Patent Application
20240034787
The present invention relates to an optic nerve protecting agent containing an anti-LRP1 antibody.
Glaucoma is a progressive neurodegenerative disease in which retinal ganglion cells constituting an optic nerve degenerate for a certain reason and visual field defects occur, and is the first leading cause of blindness in Japan and the second leading cause of blindness in the world.
Currently, the only approach for progression prevention and treatment of glaucoma is lowering intraocular pressure. In Japan, patients with normal intraocular pressure account for 70% of those with glaucoma, and there are lots of cases where symptoms of glaucoma still progress even if the intraocular pressure is controlled by an intraocular pressure lowering agent. Therefore, development of a glaucoma treatment method based on a new mechanism of action has been desired.
In glaucoma, retinal ganglion cells constituting the optic nerve are damaged. For the purpose of elucidating the mechanism of optic nerve degeneration in optic nerve diseases, the inventors of the present invention have focused on a lipoprotein derived from glial cells, being a factor that protect these retinal ganglion cells, and studied suppression of retinal nerve cell death in optic nerve degeneration such as glaucoma. As a result, it was confirmed in an in vitro system that an apolipoprotein E-containing lipoprotein (E-LP) derived from glial cells has a neuroprotective effect against apoptosis (Non Patent Literature 1, Non Patent Literature 2, and Non Patent Literature 3). Here, the apolipoprotein E-containing lipoprotein (E-LP) is an apolipoprotein-binding lipoprotein in which apolipoprotein E, being a kind of apolipoprotein, and a lipid are chemically associated with each other.
Furthermore, the inventors of the present invention have found that when E-LP is injected into the vitreous body of a mouse in which a large amount of lipoproteins are present, E-LP has a neuroprotective effect against apoptosis even in an in vivo system, and further, a complex of E-LP and a neuroprotective molecule such as low density lipoprotein receptor-related protein-1 (LRP1) that is a receptor of E-LP similarly has a neuroprotective effect against apoptosis in the in vivo system (Patent Literature 1).
In other words, the inventors of the present invention have so far clarified that E-LP provides protection of rat primary cultured retinal ganglion cells from apoptosis via LRP1 that is a receptor of E-LP, and have found that E-LP can be used as an inhibitor of a neuroprotective inhibitory effect (Patent Literature 1).
An object of the present invention is to provide an optic nerve protecting agent containing an anti-LRP1 antibody.
The inventors of the present invention have further conducted intensive studies, and elucidated that an antibody against LRP1, being a receptor of E-LP, suppresses nerve disorders induced in primary cultured retinal ganglion cells and a rat glaucoma model. This result suggests a possibility of glaucoma treatment with an antibody based on the protective effect on the retinal ganglion cells via LRP1.
Namely, the present invention relates to:
or
or
or
or
According to the present invention, an optic nerve protecting agent containing an anti-LRP1 antibody can be provided.
Hereinafter, the present invention will be described in more detail.
Specifically, a first aspect of the present invention relates to an optic nerve protecting agent containing an anti-LRP1 antibody or an anti-LRP1 antibody fragment.
As described above, the inventors of the present invention have so far found that E-LP protects rat primary cultured retinal ganglion cells from apoptosis via LRP1, being one of receptors of E-LP, and E-LP can be used as an inhibitor of a neuroprotective inhibitory effect. In the present invention, the anti-LRP1 antibody is used in place of E-LP to protect ganglion cells from apoptosis. Specifically, as shown in
The inventors of the present invention have further developed the above studies and succeeded in inducing the protection of optic nerve cells via LRP1 with the anti-LRP1 antibody or the anti-LRP1 antibody fragment. When E-LP is used, there is a concern that the antibody may bind to a target other than LRP1, but the antibody is specific to the target, and has excellent uniformity and high stability. From this point of view, the antibody according to the present invention can also be useful in pharmaceutical applications.
LRP1 also includes a protein containing an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of the protein, and being functionally equivalent to the protein. Here, the “functionally equivalent protein” is a protein having an activity equivalent to the activity of the protein, and the “functionally equivalent protein” includes a protein having, for example, a sequence identity of 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 to the amino acid sequence of the protein.
Therefore, the term “LRP1” includes a protein having an amino acid sequence having a sequence identity of 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 to the amino acid sequence represented by a sequence number specified below, and having equivalent activity.
LRP1 includes an amino acid sequence represented by any of SEQ ID NO: 1 to SEQ ID NO: 4. Here, the amino acid sequence of LRP1 can be obtained from a database known in the art. Examples of the amino acid sequence of LRP1 are shown below:
The LRP1 in the present invention includes a protein having an amino acid sequence having, for example, a sequence identity of 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% to the amino acid sequence specified by SEQ ID NO: 1, 2, 3, or 4.
Further, examples of a base sequence of a gene encoding LRP1 are shown below:
As used herein, the term “antibody” is intended to include not only an antibody itself, but also a fragment of the antibody, particularly an antigen-binding fragment. Therefore, as used herein, in a case where the term “antibody” is described, the term “antibody” is intended to include an antibody and an antibody fragment thereof. Specifically, the term “anti-LRP1 antibody” is intended to include both an anti-LRP1 antibody and an anti-LRP1 antibody fragment.
The term “antibody fragment” includes, for example, a single-chain antibody (scFv), a scFv dimer, a disulfide-stabilized V-region fragment (dsFv), Fv, Fab, Fab′, F(ab′)2, a domain antibody, and the like.
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, an IgA antibody, an IgE antibody, or an IgM antibody.
In a case where the anti-LRP1 antibody is a single-chain antibody (scFv), it can be produced by a method known in the art, and for example, it may be produced by chemical synthesis in addition to being produced by a microorganism using a phagemid, a plasmid, and the like. For example, the anti-LRP1 antibody, being a single-chain antibody, is formed by linking a heavy chain variable domain and a light chain variable domain by a flexible linker.
In a case where the anti-LRP1 antibody is an IgG antibody, the antibody may be a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody, preferably a humanized antibody or a human antibody. In a case where the antibody is an IgG antibody, it can be produced by a method known in the art. One example of the method for producing an IgG antibody will be described later, but the method is not limited thereto.
The optic nerve protecting agent containing an anti-LRP1 antibody or an anti-LRP1 antibody fragment according to the present invention protects the optic nerve in vitro or in vivo. Here, the term “optic nerve protecting agent” includes not only a case of containing other components in addition to the anti-LRP1 antibody and/or the antigen fragment thereof, but also a case of containing only the anti-LRP1 antibody and/or the antigen fragment thereof.
“Protection of optic nerve” refers to preventing degeneration of optic nerve cells, and specifically refers to inhibiting degeneration of retinal ganglion cells and reducing disorders in retinal ganglion cells, and protecting axon of retinal ganglion cells constituting the optic nerve. Here, the term “degeneration” with respect to nerves includes apoptosis, necrosis and necroptosis. The “protection of the optic nerve” can be confirmed by means known in the art, and can be confirmed, for example, by a method of hematoxylin-eosin staining (hereinafter, it is also referred to as “HE staining”) of retinal sections, immunoblotting of a marker protein of retinal ganglion cells, or electroretinogram (hereinafter, it is also referred to as “ERG”). Further, since degeneration of nerves can be detected by an immunohistological method, the “protection of optic nerve” may be confirmed using this method.
In one embodiment, the optic nerve protecting agent protects the optic nerve in a subject. As used herein, the term “subject” broadly includes animals. The animal is, for example, a vertebrate, preferably a mammal. As used herein, the term “mammal” is used to refer to any animal classified as a mammal, including, but not limited to, a human, mouse, rat, monkey, cow, horse, sheep, dog, and cat. As used herein, a preferred mammal is a human.
The anti-LRP1 antibody or the antibody fragment thereof is an antibody that specifically binds to the C2 domain of LRP1.
In one embodiment, the anti-LRP1 antibody or the antibody fragment thereof recognizes an epitope:
In the present invention, an “epitope” means a partial peptide of an LRP1 polypeptide having antigenicity and/or immunogen in the body of an animal, preferably a mammal, more preferably a human, mouse, rat or monkey. An epitope that is the partial peptide of LRP1 having antigenicity can be determined by a method known to those skilled in the art, such as immunoassay. The epitope can be determined, for example, by the following method. To start with, various partial structures of the LRP1 polypeptide are produced using known oligopeptide synthesis techniques. Specifically, it can be determined by examining a series of polypeptides sequentially shortened by an arbitrary length from the C-terminus or N-terminus of the LRP1 polypeptide, determining a rough recognition site, then synthesizing shorter peptides, and examining the reactivity with them. As another specific method, it can be determined by expressing each domain structure as a recombinant protein using each domain structure of LRP1 as a basic unit, and examining a difference in binding to these recombinant proteins.
In a case where the anti-LRP1 antibody or the antibody fragment thereof is an anti-LRP1 human IgG antibody, the anti-LRP1 antibody or the antibody fragment thereof 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, and still more preferably 1×10−8 M to 1×10−9 M.
The anti-LRP1 antibody or the antibody fragment thereof according to the present invention includes, for example, the following complementarity determining regions (CDRs):
In a preferred embodiment, the anti-LRP1 antibody or the antibody fragment thereof includes:
In another embodiment, the anti-LRP1 antibody or the antibody fragment thereof includes:
In another preferred embodiment, the anti-LRP1 antibody or the antibody fragment thereof includes:
In still another embodiment, the anti-LRP1 antibody or the antibody fragment thereof includes an amino acid sequence having a 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, or 99% or more to the amino acid sequence represented by SEQ ID NO: 19.
In still another preferred embodiment, the anti-LRP1 antibody or the antibody fragment thereof includes the amino acid sequence represented by SEQ ID NO: 19.
Another embodiment of the present invention relates to an expression vector for producing an anti-LRP1 antibody or an anti-LRP1 antibody fragment. The expression vector includes one or more nucleic acids encoding the anti-LRP1 antibody of the present invention or a part thereof. Here, the nucleic acid is operably linked to a control sequence recognized by a host cell in a case where the host cell is transfected with a vector.
The expression vector according to the present invention includes, for example, at least one nucleic acid selected from the followings:
The present invention also provides a host cell including one or more expression vectors, and a method for producing an anti-LRP1 antibody or an antigen fragment thereof using such a host cell. Here, the production method includes at least one of culturing a host cell containing an expression vector that produces an anti-LRP1 antibody or an antigen fragment thereof in a medium, and isolating the anti-LRP1 antibody or the antigen fragment thereof from the host cell or the medium. The present invention also provides a host cell genetically modified to express an anti-LRP1 antibody or an antigen fragment thereof in the absence of a vector as described above.
Yet another aspect of the invention is a composition containing the anti-LRP1 antibody or the anti-LRP1 antibody fragment or an optic nerve protecting agent previously described. Here, the term “composition” is intended to include not only a case of containing an anti-LRP1 antibody, an antigen fragment thereof, and other components other than an optic nerve protecting agent, but also a case of containing only an anti-LRP1 antibody, an antigen fragment thereof, and/or an optic nerve protecting agent (in other words, the case where other components are not contained). In one embodiment, the composition according to the present invention is a composition containing an anti-LRP1 antibody or an anti-LRP1 antibody fragment and a solvent. The composition according to the present invention is preferably a pharmaceutical composition containing an anti-LRP1 antibody or an anti-LRP1 antibody fragment at a therapeutically effective amount.
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 is, for example, instillation administration, subconjunctival injection, intravitreal administration and intraocular implant.
The composition according to the present invention can be administered orally or parenterally, preferably parenterally. Examples of a dosage form of the composition include a tablet, a capsule, a fine granule, a pill, a troche, a transfusion, an injection, eye drops, a suppository, an ointment, a patch and the like.
In one embodiment, the composition according to the present invention is a pharmaceutical composition for prevention or treatment of glaucoma. As described above, a certain percentage of glaucoma patients are patients with normal intraocular pressure, and there are many cases which still progresses even when the intraocular pressure is controlled by an intraocular pressure lowering agent. Therefore, the pharmaceutical composition of the present invention is preferably used for glaucoma patients having normal intraocular pressure. Specifically, glaucoma patients have an intraocular pressure, for example, in the range of 10 to 23 mmHg, preferably 10 to 21 mmHg, more preferably in the range of 10 to 20 mmHg.
Examples of glaucoma include primary open-angle glaucoma, normal-tension glaucoma, aqueous excessive glaucoma, ocular hypertension, acute angle-closure glaucoma, chronic angle-closure glaucoma, plateau iris syndrome, combined mechanism glaucoma, steroid-induced glaucoma, capsular glaucoma of crystalline lens, pigmentary glaucoma, amyloid glaucoma, neovascular glaucoma, malignant glaucoma, and the like.
As used herein, with respect to glaucoma, the term “treatment” is intended to include inhibiting or reducing glaucoma in addition to treating glaucoma. The term “treatment” specifically includes inhibiting a progression of optic nerve degeneration, and preventing apoptosis, necrosis and/or necroptosis of the optic nerve.
The subject includes a subject suffering from glaucoma who has received treatment by administration of an intraocular pressure lowering agent in the past, in particular, a subject suffering from glaucoma who has received treatment by administration of an intraocular pressure lowering agent in the past, and whose glaucoma symptoms are still progressing. Here, the intraocular pressure lowering agent is an intraocular pressure lowering agent known in the art, and examples thereof include an adrenaline α2 receptor agonist (Aiphagan (registered trademark) ophthalmic solution), a prostaglandin F2a derivative (Tapros (registered trademark) ophthalmic solution), a Rho kinase inhibitor (Granatec (registered trademark) ophthalmic solution), and the like, but are not limited thereto. The subject may further include a subject that has undergone a surgical procedure for the treatment of glaucoma.
In a case where the composition according to the present invention is applied to a subject for the prophylaxis or treatment of glaucoma, the composition is administered parenterally, preferably topically, more preferably intravitreally. Thus, in a case where the composition according to the present invention is a pharmaceutical composition, it is for example for parenteral administration, preferably for topical administration, more preferably for vitreous administration.
The dosage form of the composition according to the present invention is not particularly limited, but when applied to a subject for treatment of glaucoma, it is preferably eye drops or an ophthalmic injection.
The composition according to the present invention may typically contain a pharmaceutically acceptable additive, for example, a pharmaceutically acceptable excipient, binder, auxiliary agent, lubricant, solvent, diluent, stabilizer, emulsifier, preservative, carrier, solubilizing agent, tonicity agent and the like known in the art.
A dosage of the composition according to the present invention may be appropriately adjusted depending on the age, weight, disease state, and the like of the subject.
The optic nerve protecting agent and the pharmaceutical composition according to the present invention may be used as a combination agent in combination with an additional agent or pharmaceutical composition. With regard to the combination agent, active ingredients in the respective pharmaceutical compositions (that is, the first active ingredient and the second active ingredient) may be administered together, sequentially or individually, in one combined unit dosage form or in two separate unit dosage forms. A therapeutically effective amount of each active ingredient of the combination according to the present invention may be administered simultaneously, individually or in any order, sequentially or successively. In the combination agent, the first active ingredient and the second active ingredient may be individually present in each of plural units. The combination agent also includes, for example, a kit containing respective active ingredients and instructions for use.
According to the present invention, an optic nerve protecting agent and a composition containing an anti-LRP1 antibody or an anti-LRP1 antibody fragment that specifically binds to LRP1 can be provided. Since an anti-LRP1 antibody or an anti-LRP1 antibody fragment having high specificity is used, it is possible to specifically act on a factor involved in degeneration of optic nerve cells, and therefore degeneration of the optic nerve can be suppressed while reducing undesired side effects.
Furthermore, according to the present invention, an anti-LRP1 antibody or an anti-LRP1 antibody fragment can protect the optic nerve with a mechanism of action other than intraocular pressure reduction, and thus is expected to be a new therapeutic approach to glaucoma. Furthermore, due to the mechanism of avoiding degeneration of the optic nerve cells, it is expected to be able to provide another option of treatment in patients with glaucoma who have received treatment with the intraocular pressure lowering agent in the past and patients with optic nerve degeneration with normal intraocular pressure.
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited by these examples at all.
<Primary Culture of Retinal Ganglion Cells>
The primary culture of retinal ganglion cells was performed using a two-day old Sprague Dawley (SD) rat according to a method slightly modified from the method of Barres et al. (Barres et al., 1988) (Hayashi et al., J. Biol. Chem. 2009). Isolated retinal ganglion cells (RGCs) were suspended in a basic medium of RGC medium (containing 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 Hill, NJ), 50 ng/ml ciliary neurotrophic factor (CNTF; PeproTech), and 50 ng/ml basic fibroblast growth factor (bFGF; PeproTech). A 96 well plate was coated with poly-d-lysine (Sigma) and laminin (Sigma), and mounted with RGCs to 5,000 cells per well for a 96 well plate and 5,000 cells per culture insert for a microdish. The cells were subsequently cultured for at least 10 days before experiments.
<Preparation of Reconstituted Apolipoprotein E-Containing Lipoprotein>
The reconstituted apolipoprotein E-containing lipoprotein was prepared as described in the prior literature (Hayashi et al., J. Neurosci. 2007). This reconstituted lipoprotein E-containing lipoprotein was composed of 1-palmitoyl-2-oleyl-glycerophosphocholine (POPC; P3017, Sigma), cholesterol (C3045, Sigma) and recombinant human apolipoprotein E, and a molar ratio of the components was 100:10:1 or 100:0:1. A solution containing a preparation medium, plasma or reconstituted apolipoprotein E-containing lipoprotein was subjected to a non-continuous sucrose gradient. A composition of the solution used was 3 ml for a density of 1.30 g/ml, 3 ml for a density of 1.2 g/ml, 3 ml for a density of 1.1 g/ml and 6 ml for a density of 1.006 g/ml. The sucrose gradient was subjected to centrifugation using an SRP28SA1 rotor (Hitachi, Tokyo, Japan) at 100,000 g for 72 hours at 4° C. Ten fractions (1.5 ml) were collected from the top of the gradient, and subjected to immunoblotting for apolipoprotein E as follows. Fractions containing apolipoprotein E (typically fractions 5 to 7) were combined, and concentrated using an Amicon Ultra filter (50 kDa molecular weight cut-off; Millipore, Bedford, MA). The mass of lipoprotein was adjusted by a cholesterol concentration (2 μg/ml) for HDL, and by a protein concentration (100 ng/ml) for reconstituted lipoprotein. The concentrations of the cholesterol and protein were measured by a LabAssay cholesterol kit (Wako) and a BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL), respectively.
<Screening of Anti-LRP1 Antibody>
Using commercially available proteins derived from LRP1 partial domain and Human Fc (three species: C2, C3, C4) as an antigen, scFv for each cluster of LRP1 (that is, C2, C3 and C4) was obtained from a human scFv phage library.
scFvs that specifically bind to clusters 2, 3, and 4 of LRP1 (that is, C2, C3 and C4) were subjected to screening (panning) from the human scFv phagelibrary. As a result, a large number of scFvs specific to each of the LRP1 clusters 2, 3, and 4 were obtained. The results of plotting the reactivity of each clone (in the horizontal axis) are shown in
<Evaluation of Binding Site in LRP1 of Anti-LRP1 Antibody>
Four scFv clones for the LRP1 clusters were purified. These clones were selected for the stability of E. coli retention of phagemid and stability in purification as an index. The sequence of the VH and VL CDR3 of the four scFv clones, and a feed rate are shown in the following table.
SDS-PAGE of each purified scFv is shown in
Materials and conditions used in ELISA are shown in the following table.
The results shown in
<Protective Effect of scFv on Glutamate-Induced Neurotoxicity in Primary Cultured Retinal Ganglion Cells>
Apoptosis was induced with 300 μM glutamic acid and 10 μM glycine for two hours. scFv was used at a concentration of 1 μg/ml, and E-LP was used at a concentration of 100 ng/ml as a positive control. Anti-OVA (ovalbumin) scFv was used as a negative control. Nuclear aggregation 24 hours after glutamate-induced neurotoxicity was analyzed by Hoechst staining. The results regarding the protective effect on retinal ganglion cells of scFv in Group #1 (n=4 to 5), Group #2 (n=5) and Group #3 (n=5) are shown in
<Immunoblotting>
The proteins were dissolved in 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS) and 5% p-mercaptoethanol (sample buffer) and boiled for five minutes. The proteins were separated by electrophoresis on polyacrylamide gels containing 0.1% SDS, and then transferred to a polyvinylidene difluoride membrane. The membranes were incubated for one hour at room temperature with 5% skim milk in TBS-T (10 mM Tris-HCl (pH 7.4), 150 mM NaCl and Tween 20), and then probed overnight at 4° C. with a primary antibody in TBS-T containing 5% bovine serum albumin. The membranes were subsequently probed for one hour at room temperature with peroxidase-conjugated goat anti-mouse IgG (Thermo), goat anti-human IgG (Thermo), or mouse anti-goat IgG (Thermo). Immunoreactive proteins were visualized with enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) or Super Signal West Dura (Thermo). The primary antibodies used were as follows: human C2-D11 IgG (0.5 μg/ml), mouse antibody β-actin (a5441, diluention 1:10,000, Sigma), goat anti-human Brn-3a (sc-31984, diluention 1:1000, Santa Cruz). Brn-3a is a marker protein of retinal ganglion cells, and p-actin was used as a loading control. scFv (C2-D11) was administered intravitreally simultaneously with N-methyl-D-aspartic acid (NMDA). The results are shown in
<Induction and Detection of Apoptosis in Retinal Ganglion Cells (RGCs)>
RGCs were washed twice with Hanks' Balanced Salt Solution (HBSS; 14170, Invitrogen) containing 2.4 mM CaCl2, 20 mM HEPES and no magnesium, and then incubated at 37° C. for two hours with 300 μM glutamic acid and 10 μM glycine with or without E-LP, C2-D11 scFv or C2-D11 IgG in HBSS containing 2.4 mM CaCl2, 20 mM HEPES and no magnesium. After glutamic acid treatment, RGCs were cultured at 37° C. for 22 hours with a culture medium for RGC without forskolin, BDNF, CNTF or bFGF. For the detection of apoptosis, RGCs were stained with 1 μg/ml of Hoechst 33342 (346-07951, Kumamoto, DOJINDO). Fluorescence images were observed using an Olympus IX 71 microscope. Fragmented or shrunken nuclei stained with Hoechst dye were counted as apoptotic neurons, and round and smooth nuclei were counted as healthy neurons.
<NMDA-Induced Retinal Degeneration by C2-D11 Antibody in Rats>
Intravitreal injection of NMDA (Sigma) was performed with minor modifications, similar to the method described in the prior literature (Tsutsumi T. et al., Invest Ophthalmol Vis Sci. 2016, 57(14): 6461-6473). Specifically, 7-week-old rats were anesthetized with 5% isoflurane, and then maintained with 2.5% isoflurane. The pupil was dilated by instillation of phenylephrine hydrochloride and tropicamide. Subsequently, PBS as a vehicle control or 20 nmol/eye NMDA with or without 5 ng/eye E-LP, 0.15 μg/eye C2-D11 scFv or 0.75 μg/eye C2-D11 IgG in a total volume of 4 μl was injected into the vitreous cavity. Injections were 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), and the needle was inserted approximately 1.0 mm behind the corneal limbus. The eyeballs were enucleated three days after NMDA injection and retinae were removed from the sclerae, added into lysis buffer [1% Triton X-100; MP Biomedicals (Santa Ana, CA, CA, 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, 50 mM Tris-buffered saline), and then sonicated with Ultrasonic Liquid Processor Q125 (QSONICA) at 4° C. for 20 seconds (2 seconds sonication×10 times). Protein concentration of retinal sample was measured by a BCA protein assay kit (Thermo Fisher Scientific Inc.) and subjected to immunoblotting.
<Frozen Specimen Preparation and HE Staining of Rat Eyeballs>
The frozen specimen preparation and HE staining of eyeballs were performed according to the following procedure.
As a result of counting the number of cells in the ganglion cell layer (GCL) of HE stained images in
<Measurement of Thickness of Inner Plexiform Layer by Optical Coherence Tomography>
The thickness of the inner plexiform layer of the retina at the sixth day after treatment with each sample was measured by an optical coherence tomography (OCT). The thickness of the retinal inside from the inner plexiform layer is shown in
<Production of Anti-LRP1 Human IgG1 Antibody from scFv Antibody C2_D11>
As schematically shown in
<Binding Specificity of Anti-LRP1 Human IgG1 Antibody>
The binding specificity of an anti-LRP1 human IgG1 antibody to LRP1 was evaluated by ELISA and a biosensor. The results of ELISA for the anti-LRP1 human IgG1 antibody are shown in
<Intracellular Calcium Analysis in Retinal Ganglion Cells (RGCs)>
RGCs were maintained through an experiment with a heating element (Tokai Hit, Shizuoka, Japan). Cells were cultured in the microdish for at least 14 days and incubated for 30 minutes at 37° C. with 3 μM Fluo-8 acetoxy metal ester (AAT Bioquest, Sunnyvale, CA). After washing the cells twice with 500 μl HBSS containing 2.4 mM CaCl2, 20 mM HEPES, and no magnesium, 300 μM glutamic acid and 10 μM glycine with or without 100 ng/ml E-LP or 100 μg/ml C2-D11 IgG were administered. Fluorescence images were acquired every 500 msec using an ORCA-R2 digital CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) and analyzed by MetaFluor fluorescence ratio imaging software (Molecular Devices, Sunnyvale, CA). In this test, 100 μg/ml C2-D11 IgG was evaluated using 100 ng/ml apolipoprotein E-containing lipoprotein (E-LP) as a positive control and the anti-OVA antibody as a negative control. The results are shown in
<Frozen Specimen Preparation and Immunofluorescence Staining of Rat Eyeballs>
Frozen specimen preparation and immunofluorescence staining of rat eyeballs were performed in the following procedures.
In the above experiment, it was observed by fluorescent immunostaining whether C2-D11 IgG binds to LRP1 of retinal ganglion cells after being administered into the vitreous body of rats. From the results shown in
<Neuroprotective Effect of Anti-LRP1 Human IgG1 Antibody on Glutamate-Induced Retinal Ganglion Cell Death>
Glutamic acid was administered in combination with various concentrations of the anti-LRP1 human IgG1 antibody, and the rate of cell death was examined. Specifically, 300 μM glutamic acid and 10 μM glycine were used to induce apoptosis for two hours. The anti-LRP1 human IgG1 antibody was used at a concentration of 0.01 μg/ml to 10 μg/ml. Nuclear aggregation 24 hours after glutamate-induced neurotoxicity was analyzed by Hoechst staining. The results are shown in
<Production of Recombinant Protein of LRP1>
Various deficient variants of Cluster II of human low-density lipoprotein receptor-associated protein 1 (LRP1) were prepared for epitope mapping. A GST-tag was fused to each of the prepared deficient variants of Cluster II of human LRP1. A cDNA fragment of Cluster II of human low-density lipoprotein receptor-associated protein 1 (LRP1) was synthesized to encode forward linker SGGSTS, Cluster II (position, 786-1165) and backward linker ASTGS (Thermo Fisher Scientific, Waltham, MA). A cDNA fragment encoding each deficient variant of Cluster II was prepared by PCR using primer sets shown in the following table and the cDNA fragment of Cluster II as a template. An expression vector was obtained by digesting the cDNA fragment with BamHI and EcoRI and inserting it into the corresponding restriction site on pGEX-6P-1 expression vector (Cytiva, Marlborough, MA). BL21 cells having the expression vector were then cultured in MagicMedia (Thermo Fisher Scientific), and each recombinant protein (each deficient variant) was produced according to the manufacturer's protocol (Cytiva, Marlborough, MA). After lysis of E. coli by sonication, a GST fusion protein was pulled down with glutathione-sepharose (Cytiva) and used for immunoblotting.
The prepared deficient variants were subjected to electrophoresis, and gel staining was performed using Coomassie Brilliant Blue R250. The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-203477 | Dec 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/043471 | 11/26/2021 | WO |
