiological event with multiple components, multiple time and space, and multiple fields, and the existence of spatio-temporal heterogeneity at the cellular and molecular levels, the underlying mechanism is still unclear, the basic research has been in a bottleneck period for a long time, and the clinical translation is extremely limited. At present, methylprednisolone is usually used clinically to treat spinal cord injury. The application conditions of this drug are extremely limited. It must be used within 8 hours after spinal cord injury and requires high-dose pulse therapy, which has certain side effects. Therefore, deeply exploring the pathogenesis and pathophysiological process of spinal cord injury and seeking effective treatment targets conform to the medical direction of the country to focus on clinical problems and overcome difficult and complicated diseases, and have great clinical guiding significance.

[0003] Due to the existence of the blood-brain and blood-spinal cord barriers, which block the infiltration of peripheral circulating immune cells, immunologically active molecules, metabolites, signal molecules, etc. into the central nervous system, the brain and spinal cord have long been regarded as "immunologically privileged" organs. However, many recent studies have questioned the above view. The current mainstream view in the academic community is that the central nervous system no longer has "immunological privilege", so the central nervous system and the peripheral immune system cannot be simply separated and studied separately. In fact, after spinal cord injury, due to the severe damage of the blood-spinal cord barrier, a large number of peripheral immune cells, including monocytes / macrophages, neutrophils, T lymphocytes, B lymphocytes, NK cells, etc., infiltrate into the spinal cord microenvironment and jointly mediate the immune inflammatory response with the intrinsic immune cells of the spinal cord - microglia. As is well known, the composition of T lymphocytes is quite complex and heterogeneous, and they are constantly updated in the body. At the same time, there may be cell subsets at different developmental stages or functions at the same time. According to their different functions in the immune response, they can be divided into helper T cells, effector T cells, cytotoxic T cells, memory T cells, regulatory T cells, etc. Among them, helper T cells are also called CD4 + T cells, and cytotoxic T cells are called CD8 + T cells. Generally speaking, CD8 +T cells can kill specific target cells through the perforin, granzyme, and Fas / FasL signaling systems, participate in immune responses, have a killing effect on viruses, tumor cells, etc., and together with NK cells form an important defense line against viruses and tumor immunity in the body. However, a large number of infiltrating CD8 + T cells can also promote nerve cell death and inhibit axon regeneration, which is not conducive to nervous system homeostasis. Previous studies by our research group have shown that after spinal cord injury, a large number of CD8 + T cells infiltrate into the spinal cord parenchymal tissue, leading to disorder of the immune microenvironment in the spinal cord tissue, inhibiting nerve axon regeneration and motor function recovery; while inhibiting CD8 + T cell infiltration shows good neuroprotective effects.

[0004] As is well known, the migration and recruitment of T cells in the nervous system require the participation of chemokines, and the CXCL16 / CXCR6 signaling pathway, as a classical cell chemotactic signaling pathway, has been previously reported to participate in regulating the infiltration of T cells in cerebrospinal fluid. CXCL16 is a chemokine, that is, a cytokine, which induces and regulates the migration and activation of immune cells (such as T cells, B cells, monocytes, and natural killer cells, etc.). CXCR6 is the receptor of CXCL16. CXCL16 triggers downstream signal transduction on the surface of target cells by binding to CXCR6, thereby affecting cell physiological processes such as gene expression, proliferation, survival, and cell migration. This signaling pathway has been widely studied in various inflammatory diseases and immune regulation. In the nervous system, the CXCL16-CXCR6 signaling pathway has also been found to play a role in neuroimmune diseases such as multiple sclerosis. This signaling pathway may be involved in the inflammatory response of neurons and the intervention of immune cells, affecting the injury and repair processes of the nervous system. Summary of the Invention

[0005] The object of the present invention is to provide the use of anti-CXCL16 antibody in the preparation of a drug for treating spinal cord injury.

[0006] Technical Solution

[0007] The use of anti-CXCL16 antibody in the preparation of a drug for treating spinal cord injury, wherein in the drug, the anti-CXCL16 antibody is the only active ingredient.

[0008] Further, the drug includes an anti-CXCL16 antibody and a pharmaceutically acceptable carrier.

[0009] Further, the dosage form of the drug is a liquid preparation.

[0010] The inventors found through animal experimental studies that injecting CXCL16 antibody can effectively neutralize CXCL16 in the spinal cord of mice and can significantly reduce CD8 in the spinal cord of mice +T cell count and GZMB + CD8 + The number of T cells can significantly improve the hindlimb movement of mice after spinal cord injury, increase the maximum height of the iliac crest and the maximum height of the toes during mouse movement. Therefore, it is considered that anti-CXCL16 antibody can be used as a therapeutic drug for spinal cord injury.

[0011] Advantages of the present invention:

[0012] (1) The present invention discloses the use of anti-CXCL16 antibody in the preparation of a drug for treating spinal cord injury. The CXCL16 antibody can target and neutralize CXCL16 in the spinal cord injury microenvironment, inhibit the recruitment of cytotoxic CD8 + T cells, and effectively relieve secondary injury.

[0013] (2) Compared with the current drugs for treating spinal cord injury, the anti-CXCL16 antibody has low toxicity and side effects, and has a wider treatment time window and is more convenient to use. Description of the drawings

[0014] Figure 1 It is a fluorescence staining map of mouse spinal cord tissue sections after antibody injection;

[0015] Figure 2 It is the flow cytometry staining result of mouse spinal cord tissue after antibody injection;

[0016] Figure 3 It is the BMS score result of the hindlimb motor function of mice after antibody injection;

[0017] Figure 4 It is the gait analysis result of mice after antibody injection;

[0018] Figure 5 It is the statistical graph of the step length of mice after antibody injection;

[0019] Figure 6 It is the statistical graph of the maximum height of the iliac crest of mice after antibody injection;

[0020] Figure 7 It is the statistical graph of the maximum height of the toes of mice after antibody injection; [[ID=4�]]

[0021] Figure 8 It is a schematic diagram of the change in the ankle and knee joint angles of mice during movement after antibody injection;

[0022] Figure 9 It is the determination and amplitude statistics of the electromyogram of the hindlimbs of mice after antibody injection. Detailed implementation manners

[0023] The technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings and specific embodiments.

[0024] Example 1

[0025] Take C57BL / 6 female mice (Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing), and divide them into a control group IgG group and an experimental group anti-CXCL16 group. The modeling process and treatment steps are as follows: C57BL / 6 mice are fasted and water-deprived 6 hours before surgery. After inhalation anesthesia with isoflurane (Shenzhen Rewod Life Science Co., Ltd.), the back skin of the mice is shaved and disinfected with iodophor. A midline incision is made on the back, and the subcutaneous tissue, fascia, muscle, and paravertebral tissue are bluntly separated layer by layer to expose T8 and adjacent segments. The T8 lamina is carefully removed with a curved forceps to expose the spinal cord, and attention is paid to hemostasis. The spinal cord of the mice is clamped with a micro forceps (3 mm tip) for 2 seconds. After rinsing with normal saline, obvious spinal cord hemorrhage and edema can be observed, and then the incision is sutured layer by layer and disinfected, and the mice are kept warm until they wake up and then put back into the cage. Artificial urination is given every day after the operation until the bladder function returns to normal, and antibiotics and painkillers are given. From 1 week before spinal cord injury to 8 weeks after injury, the mice in the anti-CXCL16 group are injected intrathecally with 200 μg of CXCL16 mouse monoclonal antibody (once a week), while the control group is injected with IgG antibody.

[0026] BMS scores are performed from 1 to 8 weeks after injury to evaluate the recovery of hind limb motor function in mice; at 2 weeks after injury, immunofluorescence is used to detect the expression of CXCL16 in the spinal cord of mice, and flow cytometry is used to analyze the number of cytotoxic CD8 + T cells; at 8 weeks after spinal cord injury, the motor gait of mice is recorded and analyzed, and the amplitude of motor evoked potential is measured by electromyogram to evaluate the nerve conduction function. The above all behavioral tests and pathological experimental methods are as follows:

[0027] (1) BMS score

[0028] BMS scores are performed on each group of mice before and 1 to 8 weeks after modeling. The scoring is independently completed by two researchers who are familiar with the scoring rules and do not know the grouping situation. The mice are placed in an open field to observe their free activities for 4 minutes to obtain the score. The final score of each mouse is the average of the scores of the two scorers. The scoring rules are shown in Table 1.

[0029] Table 1: BMS scoring system

[0030]

[0031]

[0032] (2) Immunofluorescence staining

[0033] After the mice were euthanized, the spinal cords were removed, fixed in 4% paraformaldehyde (Wuhan Sevier) for 24 h, dehydrated in gradient ethanol, cleared in xylene, embedded in paraffin and sectioned. After dewaxing the paraffin sections in xylene and hydrating them in gradient alcohol, they were repaired under high temperature and pressure with sodium citrate repair solution (Wuhan Sevier), and then blocked with 5% BSA (Thermo Fisher Scientific) solution at room temperature for 1 hour to block non-specific binding. The primary antibody was added dropwise and incubated overnight at 4 °C in a wet box, then washed three times with PBS for 5 minutes each time. Then, the fluorescent secondary antibody was incubated at room temperature in the dark for 2 hours, and the washing with PBS was continued three times for 5 minutes each time. After counterstaining with DAPI and mounting the coverslip, photographs were taken. The information of the antibodies used is shown in Table 2.

[0034] Table 2: Antibody information

[0035]

[0036] (3) Flow cytometry analysis

[0037] After the mice were euthanized, the spinal cords were removed. Taking the injury site as the center, a 4-mm-long thoracic spinal cord was taken, minced and placed in 1.25% trypsin digestion solution. After digestion in an incubator for 20 minutes, it was resuspended with 30% Percoll solution, and the myelin fragments were removed by low-speed centrifugation at 800 g for 30 minutes. Subsequently, it was co-incubated with the flow cytometry primary antibody for 30 minutes, washed three times with PBS and then detected by machine.

[0038] (4) Gait analysis

[0039] The mice were placed on a runway 80 cm long and 4 cm wide. The anatomical landmarks of the hindlimbs were prominently shown at the iliac crest, hip joint, knee joint, ankle joint and the last toe. The mice were recorded with a camera for 6 - 10 consecutive gaits (60 fps, 4k) in the central 40-cm area. If the hindlimbs did not move, the forelimbs were used to determine the gait. Then, DeepLabCut analyzed the video offline and automatically tracked the skeletal landmarks. The maximum iliac crest and toe heights, stride length and knee-ankle angle changes in each gait cycle were measured, and a bar view of the hindlimb movement was plotted in MATLAB.

[0040] (5) Electromyogram

[0041] Electromyogram detection was performed 8 weeks after modeling. After anesthetizing the mice, the stimulating electrode was placed at the cephalic end of the exposed spinal cord, and the recording electrode was inserted 1.5 mm deep into the flexor of the biceps femoris. The reference electrode was placed at the distal end of the hindlimb tendon, and the ground wire was placed subcutaneously. The evoked potential was stimulated with 0.5 mA, 0.5 ms, 1 Hz, and the electromyogram amplitude was calculated to evaluate the hindlimb function.

[0042] The results are shown in Figures 1-9 : The data in all statistical graphs are presented in the form of mean ± standard error.

[0043] Figure 1It is a fluorescence staining map of the spinal cord tissue sections of mice after antibody injection. As can be seen from the figure, the CXCL16 positive staining in the spinal cord of the anti-CXCL16 group mice was significantly less than that of the IgG group, indicating that the injection of CXCL16 antibody could effectively neutralize CXCL16 in the spinal cord.

[0044] Figure 2 It is the flow cytometry staining result of the spinal cord tissue of mice after antibody injection. As can be seen from the figure, the injection of CXCL16 antibody significantly reduced the number of CD8 + T cells and GZMB + CD8 + T cell number.

[0045] Figure 3 It is the BMS score result of the hind limb motor function of mice after antibody injection. As can be seen from the figure, the BMS score of the uninjured front and hind limbs of mice was 9 points, and the motor ability of the hind limbs was lost at the time of injury, with a BMS score of 0 points. However, 1-8 weeks after injury, the recovery of the hind limb motor function of the mice injected with CXCL16 antibody was significantly faster than that of the control group, and the BMS score was higher.

[0046] Figure 4 It is the gait analysis result of mice after antibody injection. As can be seen from the figure, only slight ankle joint movement was shown in the hind limbs of the IgG group mice, while extensive ankle joint movement and dorsal foot standing appeared in the hind limbs of the mice injected with CXCL16 antibody, indicating that the injection of CXCL16 antibody significantly improved the hind limb movement of mice after spinal cord injury.

[0047] Figure 5 It is the statistical graph of the step length of mice after antibody injection. As can be seen from the figure, the injection of CXCL16 antibody significantly increased the movement step length of mice.

[0048] Figure 6 It is the statistical graph of the maximum height of the iliac crest of mice after antibody injection. As can be seen from the figure, the injection of CXCL16 antibody significantly increased the maximum height of the iliac crest during the movement of mice.

[0049] Figure 7 It is the statistical graph of the maximum height of the toes of mice after antibody injection. As can be seen from the figure, the injection of CXCL16 antibody significantly increased the maximum height of the toes during the movement of mice.

[0050] Figure 8 It is the schematic diagram of the ankle and knee joint angle changes during the movement of mice after antibody injection. As can be seen from the figure, the change range of the ankle joint of the anti-CXCL16 group mice during the movement was significantly larger than that of the IgG group mice, indicating that the injection of CXCL16 antibody significantly improved the hind limb motor function of mice.

[0051] Figure 9Measurement of electromyogram and amplitude statistics of the hindlimbs of mice after injecting antibodies; It can be seen from the figure that compared with the IgG group, the mice injected with CXCL16 antibody had shorter latency and higher amplitude of motor evoked potential, indicating that in-situ injection of CXCL16 antibody can improve the hindlimb nerve conduction function of spinal cord injury mice.

Claims

1. Use of an anti-CXCL16 monoclonal antibody in the preparation of a medicament for treating traumatic spinal cord injury, wherein the anti-CXCL16 monoclonal antibody is the only active ingredient in the medicament.

2. The use according to claim 1, characterized in that, The medicament comprises an anti-CXCL16 monoclonal antibody and a pharmaceutically acceptable carrier.

3. The use according to claim 1 or 2, characterized in that, The dosage form of the medicament is a liquid preparation.

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