Optic neuroprotective agents containing anti-LRP1 antibodies

Abstract

The present invention pertains to an optical nerve protecting agent containing an anti-low density lipoprotein receptor-related protein 1 (LRP1) antibody or fragment of the anti-LRP1 antibody.

Description

[Technical Field]

[0001] This invention relates to an optic nerve protective agent containing an anti-LRP1 antibody. [Background technology]

[0002] Glaucoma is a progressive neurodegenerative disease in which the retinal ganglion cells that make up the optic nerve degenerate for some reason, resulting in visual field defects. It is the leading cause of blindness in Japan and the second leading cause of blindness worldwide.

[0003] Currently, the only treatment approach to prevent the progression of glaucoma is lowering intraocular pressure (IOP). In Japan, 70% of glaucoma patients have normal IOP, and there are many cases where glaucoma symptoms progress even when IOP is controlled with antihypertensive drugs. Therefore, there has been a need for the development of glaucoma treatments based on new mechanisms of action.

[0004] In glaucoma, retinal ganglion cells that make up the optic nerve are damaged. To elucidate the mechanism of optic nerve degeneration in optic nerve diseases, the inventors focused on glial cell-derived lipoproteins, which are protective factors for these retinal ganglion cells, and conducted research on the suppression of retinal neuronal cell death in optic nerve degeneration such as glaucoma. As a result, they confirmed in vitro that glial cell-derived apolipoprotein E-containing lipoproteins (E-LPs) have a neuroprotective effect against apoptosis (Non-Patent Literature 1, Non-Patent Literature 2, and Non-Patent Literature 3). Here, apolipoprotein E-containing lipoproteins (E-LPs) are apolipoprotein-binding lipoproteins in which apolipoprotein E, a type of apolipoprotein, and lipids are chemically associated.

[0005] Furthermore, the inventors injected E-LP into the vitreous humor of mice, which contain a large amount of lipoprotein, and found that E-LP has neuroprotective effects against apoptosis in an in vivo system. They also found that a complex of E-LP with neuroprotective molecules such as Low-density lipoprotein receptor-related protein-1 (LRP1), which is the receptor for E-LP, similarly has neuroprotective effects against apoptosis in an in vivo system (Patent Document 1).

[0006] In other words, the present inventors have previously shown that E-LP protects primary cultured rat retinal ganglion cells from apoptosis via LRP1, the receptor for E-LP, and have found that E-LP can be used as an inhibitor of neuroprotective inhibitory effects (Patent Document 1). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Hayashi,H.,et al.,J.Neurosci.,February 21,2007,27(8):1933-1941 [Non-Patent Document 2] Hayashi, Hideki et al.: Neurochemistry Vol. 49 No. 2 / 3 Page 483 (2010) [Non-Patent Document 3] Hayashi, H., et al., J Biol Chem:Vol.284 No.43 pp.29605-29613(2009) [Patent Documents]

[0008] [Patent Document 1] Patent No. 6031718 [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention aims to provide an optic nerve protective agent containing an anti-LRP1 antibody. [Means for solving the problem]

[0010] The inventors further advanced their research and revealed that an antibody against LRP1, the receptor for E-LP, suppresses neuropathy induced in primary cultured retinal ganglion cells and a rat glaucoma model. This result suggests the potential of antibody-based glaucoma treatment based on LRP1-mediated retinal ganglion cell protection.

[0011] In other words, the present invention is: [1] A subject comprising an anti-LRP1 (Low density lipoprotein receptor-related protein 1) antibody or an anti-LRP1 antibody fragment. A neuroprotective agent for protecting the optic nerve in the field; [2] The optic nerve protective agent according to [1], wherein the anti-LRP1 antibody or the antibody fragment specifically binds to the C2 domain of LRP1; [3] The anti-LRP1 antibody or the antibody fragment is (a) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 1, in the region of amino acids from position 786 to 1165, (b) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 2, in the region of amino acids from position 787 to 1166, (c) In the amino acid region of LRP1 having the amino acid sequence shown in Sequence ID No. 3, from position 786 to 1165, or (d) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 4, in the region of amino acids from position 787 to 1166, A neuroprotective agent for optic nerves that recognizes an epitope, as described in [1] or [2]; [4](i) The anti-LRP1 antibody or the antibody fragment is Heavy chain complementarity determination region 1 (VH CDR1) containing an amino acid sequence having more than 80% sequence identity with the amino acid sequence shown in Sequence ID No. 9, A heavy-chain complementarity-determining region 2 (VH CDR2) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 10, and a heavy-chain complementarity-determining region 3 (VH CDR3) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 11, and a light-chain complementarity-determining region 1 (VL CDR1) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, and a light-chain complementarity-determining region 2 (VL CDR2) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, and a light-chain complementarity-determining region 3 (VL CDR3) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 14, and comprising, or (ii) the anti-LRP1 antibody or the antibody fragment, a heavy-chain variable region comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, and a light-chain variable region comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 16, and comprising, or (iii) the anti-LRP1 antibody or the antibody fragment, an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, the neuroprotective agent according to any one of [1] to [3]; [%5] The neuroprotective agent according to any one of [1] to [4], wherein the anti-LRP1 antibody or the antibody fragment is a humanized antibody or a human antibody; [%6] The neuroprotective agent according to any one of [1] to [5], wherein the anti-LRP1 antibody or the antibody fragment binds to LRP1 with a dissociation constant (Kd) of 1×10 -7 M to 1×10 -11 M; [%7] A composition comprising the neuroprotective agent according to any one of [1] to [6]; [%8] The composition according to [7], which is topically administered to a subject; [9] The composition according to [8], wherein the local administration is intravitreal administration;

[10] The composition according to [8] or [9], which is in the form of an eye drop;

[11] The composition according to any one of [8] to

[10] , which is a composition for treating glaucoma;

[12] The composition according to any one of [8] to

[11] , wherein the subject has an intraocular pressure of 10 to 21 mmHg;

[13] (i) A heavy-chain complementarity-determining region 1 (VH CDR1) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9, A heavy-chain complementarity-determining region 2 (VH CDR2) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 10, A heavy-chain complementarity-determining region 3 (VH CDR3) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 11, A light-chain complementarity-determining region 1 (VL CDR1) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, A light-chain complementarity-determining region 2 (VL CDR2) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, A light-chain complementarity-determining region 3 (VL CDR3) comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 14, and comprising, (ii) A heavy-chain variable region comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, A light-chain variable region comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 16, [[ID=2�]] and comprising, or (iii) An anti-LRP1 antibody or an anti-LRP1 antibody fragment comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19;

[14] Relates to a composition comprising the anti-LRP1 antibody or anti-LRP1 antibody fragment according to

[13] .

Advantages of the Invention

[0012] According to the present invention, an optic nerve protective agent containing an anti-LRP1 antibody can be provided. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows a schematic of the antibody-mediated neuroprotective mechanism. [Figure 2A] A schematic diagram of LRP1 and the individual ligand-binding domain proteins. [Figure 2B] This graph plots the reactivity of each clone (horizontal axis). [Figure 2C] This figure shows the amino acid sequences of the heavy and light chains of scFv. [Figure 2D] This figure shows the SDS-PAGE of each purified scFv. [Figure 2E] This figure shows the ELISA results for each purified scFv. [Figure 3A] This figure shows the protective effect of scFv (Group #1) on retinal ganglion cells against glutamate neurotoxicity. [Figure 3B] This figure shows the protective effect of scFv (Group #2) on retinal ganglion cells against glutamate neurotoxicity. [Figure 3C] This figure shows the protective effect of scFv (Group #3) on retinal ganglion cells against glutamate neurotoxicity. [Figure 4] This figure shows the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats (results of retinal immunoblotting analysis). [Figure 5] This figure shows the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats (summary of results from retinal immunoblotting analysis). [Figure 6] This figure shows the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats (HE staining of retinal tissue sections). [Figure 7]This figure shows the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats (analysis of HE-stained images of retinal tissue sections, retinal ganglion cell count). [Figure 8A] This figure shows the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats (analysis of HE-stained images of retinal tissue sections, thickness of the inner plexiform layer). [Figure 8B] This figure shows the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats (analysis of HE-stained images of retinal tissue sections, thickness of the inner plexiform layer), obtained from a test commissioned to a third-party organization. [Figure 9] This figure shows the retinal thickness from the inner plexiform layer inward, as measured by optical coherence tomography (OCT). [Figure 10] This diagram schematically shows the method for producing anti-LRP1 human IgG1 antibody from scFv. [Figure 11A] This figure shows the ELISA results for anti-LRPl human IgGl antibodies. [Figure 11B] This figure shows the ELISA results for the anti-OVA antibody (control). [Figure 12] This figure shows the binding affinity of anti-LRP1 human IgG1 antibody to immobilized LRP1-Fc. [Figure 13] This figure shows the protective effect on retinal ganglion cells, indicated by the intracellular influx of calcium induced by glutamate stimulation. [Figure 14] This figure shows the localization of anti-LRP1 human IgG1 antibody after intravitreal administration. [Figure 15] This figure shows the neuroprotective effects of various concentrations of anti-LRP1 human IgG1 antibodies. [Figure 16A] This figure shows the results of electrophoretic gel staining analysis of LRP1 recombinant protein-deficient mutants. [Figure 16B] This figure shows the results of immunoblotting analysis of LRP1 recombinant protein-deficient mutants. [Modes for carrying out the invention]

[0014] The present invention will be described in more detail below.

[0015] Specifically, the first aspect of the present invention relates to an optic nerve protective agent comprising an anti-LRP1 antibody or an anti-LRP1 antibody fragment.

[0016] As previously mentioned, the inventors have previously found that E-LP protects primary cultured rat retinal ganglion cells from apoptosis via LRP1, one of the receptors for E-LP, and that E-LP can be used as an inhibitor of neuroprotective inhibitory effects. In the present invention, an anti-LRP1 antibody is used instead of E-LP to protect ganglion cells from apoptosis. Specifically, as shown in Figure 1, the anti-LRP1 antibody specifically binds to LRP1 present on the membrane, thereby activating neuroprotective molecules and inactivating or suppressing neurodegeneration-inducing molecules, thereby preventing apoptosis of ganglion cells and protecting the nerves. Here, neuroprotective molecules include phospholipase C ("PLCγ1" in Figure 1) and protein kinase Cδ ("PKCδ" in Figure 1), and neurodegeneration-inducing molecules include NMDA-type glutamate receptor ("NMDAR" in Figure 1) and calcium ("Ca" in Figure 1). 2+ Examples include '' and GSK3β.

[0017] The inventors have further developed the above research and succeeded in inducing LRP1-mediated protection of optic nerve cells using an anti-LRP1 antibody or an anti-LRP1 antibody fragment. When using E-LP, there is a concern that it may bind to something other than the target LRP1, but the antibody is not only specific to the target, but also has excellent uniformity and high stability. From this point of view, the antibody according to the present invention can be usefully used in pharmaceutical applications.

[0018] LRP1 also includes proteins that are functionally equivalent to the protein in question, which have amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added. Here, "functionally equivalent protein" means a protein that has activity equivalent to that of the protein in question, and "functionally equivalent protein" includes proteins that have sequence identity with the amino acid sequence of the protein in question, for example, 80% or more, 83% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0019] Therefore, the term "LRP1" includes proteins that have an amino acid sequence with 80% or more, 83% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity with the amino acid sequence shown in the sequence number specified below, and that have equivalent activity.

[0020] LRP1 contains the amino acid sequence shown in one of sequence numbers 1-4. Here, the amino acid sequence of LRP1 can be obtained from a database known in the field. An example of the amino acid sequence of LRP1 is shown below: • Sequence ID No. 1 (Low-density lipoprotein receptor-related protein 1 preproprotein [Homo sapiens], NCBI Reference Sequence: NP_002323.2 (4544 amino acids)), • Sequence ID No. 2 (Low-density lipoprotein receptor-related protein 1 [Mus musculus], NCBI Reference Sequence: NP_032538.2 (4545 amino acids)), • Sequence ID No. 3 (Low-density lipoprotein receptor-related protein 1 [Macaca mulatta], NCBI Reference Sequence: XP_015007567.1 (4544 amino acids)) • Sequence ID No. 4 (Low-density lipoprotein receptor-related protein 1 [Rattus norvegicus], NCBI Reference Sequence: NP_001123962.1 (4545 amino acids))

[0021] In the present invention, LRP1 includes proteins having amino acid sequences that have sequence identity with the amino acid sequence specified by SEQ ID NOs: 1, 2, 3, or 4, for example, 80% or more, 83% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9%.

[0022] Furthermore, the base sequence of the gene encoding LRP1 is as follows, for example: • Sequence ID No. 5 (Homo sapiens LDL receptor related protein 1 (LRP1), mRNA, NCBI Reference Sequence: NM_002332.3 (14923 base pairs)), • Sequence ID No. 6 (Mus musculus low density lipoprotein receptor-related protein 1 (Lrp1), mRNA, NCBI Reference Sequence: NM_008512.2 (14907 base pairs)) • Sequence ID No. 7 (PREDICTED: Macaca mulatta LDL receptor related protein 1 (LRP1), mRNA, NCBI Reference Sequence: XM_015152081.2 (14900 base pairs)) • Sequence ID No. 8 (Rattus norvegicus LDL receptor related protein 1 (Lrp1), mRNA, NCBI Reference Sequence: NM_001130490.1 (14714 base pairs)) These are some examples.

[0023] In this specification, the term "antibody" includes not only the antibody itself but also fragments of the antibody, particularly antigen-binding fragments. Therefore, when "antibody" is used in this specification, the term "antibody" includes the antibody and its antibody fragments. Specifically, the term "anti-LRP1 antibody" includes both the anti-LRP1 antibody and the anti-LRP1 antibody fragment.

[0024] The term "antibody fragment" includes, for example, single-chain antibodies (scFv), scFv dimers, disulfide-stabilized V-region fragments (dsFv), Fv, Fab, Fab', F(ab')2, and domain antibodies.

[0025] In one embodiment, the anti-LRP1 antibody may be a single-chain antibody (scFv). In another embodiment, the anti-LRP1 antibody may be an IgG antibody, IgA antibody, IgE antibody, or IgM antibody.

[0026] If the anti-LRP1 antibody is a single-chain antibody (scFv), it can be produced by methods known in the art, for example, by microorganisms using phagemids and plasmids, or by chemical synthesis. For example, in a single-chain anti-LRP1 antibody, the heavy chain variable domain and the light chain variable domain are linked by a flexible linker.

[0027] If the anti-LRP1 antibody is an IgG antibody, it may be a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody, preferably a humanized antibody or a human antibody. If the antibody is an IgG antibody, it can be produced by methods known in the art. An example of a method for producing an IgG antibody will be described later, but is not limited thereto.

[0028] The optic neuroprotective agent according to the present invention, comprising an anti-LRP1 antibody or an anti-LRP1 antibody fragment, protects the optic nerve in vitro or in vivo. Here, the term "optic neuroprotective agent" includes cases where it comprises only the anti-LRP1 antibody and / or its antigen fragment, as well as cases where it comprises other components in addition to the anti-LRP1 antibody and / or its antigen fragment.

[0029] "Protection of the optic nerve" refers to preventing the degeneration of optic nerve cells, specifically, suppressing degeneration and mitigating damage to retinal ganglion cells, as well as protecting the axons of retinal ganglion cells that constitute the optic nerve. Here, the term "degeneration" in relation to nerves includes apoptosis, necrosis, and necroptosis. "Protection of the optic nerve" can be confirmed by means known in the field, for example, by hematoxylin-eosin staining (hereinafter also referred to as "HE staining") of retinal sections, immunoblotting of marker proteins of retinal ganglion cells, and electroretinography (hereinafter also referred to as "ERG"). Furthermore, since nerve degeneration can be detected by immunohistochemical methods, "protection of the optic nerve" may also be confirmed using these methods.

[0030] In one embodiment, the optic nerve protectant protects the optic nerve in the subject. In this specification, “subject” broadly encompasses animals. Animals are, for example, vertebrates, preferably mammals. In this specification, the term “mammal” is used to refer to any animal classified as a mammal, including, but not limited to, humans, mice, rats, monkeys, cattle, horses, sheep, dogs, and cats. In this specification, a preferred mammal is human.

[0031] An anti-LRP1 antibody or its antibody fragment is an antibody that specifically binds to the C2 domain of LRP1.

[0032] In one embodiment, an anti-LRP1 antibody or an antibody fragment thereof (a) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 1, in the region of amino acids from position 786 to 1165, (b) In the amino acid region of LRP1 having the amino acid sequence shown in Sequence ID No. 2, from position 787 to 1166, or (c) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 3, in the region of amino acids from position 786 to 1165, (d) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 4, in the region of amino acids from position 787 to 1166, Recognize the epitope.

[0033] In the present invention, an "epitope" means a partial peptide of an LRP1 polypeptide having antigenicity and / or immunogenicity in the body of an animal, preferably a mammal, more preferably a human, mouse, rat or monkey. An epitope, which is a partial peptide of LRP1 having antigenicity, can be determined by methods known to those skilled in the art, such as an immunoassay. The epitope can be determined, for example, by the following methods. First, various partial structures of the LRP1 polypeptide are prepared using known oligopeptide synthesis techniques. Specifically, a series of polypeptides sequentially shortened by an appropriate length from the C-terminus or N-terminus of the LRP1 polypeptide are considered, and after determining a rough recognition site, shorter peptides are synthesized and their reactivity is examined to determine the epitope. As another specific method, each domain structure of LRP1 is used as a basic unit, each domain structure is expressed as a recombinant protein, and the epitope can be determined by examining the differences in binding properties to these recombinant proteins.

[0034] When the anti-LRP1 antibody or its antibody fragment is an anti-LRP1 human IgG antibody, the anti-LRP1 antibody or its antibody fragment binds to LRP1 with a dissociation constant (Kd) of, for example, 1×10 -5 M to 1×10 -12 M, preferably 1×10 -7 M to 1×10 -11 M, more preferably 1×10 -8 M to 1×10 -10 M, even more preferably 1×10 -8 M to 1×10 -9 M.

[0035] The anti-LRP1 antibody or its antibody fragment according to the present invention includes, for example, the following complementarity determining regions (CDRs; complementarity determining region): Heavy chain complementarity determination region 1 (VH CDR1) containing amino acid sequences having sequence identity of 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence shown in SEQ ID NO: 9, Heavy chain complementarity determination region 2 (VH CDR2) containing amino acid sequences having sequence identity of 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence shown in SEQ ID NO: 10, Heavy chain complementarity determination region 3 (VH CDR3) containing amino acid sequences having sequence identity of 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence shown in SEQ ID NO: 11, Light chain complementarity determination region 1 (VL CDR1) containing amino acid sequences having sequence identity of 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence shown in SEQ ID NO: 12, Light chain complementarity determination region 2 (VL CDR2) containing amino acid sequences having sequence identity of 80%, 83%, 85%, 90%, 93%, 95%, 97%, 98%, and 99% or more with the amino acid sequence shown in SEQ ID NO: 13, Light chain complementarity determination region 3 (VL CDR3) containing amino acid sequences that have sequence identity of 80%, 83%, 85%, 90%, 93%, 95%, 97%, 98%, and 99% or more with the amino acid sequence shown in SEQ ID NO: 14.

[0036] In a preferred embodiment, the anti-LRP1 antibody or the antibody fragment thereof Heavy chain complementarity determination region 1 (VH CDR1), which includes the amino acid sequence shown in SEQ ID NO: 9, Heavy chain complementarity determination region 2 (VH CDR2), which includes the amino acid sequence shown in SEQ ID NO: 10, Heavy chain complementarity determination region 3 (VH CDR3), which includes the amino acid sequence shown in SEQ ID NO: 11, Light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence shown in SEQ ID NO: 12, Light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence shown in SEQ ID NO: 13, Light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence shown in SEQ ID NO: 14 and Includes.

[0037] [Table 1]

[0038] In another embodiment, an anti-LRP1 antibody or an antibody fragment thereof • A heavy chain variable region containing amino acid sequences having sequence identity of 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence shown in SEQ ID NO: 15, • Light chain variable regions containing amino acid sequences having sequence identity of 80%, 83%, 85%, 90%, 93%, 95%, 97%, 98%, and 99% or more with the amino acid sequence shown in SEQ ID NO: 16. Includes.

[0039] In another preferred embodiment, the anti-LRP1 antibody or its antibody fragment is • A heavy chain variable region containing the amino acid sequence shown in Sequence ID No. 15, • Light chain variable region containing the amino acid sequence shown in Sequence ID No. 16 It is preferable that the heavy chain variable region and the light chain variable region are connected by a linker.

[0040] [Table 2]

[0041] In yet another embodiment, the anti-LRP1 antibody or antibody fragment contains an amino acid sequence having sequence identity of 80%, 83%, 85%, 90%, 93%, 95%, 97%, 98%, and 99% or more with the amino acid sequence shown in SEQ ID NO: 19.

[0042] In yet another preferred embodiment, an anti-LRP1 antibody or an antibody fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 19.

[0043] [Table 3]

[0044] Another aspect of the present invention relates to an expression vector for producing an anti-LRP1 antibody or an anti-LRP1 antibody fragment. The expression vector contains one or more nucleic acids encoding the anti-LRP1 antibody or a portion thereof of the present invention, wherein the nucleic acids are operably linked to a control sequence that is recognized by the host cell when the host cell is transfected with the vector.

[0045] The expression vector according to the present invention comprises, for example, at least one nucleic acid selected from the following: DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 15 and DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 16, DNA having the polynucleotide sequence represented by Sequence ID No. 17 and DNA having the polynucleotide sequence represented by Sequence ID No. 18, DNA that hybridizes under constricted conditions with a polynucleotide sequence having a polynucleotide sequence complementary to the polynucleotide sequence represented by SEQ ID NO: 17 and the polynucleotide sequence represented by SEQ ID NO: 18, and encodes a protein that specifically binds to LRP1. • A DNA encoding a protein that has 80% or more sequence identity with the polynucleotide sequence represented by SEQ ID NO: 17 and 80% or more sequence identity with the polynucleotide sequence represented by SEQ ID NO: 18, and that specifically binds to LRP1. DNA containing the degenerate isomer of the polynucleotide sequence represented by SEQ ID NO: 17 and the degenerate isomer of the polynucleotide sequence represented by SEQ ID NO: 18. • DNA encoding a protein having the amino acid sequence represented by Sequence ID No. 19, • DNA having a polynucleotide sequence represented by Sequence ID No. 20, • DNA that hybridizes under constricted conditions with a polynucleotide sequence complementary to the polynucleotide sequence represented by Sequence ID No. 20, and encodes a protein that specifically binds to LRP1. • A DNA that comprises a polynucleotide sequence having 80% or more sequence identity with the polynucleotide sequence represented by Sequence ID No. 20, and which encodes a protein that specifically binds to LRP1. DNA encoding a protein having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of a protein encoded by the polynucleotide sequence represented by Sequence ID No. 20, wherein DNA encoding a protein that specifically binds to LRP1, DNA containing degenerate isomers of the polynucleotide sequence represented by SEQ ID NO: 20.

[0046] The present invention also provides host cells comprising one or more expression vectors, and a method for producing an anti-LRP1 antibody or its antigenic fragment using such host cells, wherein the production method comprises at least one of culturing the host cells comprising expression vectors that produce an anti-LRP1 antibody or its antigenic fragment in a culture medium, and isolating the anti-LRP1 antibody or its antigenic fragment from the host cells or culture medium. The present invention also provides host cells that have been genetically engineered to express an anti-LRP1 antibody or its antigenic fragment in the absence of such vectors.

[0047] Another aspect of the present invention is the aforementioned anti-LRP1 antibody or anti-LRP1 antibody fragment or vision This is a composition containing a neuroprotective agent. Here, the term "composition" includes anti-LRP1 antibody, its antigenic fragment, vision In addition to other components besides neuroprotective agents, if any, anti-LRP1 antibody, its antigenic fragment and / or vision This also includes cases where the composition contains only a neuroprotective agent (i.e., no other components). In one embodiment, the composition according to the present invention is a composition comprising an anti-LRP1 antibody or an anti-LRP1 antibody fragment and a solvent. Preferably, the composition according to the present invention is a pharmaceutical composition containing an anti-LRP1 antibody or an anti-LRP1 antibody fragment in a therapeutically effective amount.

[0048] In one embodiment, the composition according to the present invention is a composition for topical administration, which is administered topically to a subject. Topical administration includes, for example, eye drops, subconjunctival injection, intravitreous administration, and intraocular implantation.

[0049] The composition according to the present invention can be administered orally or parenterally, and is preferably administered parenterally. Examples of dosage forms of the composition include tablets, capsules, granules, pills, lozenges, infusions, injections, eye drops, suppositories, ointments, and patches.

[0050] In one embodiment, the composition according to the present invention is a pharmaceutical composition for the prevention or treatment of glaucoma. As mentioned above, a certain percentage of glaucoma patients have normal intraocular pressure, and there are many cases in which the disease progresses even when intraocular pressure is controlled with intraocular pressure-lowering agents. Therefore, the pharmaceutical composition of the present invention is preferably used in glaucoma patients who have normal intraocular pressure. Specifically, glaucoma patients have intraocular pressure in the range of, for example, 10 to 23 mmHg, preferably 10 to 21 mmHg, and more preferably 10 to 20 mmHg.

[0051] Examples of glaucoma include primary open-angle glaucoma, normal-tension glaucoma, aqueous humor hyperproductive glaucoma, ocular hypertension, acute angle-closure glaucoma, chronic angle-closure glaucoma, plateau iris syndrome, mixed glaucoma, steroid glaucoma, lens-capsular glaucoma, pigment glaucoma, amyloid glaucoma, neovascular glaucoma, and malignant glaucoma.

[0052] In this specification, with respect to glaucoma, the term "treatment" includes not only treating glaucoma but also suppressing or mitigating glaucoma. Specifically, the term "treatment" includes suppressing the progression of optic nerve degeneration and preventing apoptosis, necrosis, and / or necroptosis of the optic nerve.

[0053] The subjects include individuals with glaucoma who have previously received treatment with intraocular pressure-lowering agents, and in particular, individuals with glaucoma who have previously received treatment with intraocular pressure-lowering agents and whose glaucoma symptoms are still progressing. Here, the intraocular pressure-lowering agents are known in this field and include, but are not limited to, adrenergic α2 receptor agonists (Alphagan® eye drops), prostaglandin F2α derivatives (Tapros® eye drops), and Rho kinase inhibitors (Granatec® eye drops). The subjects may also include individuals who have undergone surgical procedures for the treatment of glaucoma.

[0054] When the composition according to the present invention is applied to a subject for the prevention or treatment of glaucoma, the composition is administered parenterally, preferably topically, and more preferably intravitreally. Therefore, if the composition according to the present invention is a pharmaceutical composition, the pharmaceutical composition is, for example, for parenteral administration, preferably topically, and more preferably intravitreally.

[0055] The dosage form of the composition according to the present invention is not particularly limited, but when applied to a subject for the treatment of glaucoma, it is preferably an eye drop or an ophthalmic injection.

[0056] The compositions according to the present invention may typically contain pharmaceutically acceptable additives, such as pharmaceutically acceptable excipients, binders, auxiliaries, lubricants, solvents, diluents, stabilizers, emulsifiers, preservatives, carriers, solubilizers, isotonic agents, and the like, which are known in the art.

[0057] The dosage of the composition according to the present invention may be adjusted as appropriate depending on the age, weight, and medical condition of the subject.

[0058] The optic nerve protective agents and pharmaceutical compositions according to the present invention may be used as combination agents in combination with further agents or pharmaceutical compositions. With respect to combination agents, the active ingredients in each pharmaceutical composition (i.e., the first active ingredient and the second active ingredient) may be administered together, sequentially, or individually, in one combination unit dosage form or in two separate unit dosage forms. A therapeutically effective amount of each active ingredient of the combination agent according to the present invention may be administered simultaneously, individually, or in any order, sequentially or consecutively. In the combination agent, the first active ingredient and the second active ingredient may exist individually in multiple units. Combination agents also include, for example, kits containing each active ingredient and instructions for use.

[0059] According to the present invention, it is possible to provide optic nerve protective agents and compositions containing an anti-LRP1 antibody or an anti-LRP1 antibody fragment that specifically binds to LRP1. By using a highly specific anti-LRP1 antibody or anti-LRP1 antibody fragment, it is possible to specifically act on factors involved in the degeneration of optic nerve cells, and therefore, it is possible to suppress optic nerve degeneration while reducing undesirable side effects.

[0060] Furthermore, according to the present invention, anti-LRP1 antibodies or anti-LRP1 antibody fragments can protect the optic nerve through mechanisms other than intraocular pressure reduction, and are therefore expected to represent a new therapeutic approach for glaucoma. Moreover, due to the mechanism of avoiding the degeneration of optic nerve cells, it is expected to provide an alternative treatment option for glaucoma patients who have previously received treatment with intraocular pressure-lowering agents, and for patients with optic nerve damage with normal intraocular pressure. [Examples]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way by these examples.

[0062] <Primary culture of retinal ganglion cells> Primary cultures of retinal ganglion cells were performed using 2-day-old Sprague Dawley (SD) rats, following a slightly modified method (Hayashi et al., J. Biol. Chem. 2009) based on the method of Barres et al. (1988). Isolated retinal ganglion cells (RGCs) were mixed in RGC medium (1 mM glutamine, 5 μg / ml insulin, 60 μg / ml N-acetylcysteine, 62 ng / ml progesterone, 16 μg / ml putrescine, 40 ng / ml sodium selenite, 0.1 mg / ml bovine serum albumin, 40 ng / ml triiodothyronine, 0.1 mg / ml transferrin, 1 mM sodium pyruvate, 2%, B27 supplement (Invitrogen, Carlsbad, CA), 10 μM forskolin (Sigma, St. Louis, MO), 50 ng / ml brain-derived neurotrophic factor (BDNF; PeproTech, Rocky) RGCs were suspended in basal medium in 50 ng / ml ciliary neurotrophic factor (CNTF; PeproTech) and 50 ng / ml basic fibroblast growth factor (bFGF; PeproTech) (Hill, NJ). 96-well plates were coated with poly-d-lysine (Sigma) and laminin (Sigma), and RGCs were mounted so that 5,000 cells were placed in each well of the 96-well plates and 5,000 cells were placed in each culture insert of the microdishes. The cells were then cultured for at least 10 days before the experiment.

[0063] <Preparation of reconstituted apolipoprotein E-containing lipoprotein> The reconstituted apolipoprotein E-containing lipoprotein was prepared as described in the previous literature (Hayashi et al., J. Neurosci. 2007). AppointmentThe lipoprotein E-containing lipoprotein was composed of 1-palmitoyl-2-oleyl-glycerophosphocholine (POPC; P3017, Sigma), cholesterol (C3045, Sigma), and recombinant human apolipoprotein E, with molar ratios of 100:10:1 or 100:0:1. Preparation media, plasma, or solutions containing the reconstituted apolipoprotein E-containing lipoprotein were subjected to a discontinuous sucrose gradient. The solutions used were 3 ml at a density of 1.30 g / ml, 3 ml at a density of 1.2 g / ml, 3 ml at a density of 1.1 g / ml, and 6 ml at a density of 1.006 g / ml. The sucrose gradient was centrifuged at 100,000 g at 4°C for 72 hours using an SRP28SA1 rotor (Hitachi, Tokyo, Japan). Ten fractions (1.5 ml each) were collected from the top of the slope and subjected to immunoblotting for apolipoprotein E as described below. The fractions containing apolipoprotein E (typically fractions 5-7) were combined and concentrated using an Amicon Ultra filter (50 kDa molecular weight cutoff; Millipore, Bedford, MA). The amount of lipoprotein was adjusted by cholesterol concentration (2 μg / ml) for HDL and by protein concentration (100 ng / ml) for reconstituted lipoprotein. These cholesterol and protein concentrations were measured using a LabAssay cholesterol kit (Wako) and a BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL), respectively.

[0064] <Screening of anti-LRP1 antibodies> Using commercially available LRP1 partial domains and human fc-derived proteins (three types: C2, C3, and C4) as antigens, scFv for each LRP1 cluster (i.e., C2, C3, and C4) was obtained from a synthetic human scFv phage display library. A schematic diagram of the antigens used, i.e., LRP1, is shown in Figure 2A.

[0065] We screened (panned) a human scFv synthetic library for scFvs that specifically bind to LRP1 clusters 2, 3, and 4 (i.e., C2, C3, and C4). As a result, we obtained numerous scFvs specific to each of the LRP1 clusters 2, 3, and 4. The reactivity of each clone (horizontal axis) is plotted in Figure 2B. From the clones after three panning cycles (93 clones each), we obtained multiple phage clones (52, 45, and 47 clones, respectively) that specifically react to each domain of LRPl (ligand-binding clusters C2, C3, and C4). Each scFv was sequenced, and it was confirmed that most of them had different CDR3 sequences. The scFv library used for screening was derived from DP47-DPL16 (PDB:3GHE), a library in which random sequences were introduced into the CDR3 regions of both the heavy and light chains. Figure 2C shows the amino acid sequences of the heavy chain ("H chain" in the figure) and light chain ("L chain" in the figure) of scFv, with the randomized portion in each chain indicated by "X".

[0066] <Evaluation of the LRP1 binding site of anti-LRP1 antibodies> Four scFv clones were purified from the LRP1 cluster. These clones were selected based on their stability in E. coli retention of phagemids and their stability during purification. The sequences of the VH CDR3 and VL CDR3 of the four scFv clones, as well as their supply, are shown in the table below.

[0067] [Table 4]

[0068] Figure 2D shows the SDS-PAGE of each purified scFv. Each purified scFv had a purity of 90% or higher. Figure 2E shows the ELISA results for C2-D11, C2-F8, C2-G3, and C2-G9 among the purified scFv.

[0069] The materials and conditions used in the ELISA are shown in the table below.

[0070] [Table 5]

[0071] The results shown in Figure 2E reveal that these scFv molecules exhibit specific reactivity towards C2-Fc (C2 domain) at a low concentration of 100 ng / ml.

[0072] <Protective effect of scFv on glutamate damage in primary cultured retinal ganglion cells> Apoptosis was induced for 2 hours using 300 μM glutamate and 10 μM glycine. The scFv used was at a concentration of 1 μg / ml, and E-LP was used as a positive control at a concentration of 100 ng / ml. Anti-OVA (ovalbumin) scFv was used as a negative control. Nucleus aggregation 24 hours after glutamate-induced damage was analyzed by Hoechst staining. The results regarding the protective effect of scFv on retinal ganglion cells in Group #1 (n=4~5), Group #2 (n=5), and Group #3 (n=5) are shown in Figures 3A, 3B, and 3C, respectively. These results confirmed that, among the scFv clones (D11, F8, G3, G9, G5, A7) that bind to the ligand-binding cluster C2, D11 exhibits a protective effect on retinal ganglion cells. On the other hand, scFv clones that bind to C3 and C4 did not show any neuroprotective effects.

[0073] <Immunoblotting> The proteins were dissolved in 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS), and 5% β-mercaptoethanol (sample buffer) and boiled for 5 minutes. The proteins were separated by electrophoresis on a polyacrylamide gel containing 0.1% SDS and then transferred to a polyvinylidene difluoride membrane. The membrane was incubated with 5% skim milk in TBS-T (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) at room temperature for 1 hour, and then explored overnight at 4°C with primary antibodies in TBS-T containing 5% bovine serum albumin. Subsequently, the membrane was probed at room temperature for 1 hour with peroxidase-conjugated goat anti-mouse IgG (Thermo), goat anti-human IgG (Thermo), or mouse anti-goat IgG (Thermo). Immunoreactive proteins were visualized using enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) or Super Signal West Dura (Thermo). The primary antibodies used were: human C2-D11 IgG (0.5 μg / ml), mouse antibody β-actin (a5441, dilution 1:10,000, Sigma), and goat anti-human Brn-3a (sc-31984, dilution 1:1000, Santa Cruz). Brn-3a is a marker protein for retinal ganglion cells, and β-actin was used as a loading control. scFv(C2-D11) was administered intravitreously simultaneously with N-methyl-D-aspartic acid (NMDA). The results are shown in Figures 4 and 5.

[0074] Figure 4 shows the results of immunoblotting analysis of the retina, demonstrating the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats. Figure 5 summarizes the results of the immunoblotting analysis of Figure 4 (3 to 6 cases) to show the protective effect of scFv(C2-D11) on retinal ganglion cells in NMDA-induced optic neuropathy rats (summary of immunoblotting analysis results of the retina). From Figures 4 and 5, the protective effect of scFv(C2-D11) on retinal ganglion cells was confirmed.

[0075] <Induction and detection of apoptosis in retinal ganglion cells (RGCs)> RGCs were washed twice with Hanks equilibrium salt solution (HBSS; 14170, Invitrogen) containing 2.4 mM CaCl2 and 20 mM HEPES, without magnesium. They were then incubated at 37°C for 2 hours in HBSS containing 2.4 mM CaCl2 and 20 mM HEPES, without magnesium, with 300 μM glutamic acid and 10 μM glycine, either containing or not containing E-LP, C2-D11 scFv, or C2-D11 IgG. After glutamic acid treatment, RGCs were cultured at 37°C for 22 hours in RGC culture medium without forskolin, BDNF, CNTF, and bFGF. For the detection of apoptosis, RGCs were stained with 1 μg / ml Hoechst 33342 (346-07951, Kumamoto, DOJINDO). Fluorescence images were observed using an Olympus IX71 microscope. Fragmented or reduced nuclei stained with Hoechst dye were counted as apoptotic neurons, while round and smooth nuclei were counted as healthy neurons.

[0076] <Rat ni ok ruN MDA-induced retinal damage Protective effect of C2-D11 antibody against > Intravitreal injection of NMDA (Sigma-A) was performed in a manner similar to that described in previous literature (Tsutsumi T. et al., Invest Ophthalmol Vis Sci. 2016, 57(14):6461-6473), with minor modifications. Specifically, 7-week-old rats were anesthetized with 5% isoflurane and then maintained with 2.5% isoflurane. The pupils were dilated with phenylephrine hydrochloride and tropicamide eye drops. Subsequently, 20 nmol / eye NMDA, 0.15 μg / eye C2-D11 scFv, or 0.75 μg / eye C2-D11 IgG were injected into the vitreous cavity in a total volume of 4 μl, with or without the presence of PBS or 5 ng / eye E-LP as a solvent control. Injection was performed under a microscope using a 34-gauge needle (Nanopass, Terumo, Tokyo, Japan) connected to a microsyringe (80008, Hamilton, Reno, NV) equipped with an injection pump (Fusion 200, Chemyx Inc., Stafford, TX), with the needle inserted approximately 1.0 mm posterior to the corneal margin. Three days after NMDA injection, the eyeball was removed, and the retina was extracted from the sclera. The retina was then added to a lysis buffer [1% Triton X-100; MP Biomedicals (Santa Ana, California, USA), 0.1% sodium deoxycholate; Wako (Osaka, Japan), 1% EDTA; DOJINDO (Kumamoto, Japan), complete protease inhibitor cocktail; Roche (Basel, Switzerland), phosstop phosphatase inhibitor cocktail; Roche, in 50 mM Tris-buffered saline] and sonicated at 4°C for 20 seconds (2 seconds x 10 times) using an ultrasonic device Q125 (QSONICA). The protein concentration of the retinal sample was measured using the BCA protein assay kit (Thermo Fisher Scientific) and subjected to immunoblotting.

[0077] <Preparation of frozen rat eyeballs and HE staining> The preparation of frozen eyeball specimens and HE staining were performed using the following procedure. (1) The eyeball was removed on the third day after administration of NMDA+ / C2-D11 scFv. (2) Gently grasp the eyeball with ring tweezers, cut the conjunctiva with scissors, then cut a longer portion of the optic nerve, and immerse it in Super Fix at 4°C for 2 hours. (3) After fixation for 2 hours, an eye cup was fabricated. (4) Return to Super Fix and fix for 22 hours (overnight, 4°C). (5) After 22 hours, the material was transferred to 20% sucrose and immersed at room temperature until it sank. Next, it was transferred to 30% sucrose and the process was repeated up to 40% sucrose. (6) Remove the eye cup which had sunk in 40% sucrose. (7) OCT compound was placed in the cryomold, and the eye cup was submerged in it. (8) The sample was embedded in isopentane cooled with liquid nitrogen. (9) The temperature of the cryostat sample stage was set to -20°C. (10) The section was cut to a thickness of 10 μm. (11) Thin sections were stained with HE. (12) After HE staining, the number of retinal ganglion cells (RGCs) and the thickness of the internal plexiform layer (IPL) were measured at a position 1 mm to 1.5 mm from the optic nerve head. The results are shown in Figures 6, 7, and 8. In Figure 6, GCL represents the ganglion cell layer and IPL represents the internal plexiform layer.

[0078] The number of cells in the ganglion cell layer (GCL) of the HE-stained images in Figure 6 was counted (4 to 6 cases), and the results showed that scFv(C2-D11) had a protective effect against the reduction in GCL cell number caused by NMDA (Figure 7). In addition, the thickness of the inner plexiform layer (IPL) of the HE-stained images in Figure 6 was measured (4 to 6 cases), and the results showed that scFv(C2-D11) had an inhibitory effect (protective effect) against the reduction in IPL thickness caused by NMDA (Figure 8A). Furthermore, the results obtained from a similar experiment commissioned to a third-party institution are shown in Figure 8B. The results in Figure 8B show that good reproducibility was obtained.

[0079] <Measurement of inner repellent layer thickness using optical coherence tomography> The thickness of the inner plexiform layer of the retina 6 days after treatment with each sample was measured using an optical coherence tomography (OCT). The retinal thickness from the inner plexiform layer is shown in Fig. 9. The thickness of the inner plexiform layer in the NMDA administration group and the NMDA + OVA administration group was significantly decreased compared with that in the PBS administration group, and no decrease was observed in the NMDA + LP administration group and the NMDA + C2-D11 scFv administration group. From this, the retinal protective effect of C2-D11 scFv was confirmed.

[0080] <Production of anti-LRP1 human IgG1 antibody from scFv antibody C2_D11> As outlined in Fig. 10, an anti-LRP1 human IgG1 antibody was produced from C2_D11. From the scFv sequence of C2_D11, the amino acid sequences corresponding to VL and VH were analyzed from the gene sequence of the phagemid vector, and each of VL and VH was incorporated into a gene expression vector having the Fc region of human IgG1. Each plasmid was double-transfected into a eukaryotic cell expression system (HEK-293) to establish cells that transiently express the antibody. The expressed antibody was isolated and purified from the culture supernatant after culturing for about one week by affinity purification with protein A. Note that pY03’-FLAG-C2-D11, which is a phagemid capable of expressing a fusion protein with the g3 protein of M13 phage, was deposited on November 20, 2020, under the deposit number NITE BP-03322 at the Patent Microorganisms Depositary, Biotechnology Center, National Institute of Technology and Evaluation (NPMD) (address: Room 122, 2-5-⑧ Kazusa Kamashima, Kisarazu City, Chiba Prefecture).

[0081] <Binding specificity of anti-LRP1 human IgG1 antibody> The binding specificity of anti-LRPl human IgGl antibodies to LRP1 was evaluated using ELISA and biosensors. The ELISA results for anti-LRPl human IgGl antibodies are shown in Figure 11A, and the ELISA results for the control anti-OVA antibody are shown in Figure 11B. After immobilizing the C2 domain of LRP-1 on an ELISA plate, purified antibody was added to the plate, and the amount of bound antibody was quantified by colorimetric analysis. Furthermore, the binding affinity of anti-LRPl human IgGl antibodies to immobilized LRP1-Fc is shown in Figure 12. The C2 domain protein of LRP-1 was immobilized on a biosensor chip, and antibodies with C2_D11 in the VL and VH positions were continuously added at the concentrations shown in Figure 12. The affinity of the antibody to LRP-1 was calculated by determining the on-rate (Kon) and off-rate (Koff) values ​​for each binding. These results indicate that the dissociation constant (Kd) of the anti-LRPl human IgGl antibody prepared from C2_D11 was 0.9 nM, demonstrating strong affinity and binding specificity for LRPl.

[0082] <Intracellular calcium analysis in retinal ganglion cells (RGCs)> RGC was maintained through experiments using a heating element (Tokai Hit, Shizuoka, Japan). Cells were cultured in microdishes for at least 14 days and incubated with 3 μM Fluo-8 acetoxymetal ester (AAT Bioquest, Sunnyvale, CA) for 30 minutes at 37°C. After washing the cells twice with 500 μl of magnesium-free HBSS containing 2.4 mM CaCl2 and 20 mM HEPES, the cells were administered 300 μM glutamate and 10 μM glycine, with or without 100 ng / ml E-LP or 100 μg / ml C2-D11 IgG. Fluorescence images were acquired every 500 msec using an ORCA-R2 digital CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed using MetaFluor fluorescence ratio imaging software (Molecular Devices, Sunnyvale, CA). In this study, 100 μg / ml ApoE-containing lipoprotein (E-LP) was used as a positive control and an anti-OVA antibody as a negative control to evaluate 100 μg / ml C2-D11 IgG. The results are shown in Figure 13.

[0083] Figure 13 shows the evaluation of retinal ganglion cell protective effects using glutamate-induced intracellular calcium influx as an indicator. Intracellular calcium influx was observed with 300 μM glutamate, and as a result, 100 μg / ml C2-D11 IgG exhibited a stronger inhibitory effect on intracellular calcium influx than E-LP. Since excessive calcium influx induced by 300 μM glutamate induces apoptosis, these results are thought to indicate one of the neuroprotective mechanisms of C2-D11 IgG.

[0084] <Preparation of frozen rat eyeballs and immunofluorescence staining> The frozen specimens of mouse eyeballs and their immunofluorescence staining were performed using the following procedure. (1) Thin sections were prepared in the same manner as the HE staining method described above. (2) A 15% blocking buffer (PBS containing 15% goat serum and 0.05% Tween 20) was added and allowed to react at room temperature for 1 hour. (3) Washed with PBS for 15 minutes. (4) The primary antibody (human anti-LRP1 IgG in 1% blocking buffer) was added and incubated overnight at 4°C. (5) It was washed with PBS for 15 minutes. (6) The secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit IgG (diluted 1:500, Invitrogen)) and Alexa Fluor 594-conjugated goat anti-human IgG (diluted 1:500, Invitrogen)) were added and reacted at room temperature for 1 hour. (7) With PBS containing Heochst33342 (diluted 1:2000) 10 It was washed. (8) After mounting, it was observed with a fluorescence microscope. The results are shown in Fig. 14.

[0085] The above experiment observes whether C2-D11 IgG binds to LRP1 of retinal ganglion cells by fluorescence immunostaining after administration into the rat vitreous body. From the results shown in Fig. 14, it was revealed that C2-D11 IgG and LRP1 are co-localized in the ganglion cell layer (arrow in the figure). On the other hand, it was shown that the anti-OVA antibody is not co-localized. From this result, it was shown that C2-D11 IgG binds to LRP1 on retinal ganglion cells and exhibits a neuroprotective effect.

[0086] <Neuroprotective effect of anti-LRP1 human IgG1 antibody against glutamate-induced retinal ganglion cell death> Glutamate and anti-LRP1 human IgG1 antibody at various concentrations were administered in combination, and the rate of cell death was examined. Specifically, apoptosis was induced for 2 hours using 300 μM glutamate and 10 μM glycine. The anti-LRP1 human IgG1 antibody used was at a concentration of 0.01 - 10 μg / ml. Aggregation of nuclei 24 hours after induction of glutamate injury was analyzed by Hoechst staining. The results are shown in Fig. 15. From Fig. 15, a neuroprotective effect was observed with the 10 μg / mL IgG antibody (n = 5).

[0087] <Production of recombinant protein of LRP1> For epitope mapping, various cluster II knockout mutants of human low-density lipoprotein receptor-associated protein 1 (LRP1) were prepared. Each prepared human LRP1 cluster II knockout mutant was fused with a GST tag. The human LRP1 cluster II cDNA fragments were synthesized to encode the forward linker SGGSTS, cluster II (positions 786-1165), and backward linker ASTGS (Thermo Fisher Scientific, Waltham, MA). The cDNA fragments encoding each cluster II knockout mutant were prepared by PCR using the primer sets and cluster II cDNA fragments shown in the table below as templates. Expression vectors were obtained by digesting the cDNA fragments with BamHI and EcoRI and inserting them into the corresponding restriction sites of the pGEX-6P-1 expression vector (Cytiva, Marlborough, MA). Next, BL21 cells containing expression vectors were cultured in MagicMedia (Thermo Fisher Scientific), and recombinant proteins (each knockout mutant) were produced according to the manufacturer's protocols (Cytiva, Marlborough, MA). After lysing of E. coli by sonication, the GST fusion protein was pulled down with glutathione-Sepharose (Cytiva) and used for immunoblotting.

[0088] [Table 6]

[0089] <Identification of epitopes> The prepared knockout mutants were subjected to electrophoresis and gel staining with Coomassie Brilliant Blue R250. The results are shown in Figure 16A. Immunoblotting of the prepared knockout mutants was performed using C2-D11 IgG in 5% BSA / TBS-T, in the same manner as described in the <Immunoblotting> section above. The secondary antibody used was goat anti-human IgG conjugated with HRP. The results are shown in Figure 16B. From Figure 16B, the C2-D11 epitope was identified.

Claims

1. It comprises an anti-LRP1 (Low Density Lipoprotein Receptor-Related Protein 1) antibody or an anti-LRP1 antibody fragment that specifically binds to LRP1, (i) The anti-LRP1 antibody or the antibody fragment is Heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence shown in SEQ ID NO: 9, The heavy chain complementarity determination region 2 (VH CDR2) includes the amino acid sequence shown in SEQ ID NO: 10, The heavy chain complementarity determination region 3 (VH CDR3) includes the amino acid sequence shown in SEQ ID NO: 11, Light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence shown in SEQ ID NO: 12, Light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence shown in Sequence ID No. 13, Light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence shown in SEQ ID NO: 14 and A nerve-protective agent containing optic nerve protectants.

2. (ii) The anti-LRP1 antibody or the antibody fragment is A heavy chain variable region containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence shown in Sequence ID No. 15, A light chain variable region containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence shown in Sequence ID No. 16, Includes, or (iii) The anti-LRP1 antibody or the antibody fragment is The optic nerve protective agent according to claim 1, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

19.

3. The optic nerve protective agent according to claim 1 or 2, wherein the anti-LRP1 antibody or the antibody fragment specifically binds to the C2 domain of LRP1.

4. The anti-LRP1 antibody or the antibody fragment is (a) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 1, in the region of amino acids from position 786 to 1165, (b) In the amino acid sequence of LRP1 having the amino acid sequence shown in Sequence ID No. 2, in the region of amino acids from position 787 to 1166, (c) In the amino acid region of LRP1 having the amino acid sequence shown in Sequence ID No. 3, from position 786 to 1165, or (d) In the amino acid sequence of LRP1 having the amino acid sequence shown in SEQ ID NO: 4, in the region of amino acids from position 787 to 1166, An optic nerve protective agent according to any one of claims 1 to 3, which recognizes an epitope.

5. The optic nerve protective agent according to any one of claims 1 to 4, wherein the anti-LRP1 antibody or the antibody fragment is a humanized antibody or a human antibody.

6. The anti-LRP1 antibody or the antibody fragment is 1 × 10 －７ M~1 x 10 －１１ An optic nerve protective agent according to any one of claims 1 to 5, which binds to LRP1 at a dissociation constant (Kd) of M.

7. A composition comprising an optic nerve protective agent according to any one of claims 1 to 6.

8. The composition according to claim 7, which is administered locally to a target.

9. The composition according to claim 8, wherein the local administration is intravitreous administration.

10. The composition according to claim 8 or 9, in the form of eye drops.

11. A composition for the treatment of glaucoma, according to any one of claims 8 to 10.

12. The composition according to any one of claims 8 to 11, wherein the subject has an intraocular pressure of 10 to 21 mmHg.

13. (i) Heavy chain complementarity determination region 1 (VH CDR1) containing the amino acid sequence shown in Sequence ID No. 9, The heavy chain complementarity determination region 2 (VH CDR2) includes the amino acid sequence shown in SEQ ID NO: 10, The heavy chain complementarity determination region 3 (VH CDR3) includes the amino acid sequence shown in SEQ ID NO: 11, Light chain complementarity determination region 1 (VL CDR1) containing the amino acid sequence shown in SEQ ID NO: 12, Light chain complementarity determination region 2 (VL CDR2) containing the amino acid sequence shown in Sequence ID No. 13, Light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence shown in SEQ ID NO: 14 and An anti-LRP1 antibody or an anti-LRP1 antibody fragment that specifically binds to LRP1, comprising [the specified substance].

14. (ii) A heavy chain variable region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 15, A light chain variable region containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence shown in Sequence ID No. 16, Includes, or (iii) The anti-LRP1 antibody or anti-LRP1 antibody fragment according to claim 13, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

19.

15. A composition comprising the anti-LRP1 antibody or anti-LRP1 antibody fragment described in claim 13 or 14.

Images