Fusion proteins comprising an anti-dopamine transporter antibody and a bcl-2 protein and uses thereof

Abstract

The application provides a fusion protein containing an anti-dopamine transporter antibody and a Bcl-2 protein and application thereof. The fusion protein of the application achieves the effect of treating Parkinson's disease through various efficacy experiments.

Description

Technical Field

[0001] This invention relates to a fusion protein comprising an anti-dopamine transporter antibody and Bcl-2 protein and its applications. Background Technology

[0002] Parkinson's disease is a motor disorder that commonly affects people in their fifties and sixties. Its main symptoms include resting tremors, muscle rigidity, bradykinesia, postural instability leading to balance problems. Early symptoms often include rhythmic tremors in one hand, slowed movements, and muscle rigidity that gradually spreads throughout the body, eventually requiring a wheelchair for mobility. Parkinson's disease is caused by the death of neurons in the substantia nigra pars compacta of the midbrain that release dopamine, a neurotransmitter. This leads to dopamine deficiency, which in turn affects the striatum in the basal ganglia, which relies on substantia nigra cells for dopamine transmission. This impairs the regulation of information between the cerebral cortex, thalamus, and extrapyramidal system, resulting in dysfunctional coordination between muscles. Like Alzheimer's disease, Parkinson's disease is incurable. Currently, levodopamine supplementation is used clinically to alleviate symptoms in Parkinson's disease patients, but its use often causes side effects. When symptoms are more severe and cannot be controlled by medication, deep brain stimulation by implanting electrodes in the brain can be considered, but this is a highly invasive and uncertain brain surgery. Because neurons are constantly dying, neither medication nor surgery can at most alleviate certain symptoms, but has any therapeutic function.

[0003] Currently, clinical drug treatments for Parkinson's disease have limited efficacy and serious side effects, making it difficult for many patients to continue treatment. More importantly, drugs only relieve symptoms but fail to address the root causes of neurodegeneration and death. Therefore, developing a new drug that can truly cure Parkinson's disease is a crucial issue that this invention aims to address.

[0004] To address the aforementioned problems, those skilled in the art urgently need to develop novel pharmaceutical compositions for the treatment of Parkinson's disease to benefit the vast population in need. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a fusion protein comprising an anti-dopamine transporter (DAT) antibody and Bcl-2 protein.

[0006] In embodiments of the present invention, the anti-dopamine transporter antibody is a single-chain variable fragment (scFv) or a nanobody (Nb).

[0007] In an embodiment of the present invention, the anti-dopamine transporter antibody comprises an amino acid sequence as shown in Sequence Identification Number: 2.

[0008] In an embodiment of the present invention, the Bcl-2 protein comprises the amino acid sequence shown as sequence identification number 3.

[0009] In embodiments of the present invention, the amino acid sequence of the fusion protein is as shown in Sequence Identification Number: 1 or Sequence Identification Number: 6.

[0010] In an embodiment of the present invention, the fusion protein further comprises a green fluorescent protein, wherein the green fluorescent protein is Aequorea australis green fluorescent protein 1 (AausFP1), which is linked to the N-terminus of the Bcl-2 protein.

[0011] In embodiments of the present invention, the amino acid sequence of AausFP1 is as shown in sequence identification number 4 or sequence identification number 7.

[0012] In an embodiment of the present invention, the fusion protein further comprises a GS linker, wherein the GS linker comprises an amino acid sequence as shown in Sequence Identification Number: 5, and the GS linker connects the C-terminus of the anti-dopamine transporter antibody to the N-terminus of AausFP1.

[0013] In embodiments of the present invention, the fusion protein is expressed on the outer membrane of mitochondria.

[0014] In an embodiment of the present invention, the fusion protein is isolated from umbilical cord mesenchymal stem cells (UMSC) by encapsulating a vector expressing the fusion protein with lentiviral particles produced by human embryonic kidney cells via lentiviral transduction.

[0015] In an embodiment of the present invention, the nanobody comprises complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3).

[0016] In an embodiment of the present invention, the amino acid sequence of CDR1 is as shown in sequence identification number 8, the amino acid sequence of CDR2 is as shown in sequence identification number 9, and the amino acid sequence of CDR3 is as shown in sequence identification number 10.

[0017] Another object of the present invention is to provide a pharmaceutical composition comprising the fusion protein as described above and a pharmaceutically acceptable carrier.

[0018] Another object of the present invention is to provide the use of the fusion protein as described above for the preparation of pharmaceuticals for the treatment of Parkinson's disease.

[0019] In embodiments of the present invention, the fusion protein retains neurites in human neuroblastoma cells that overexpress α-synuclein (α-Syn).

[0020] In embodiments of the present invention, the pharmaceutical product improves motor function and survival rate in individuals suffering from Parkinson's disease.

[0021] In summary, the fusion protein of the present invention achieves the therapeutic effect on Parkinson's disease through the results illustrated in the following examples.

[0022] The following will further illustrate the embodiments of the present invention. The examples listed below are for illustrative purposes only and are not intended to limit the scope of the present invention. Any person skilled in the art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the contents defined in the appended claims. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of the fusion protein of the present invention, wherein DAT scFv represents anti-dopamine transporter (DAT) antibody, scFv represents single chain variable fragment, GS linker represents GS linker, AausFP1 represents Aequorea australis green fluorescent protein 1, and Bcl-2 anchor represents Bcl-2 protein.

[0024] Figure 2The image shows mitochondria targeting the fusion protein generated from umbilical cord mesenchymal stem cells (UMSCs). In this diagram, DAT scFv represents an anti-dopamine transporter antibody, scFv represents a single-chain variable fragment, AausFP represents Aequorea australis green fluorescent protein 1, and Bcl-2 protein was used as the mitochondrial-targeting anchor. The vector expressing the fusion protein was coated with lentiviral particles generated from the human embryonic kidney cell line HEK-293T. After 7 days of lentiviral transduction, UMSCs were harvested and lysed. Mitochondria expressing the fusion protein were isolated using a trehalose-containing buffer for subsequent experiments.

[0025] Figures 3A-3D Characterization of mitochondria expressing the fusion protein. (3A) Gating strategy for mitochondria expressing the fusion protein. UMSC-derived mitochondria expressing the fusion protein were analyzed by flow cytometry using size-defined beads, a specific antibody against the scFv fragment, and a fluorescent signal of AausFP (i.e., AausFP1). (3B, 3C) Expression levels of scFv on DAT-targeted mitochondria were detected by flow cytometry (3B) and dot plotting (3C) using specific antibodies against scFv and cytochrome c. (3D) ATP production was measured using the ATPlite kit according to user instructions.

[0026] Figures 4A-4C Treatment of mitochondria expressing the fusion protein showed that it promoted the preservation of neurites in human neuroblastoma cell line SH-SY5Y cells overexpressing α-synuclein (α-Syn). (4A) A procedure to assess the effect of mitochondria expressing the fusion protein on neuron-like morphology in SH-SY5Y cells overexpressing α-synuclein. UMSC-derived mitochondria or mitochondria expressing the fusion protein (1... Parental cells and SH-SY5Y cells overexpressing α-synuclein were treated with retinoic acid / brain-derived neurotrophic factor (BDNF)-induced differentiation (4 x 10^4 cells). The medium was replaced after 2 hours or mitochondrial treatment and cultured for another 7 days. Neuronal-like neurites (roots, type I nodes, and terminals) of these cells were identified at the specified number of days (4B). Alternatively, these cells were collected according to user instructions, and ATP-generating activity was measured using the ATPlite kit; ns indicates no statistical significance (4C).

[0027] Figures 5A-5D The mitochondria expressing the fusion protein improve motor function in an A53T-Parkinson's disease (PD) transgenic mouse model. (5A) Representative procedure for assessing the effects of mitochondria expressing the fusion protein on A53T-PD mice. Male A53T mice were injected weekly via tail vein with or without mitochondria expressing the fusion protein (5 mg / kg) for three months. Their survival was recorded (5B). At 12 months of age, the swirl bar test (5C) and open field test (OFT) (5D) were performed to assess the motor function of these mice.

[0028] Figure 6 The results showed that in the A53T-PD mouse model, mitochondria expressing the fusion protein were concentrated in the brain. 52-week-old A53T transgenic mice were injected via tail vein with either unmodified mitochondria stained with DiR750 dye or mitochondria expressing the fusion protein (3 mg / kg). Twenty-four hours later, the mice were sacrificed, and the fluorescence signal of DiR750 in brain tissue was detected using an IVIS imaging system at an excitation wavelength of 680 nm and an emission wavelength of 750 nm.

[0029] Figure 7A and 7B Treatment with mitochondria expressing the fusion protein showed that it promoted the preservation of neurites in the human neuroblastoma cell line SH-SY5Y, which overexpresses α-synuclein (α-Syn). This was achieved using UMSC-derived mitochondria or mitochondria expressing the fusion protein (1...). Parental cells and SH-SY5Y cells overexpressing α-synuclein were treated with retinoic acid / brain-derived neurotrophic factor (BDNF)-induced differentiation (4 × 10^4 cells). The medium was replaced after 2 hours or mitochondrial treatment and cultured for another 7 days. (7A) At the specified number of days, neuron-like neurites (roots, type I nodes, and terminals) of these cells were identified. Alternatively, these cells were collected according to user instructions, and ATP-generating activity was measured using the ATPlite kit (7B). Detailed Implementation

[0030] definition

[0031] The numerical values ​​used in this article are approximate, and all experimental data are expressed as approximations. Within a 20% range, it is preferable to be in Within a 10% range, the optimal value is... Within 5%.

[0032] Unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification (especially in the claims below) shall be understood to include both singular and plural forms.

[0033] As used in this article, “treating” or “treatment” means alleviating, reducing, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, and lowering, stopping, or reversing the progression of the severity of a condition or symptom that is being treated.

[0034] According to the present invention, pharmaceutical products can be manufactured in dosage forms suitable for parenterally or orally administration using techniques known to those skilled in the art. These include, but are not limited to, injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0035] The pharmaceutical products according to the present invention can be administered via non-parenteral routes selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0036] The pharmaceutical products according to the present invention may comprise pharmaceutically acceptable carriers widely used in pharmaceutical manufacturing techniques. For example, such pharmaceutically acceptable carriers may comprise one or more agents selected from the group consisting of: solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these agents fall within the scope of expertise and practice of those skilled in the art.

[0037] According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), sugar solution, aqueous solution containing alcohol, and combinations thereof.

[0038] According to the present invention, the amino acid sequence of the fusion protein is as shown in Sequence Identification Number: 1 or Sequence Identification Number: 6. The fusion protein contains an anti-dopamine transporter (DAT) antibody, which contains the amino acid sequence shown in Sequence Identification Number: 2.

[0039] According to the present invention, the fusion protein includes Bcl-2 protein, and the amino acid sequence of Bcl-2 protein is as shown in sequence identification number 3.

[0040] According to the present invention, the fusion protein may contain green fluorescent protein, wherein the green fluorescent protein is Aequorea australis green fluorescent protein 1 (AausFP1), and the amino acid sequence of AausFP1 is as shown in sequence identification number 4 or sequence identification number 7.

[0041] According to the present invention, the fusion protein may include a GS linker. The amino acid sequence of the GS linker is shown as sequence identification number 5.

[0042] According to the present invention, the anti-dopamine transporter antibody can be a single-chain variable fragment (scFv) or a nanobody (Nb). When the anti-dopamine transporter antibody is a nanobody, the amino acid sequence of its complementarity determining region 1 (CDR1) is as shown in sequence identification number 8; the amino acid sequence of its complementarity determining region 2 (CDR2) is as shown in sequence identification number 9; and the amino acid sequence of its complementarity determining region 3 (CDR3) is as shown in sequence identification number 10.

[0043] Figure 1 This is a schematic diagram of the structure of the fusion protein of the present invention, where DAT scFv represents the anti-dopamine transporter (DAT) antibody, scFv represents the single-chain variable fragment, GS linker represents the GS linker, AausFP1 represents the green fluorescent protein 1 of the Australian aequorea australis, and Bcl-2 anchor represents the Bcl-2 protein. The fusion protein consists of the anti-dopamine transporter antibody, the GS linker, AausFP1, ​​and the Bcl-2 protein in the following order from N-terminus to C-terminus.

[0044] The present invention is further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the appended claims.

[0045] Example 1. Generation of mitochondria expressing a fusion protein from umbilical cord mesenchymal stem cells (UMSCs).

[0046] Figure 2The image shows mitochondria targeting the fusion protein generated from umbilical cord mesenchymal stem cells (UMSCs). In this diagram, DAT scFv represents an anti-dopamine transporter antibody, scFv represents a single-chain variable fragment, AausFP represents Aequorea australis green fluorescent protein 1, and Bcl-2 protein was used as the mitochondrial-targeting anchor. The vector expressing the fusion protein was coated with lentiviral particles generated from the human embryonic kidney cell line HEK-293T. After 7 days of lentiviral transduction, UMSCs were harvested and lysed. Mitochondria expressing the fusion protein were isolated using a trehalose-containing buffer for subsequent experiments.

[0047] Example 2. Isolation of mitochondria expressing fusion protein from umbilical cord mesenchymal stem cells (UMSC)

[0048] Figures 3A-3D Characterization of mitochondria expressing the fusion protein. (3A) Gating strategy for mitochondria expressing the fusion protein. UMSC-derived mitochondria expressing the fusion protein were analyzed by flow cytometry using size-defined beads, a specific antibody against the scFv fragment, and fluorescence signals of AausFP (i.e., AausFP1). (3B, 3C) Expression levels of scFv on DAT-targeted mitochondria were detected by flow cytometry (3B) and dot plotting (3C) using specific antibodies against scFv and cytochrome c. (3D) ATP production was measured using the ATPlite kit according to user instructions.

[0049] The results of this embodiment confirm that mitochondria expressing fusion proteins can indeed be isolated from umbilical cord mesenchymal stem cells (UMSCs).

[0050] Example 3. Mitochondria expressing the fusion protein retain neural synapses in human neuroblastoma cell line SH-SY5Y cells that overexpress α-synuclein (α-Syn).

[0051] Figures 4A-4C Treatment of mitochondria expressing the fusion protein showed that it promoted the preservation of neurites in human neuroblastoma cell line SH-SY5Y cells overexpressing α-synuclein (α-Syn). (4A) A procedure to assess the effect of mitochondria expressing the fusion protein on neuron-like morphology in SH-SY5Y cells overexpressing α-synuclein. UMSC-derived mitochondria or mitochondria expressing the fusion protein (1... Parental cells and SH-SY5Y cells overexpressing α-synuclein were treated with retinoic acid / brain-derived neurotrophic factor (BDNF)-induced differentiation (4 × 10^4 cells). The medium was replaced after 2 hours or mitochondrial treatment and cultured for another 7 days. Neuronal-like neurites (roots, type I nodes, and terminals) of these cells were identified at the specified number of days (4B). Alternatively, these cells were collected according to user instructions, and ATP-generating activity was measured using the ATPlite kit; ns indicates no statistical significance (4C).

[0052] The results of this embodiment confirm that mitochondria expressing the fusion protein retain neurites in human neuroblastoma cell line SH-SY5Y cells that overexpress α-synuclein.

[0053] Example 4. Intravenous injection of mitochondria expressing fusion protein improves motor function and survival rate in A53T-Parkinson's disease (PD) mice.

[0054] Figures 5A-5D This demonstrates that mitochondria expressing the fusion protein improve motor function in an A53T-Parkinson's disease (PD) transgenic mouse model. (5A) Representative procedure for assessing the effects of mitochondria expressing the fusion protein on A53T-PD mice. Male A53T mice were administered mitochondria expressing the fusion protein (5 mg / kg) weekly via tail vein injection or without injection for three months. Their survival rate was recorded (5B). At 12 months of age, the rotation bar test (5C) and open field test (OFT) (5D) were performed to assess the motor function of these mice.

[0055] The results of this embodiment confirm that intravenous injection of mitochondria expressing the fusion protein can improve motor function and survival rate in A53T-Parkinson's disease mice.

[0056] Example 5. Biodistribution of mitochondria expressing the fusion protein in A53T-Parkinson's disease (PD) mice.

[0057] Figure 6 The results showed that in the A53T-PD mouse model, mitochondria expressing the fusion protein were concentrated in the brain. 52-week-old A53T transgenic mice were injected via tail vein with either unmodified mitochondria stained with DiR750 dye or mitochondria expressing the fusion protein (3 mg / kg). Twenty-four hours later, the mice were sacrificed, and the fluorescence signal of DiR750 in brain tissue was detected using an IVIS imaging system at an excitation wavelength of 680 nm and an emission wavelength of 750 nm.

[0058] The results of this embodiment confirm that, in the A53T-PD mouse model, mitochondria expressing the fusion protein are concentrated in the brain.

[0059] Example 6. Treatment of mitochondria expressing fusion proteins promotes neurite spur preservation in human neuroblastoma cell line SH-SY5Y cells overexpressing α-synuclein (α-Syn).

[0060] In this embodiment, the anti-dopamine transporter (DAT) antibody of the fusion protein is a nanobody (Nb).

[0061] Figure 7A and 7B Treatment with mitochondria expressing the fusion protein showed that it promoted the preservation of neurites in the human neuroblastoma cell line SH-SY5Y, which overexpresses α-synuclein (α-Syn). This was achieved using UMSC-derived mitochondria or mitochondria expressing the fusion protein (1...). Parental cells and SH-SY5Y cells overexpressing α-synuclein were treated with retinoic acid / brain-derived neurotrophic factor (BDNF)-induced differentiation (4 × 10^4 cells). The medium was replaced after 2 hours or mitochondrial treatment and cultured for another 7 days. (7A) At the specified number of days, neuron-like neurites (roots, type I nodes, and terminals) of these cells were identified. Alternatively, these cells were collected according to user instructions, and ATP-generating activity was measured using the ATPlite kit (7B).

[0062] The results of this embodiment confirm that mitochondria expressing the fusion protein retain neurites in human neuroblastoma cell line SH-SY5Y cells that overexpress α-synuclein.

[0063] In summary, the fusion protein of the present invention achieves the therapeutic effect on Parkinson's disease through the results illustrated in the above embodiments.

[0064] The above description is illustrative only and not restrictive. Any equivalent modifications or alterations made without departing from the spirit and scope of this invention should be included in the appended claims.

Claims

1. A fusion protein comprising an anti-dopamine transporter antibody and Bcl-2 protein.

2. The fusion protein according to claim 1, wherein the anti-dopamine transporter antibody is a single-chain variable fragment or a nanobody.

3. The fusion protein of claim 1, wherein the anti-dopamine transporter antibody comprises an amino acid sequence as shown in Sequence Identification Number:

2.

4. The fusion protein according to claim 3, wherein the Bcl-2 protein comprises the amino acid sequence shown in sequence identification number 3.

5. The fusion protein according to claim 1, wherein the amino acid sequence is as shown in sequence identification number 1 or sequence identification number 6.

6. The fusion protein according to claim 1, further comprising a green fluorescent protein, wherein the green fluorescent protein is Aequorea australis green fluorescent protein 1 (AausFP1), and the AausFP1 is linked to the N-terminus of the Bcl-2 protein.

7. The fusion protein according to claim 6, wherein the amino acid sequence of AausFP1 is as shown in sequence identification number 4 or sequence identification number 7.

8. The fusion protein of claim 6, further comprising a GS linker, wherein the GS linker comprises an amino acid sequence as shown in sequence identification number 5, the GS linker connecting the C-terminus of the anti-dopamine transporter antibody and the N-terminus of the AausFP1.

9. The fusion protein according to claim 1, wherein it is expressed on the outer membrane of mitochondria.

10. The fusion protein of claim 1, wherein the fusion protein is isolated from umbilical cord mesenchymal stem cells (UMSC) by lentiviral transduction, wherein the vector expressing the fusion protein is coated with lentiviral particles produced by human embryonic kidney cells.

11. The fusion protein according to claim 2, wherein the nanobody comprises complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3).

12. The fusion protein according to claim 11, wherein the amino acid sequence of CDR1 is as shown in sequence identification number 8, the amino acid sequence of CDR2 is as shown in sequence identification number 9, and the amino acid sequence of CDR3 is as shown in sequence identification number 10.

13. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. Use of a fusion protein according to any one of claims 1 to 12 in the preparation of a pharmaceutical product for treating Parkinson's disease.

15. The use according to claim 14, wherein the fusion protein retains neurites in human neuroblastoma cells that overexpress α-synuclein (α-Syn).

16. The use according to claim 14, wherein the pharmaceutical product improves motor function and survival rate in individuals suffering from Parkinson's disease.

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