Pharmaceutical composition containing human-derived anti-collapsen reaction-mediating protein 2 (CRMP2) antibody

Abstract

This provides novel drugs for the treatment of neurodegenerative diseases such as tauopathies, particularly Alzheimer's disease (AD). [Solution] Collapsin reaction-mediated protein 2 (CRMP2) is established as a therapeutic target in neurodegenerative diseases. For CRMP2-targeted therapy, a novel human-derived anti-CRMP2 antibody and a pharmaceutical composition comprising its fragments, derivatives, and variants are provided.

Description

[Technical Field]

[0001] The present invention relates, in general terms, to pharmaceutical compositions comprising novel human-derived anti-collapsen reaction-mediated protein 2 (CRMP2) antibodies and equivalent antigen-binding molecules, as well as to their use, particularly for treating neurodegenerative diseases. [Background technology]

[0002] The pathogenesis of neurodegenerative diseases is extremely complex and diverse. Neurodegenerative diseases are chronic diseases characterized by the progressive loss of neurons in the brain and spinal cord, with the most prominent and common group being dementia-related tauopathies such as Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD). The underlying biology of these diseases includes, for example, the aggregation of soluble amyloid species into insoluble amyloid plaques, as observed in Alzheimer's disease (AD), as well as hyperphosphorylation of tau accompanied by the formation of intracellular neurofibrillary tangles, as observed in AD, PiD, PSP, and FTD, and neuronal death accompanied by various related processes including neuroinflammation, synaptic and circuit dysfunction, mitochondrial dysfunction, and bioenergetic dysfunction. The earliest neuropathological changes in AD include neuronal degeneration, likely resulting from the deposition of β-amyloid plaques and neurofibrillary tangles in the brain. Neuronal degeneration, including neuronal dysfunction and loss of functional synapses, can lead to cognitive impairment and memory impairment, primarily in older adults.

[0003] As recently summarized by Yu et al. in "Novel Therapeutic Approaches for Alzheimer's Disease: An Updated Review" Int.J.Mol.Sci.2021 Jul 30;22(15):8208.doi:10.3390 / ijms22158208, despite the severe and chronic effects of AD, current treatments fail to achieve satisfactory therapeutic effects or halt disease progression. Currently, there are only eight drugs approved by the FDA for the treatment of AD: donepezil, rivastigmine, galantamine, tacrine, memantine, aducanuma brecanemab, and donanemab. The first four drugs are acetylcholinesterase inhibitors (AChEIs), while memantine is an N-methyl-D-aspartate receptor (NMDAR) antagonist, and aducanumab, lecanemab, and donanemab are human anti-Aβ antibodies that target aggregated Aβ. US and European guidelines list AChEIs as the first-line drug treatment for mild to moderate AD. However, in mild to moderate AD, AChEIs show only moderate efficacy against cognitive deficits, and their efficacy against functional capacity is not significant. Memantine has very limited efficacy against cognitive symptoms and does not lead to functional improvement. Novel therapeutic approaches for AD are being studied in various forms, including pharmacological and non-pharmacological interventions such as anti-Aβ therapy, anti-tau therapy, anti-neuroinflammatory therapy, neuroprotective agents, and brain stimulation.

[0004] Therefore, new drugs are still needed to treat neurodegenerative diseases such as tauopathies, especially Alzheimer's disease (AD). [Overview of the project]

[0005] According to the present invention, solutions to the problems defined above are characterized in the claims and provided by embodiments disclosed herein and illustrated in the following examples and drawings. Accordingly, the present invention relates to a pharmaceutical composition comprising an anti-CRMP2 antibody, a CRMP2 binding fragment and / or an equivalent CRMP2 binding molecule, which preferably exhibits substantially the same binding and biological properties as illustrated in the accompanying examples and drawings using a lead antibody called NI-504.3E7.

[0006] As described in the "Background Technology" section, the success of current drug therapies to treat AD, the most common neurodegenerative disease, has been mixed or insignificant. Therefore, we explored alternative target proteins that may potentially play a role in the onset and / or progression of this disease, and in which targeting one or more forms of the target protein, such as specific conformations, denatured and natively folded forms, monomers and multimers, phosphorylated and unphosphorylated forms, etc., would lead to disease-modifying effects.

[0007] After diligently researching several hypotheses and investigating various types of disease models, the inventors finally arrived at a novel therapeutic strategy and selected collagen response-mediated protein 2 (CRMP2) as a novel therapeutic target for neurodegenerative diseases such as Alzheimer's disease (AD).

[0008] Collapsin reaction-mediating proteins (CRMPs) represent a family of cytoplasmic proteins (CRMP1-5) that are highly expressed in the developing brain. CRMPs act as signaling molecules that lead to neuronal differentiation by regulating microtubule polymerization and stabilization, actin bundling, and endocytosis. (See Brustovetsky et al., Cells 10(2021), 2781). CRMP2 is a cytoplasmic protein involved in axon guidance and neurite extension via the semaphorin 3A pathway. In contrast to other members of the CRMP family, CRMP2 maintains high levels of expression even in adulthood. CRMP2 is a phosphoprotein that binds to tubulin heterodimers to promote microtubule assembly and stabilization, but phosphorylation of CRMP2 negatively regulates its ability to bind to and stabilize tubulin. Therefore, phosphorylation affects the affinity of CRMP2 for assembled microtubules and tubulin dimers, resulting in axon extension or retraction. Brustovetsky et al., Cells 10 (2021), 2781, provides a more detailed explanation of how increased CRMP2 phosphorylation correlates with decreased CRMP2 binding to dynamin-related protein 1 (Drp1), Miro 2, and kinesin 1 light chain (KLC1). The switching between inactive (phosphorylated) and active (unphosphorylated) CRMP2 is a continuous physiological adaptation mechanism to prevent abnormal neuronal sprouting. Overall, a balance exists between active and inactive CRMP2. Furthermore, CRMP2 is extremely important for neurogenesis and has been reported to be associated with numerous neurodegenerative diseases, leading to its proposal as a specific marker for Alzheimer's disease (AD). See, for example, Soutar et al., Curr Alzheimer Res 6 (2009), 269-278.

[0009] However, until now, CRMP2 has not been considered a possible target for inhibiting pathological processes in the brain. This is probably because pathological forms of CRMP2 were not as well known as known target proteins in neurodegeneration, such as Aβ and hyperphosphorylated tau and their aggregates.

[0010] Therefore, the results of the in vivo experiments conducted by the inventors are all the more surprising in that targeting CRMP2 with an anti-CRMP2 antibody leads to significant therapeutic effects in various animal models of neurodegenerative diseases (one of which is illustrated in the examples). In particular, the inventors were able to demonstrate that an anti-CRMP2 antibody called NI-504.3E7 reduces the load and mean size of amyloid-β (Aβ) plaques by approximately 60% in TTBK1 / APP transgenic mice. See Example 11 and Figure 10. In addition to reducing amyloid plaque load in transgenic mouse models of AD, targeting CRMP2 has also been shown to improve long-term potentiation and cognitive function in aged mice; see Examples 13 and 14.

[0011] Characterization of antibody NI-504.3E7 in biochemical and in vitro cell assays revealed that this antibody selectively binds to human CRMP2 compared to the other four CRMP members (Examples 2 and 3 and Figure 2), can bind with high affinity to full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2) (Example 4), reduces pCRMP2 levels in a concentration-dependent manner (Example 5 and Figure 4), and reverses the increase in spine density induced by pCRMP2 aggregates and monomeric CRMP, respectively (see Example 9 and Figure 8).

[0012] While we do not intend to dwell on theory, considering these properties of this antibody, we hypothesize that by interfering with the levels of pCRMP2 and CRMP2, and the ratio between them, respectively, the adverse effects of CRMP / CRMP2 in the brain caused by changes in the abundance and phosphorylation state of CRMP2 can be reversed.

[0013] For example, the phosphorylation status of CRMP2 has been reported to alter in various neuropathologies, including Huntington's disease (HD) and Alzheimer's disease (AD). In particular, high levels of phosphorylated CRMP2 have been reported in association with neurofibrillary tangles in the human AD brain, suggesting that the accumulation of phosphorylated CRMP2 may be an early event of AD. Not only are levels of phosphorylated CRMP2 increased, but CRMP2 has also been shown to be hyperphosphorylated, meaning that the relative levels of CRMP2 phosphorylation are indeed increased in brain regions associated with AD pathology. See Cole et al., J. Neurochem. 103 (2007), 1132-44.

[0014] While we do not intend to dwell on theory here, based on experimental evidence, we believe that a decrease in CRMP2 levels and an increase in (excessive) phosphorylated CRMP2 levels may not only be markers for neurodegenerative diseases as previously described, but may actually be actively involved in the onset and manifestation of these diseases.

[0015] In this regard, further experiments conducted in accordance with the present invention revealed that the exemplary antibody NI-504.3E7 can detect CRMP2 intracellularly using fully differentiated SH-SY5Y cells, as demonstrated in Example 12.

[0016] In addition, to rule out the possibility that the observed biological activity depends on the specific amino acid sequence of the variable region of antibody NI-504.3E7, several variants with modifications in the framework region and variants with modifications in the CDRs were constructed and tested. As shown in Table VII, several amino acid modifications affect the absolute EC 50 values, but all values remain in the nanomolar range for both CRMP and pCRMP2.

[0017] Thus, in a general aspect, the present invention relates to CRMP2 as a therapeutic target in the treatment of neurodegenerative diseases, and for use as a medicament, preferably for preventing, delaying the progression of, or treating neurodegenerative diseases or cognitive impairments, and / or improving memory and learning abilities, and any molecule capable of interfering with CRMP2 signaling of the same and similar types as demonstrated for antibody NI-504.3E7. Preferably, such a molecule is a CRMP2-binding molecule that exhibits binding characteristics and biological activities as demonstrated for antibody NI-504.3E7 in the described biochemical assays and in vitro cell assays. Such a molecule can be obtained by screening a compound library using the assays described in the examples, designed in silico, or derived from antibody NI-504.3E7.

[0018] However, due to the lack of immunogenicity and use in proof-of-concept, the use of human or human-derived antibodies is particularly preferred.

[0019] As exemplified in the examples, in a complex antibody discovery process, human monoclonal anti-CRMP2 antibodies were cloned and identified. See Example 1. Those antibodies were tested for their binding specificities and binding affinities. As can be derived from Table VI of Example 1, some of the cloned antibodies had an EC 50Although it showed specific binding to the CRMP2 peptide in terms of values, it is noteworthy that the EC was less than 20 nM in the ELISA assay. 50 Only three antibodies showed specific binding to the CRMP2 protein. These three antibodies, NI-504.3E7, NI-504.10, and NI-504.12, further demonstrated binding signals in human brain tissue in immunohistochemistry (IHC) assays. Specifically, only NI-504.3E7 and NI-504.12 showed strong binding signals in human hippocampal tissue at a concentration of 0.2 μg / mL. Of these antibodies, only NI-504.3E7 showed selective binding to CRMP2, specifically to both phosphorylated and unphosphorylated CRMP2, while the other two antibodies also recognized CRMP1 and CRMP4.

[0020] More specifically, the antibody NI-504.3E7 exhibits high affinity, meaning it has a 4 nM EC2 value relative to full-length CRMP2 when measured in an ELISA assay. 50 It was shown that binding occurred, but only weak binding occurred to CRMP1, meaning that when determined in an ELISA assay, the EC2 was 69 nM. 50 Binding was only observed at [location]. The binding of antibody NI-504.3E7 to CRMP3, CRMP4, and CRMP5 was very weak, and EC 50 The value could not be determined. Similar results were obtained by Western blot analysis, which showed that antibody NI-504.3E7 exhibited strong binding to full-length CRMP2 and weak binding to CRMP1, but no binding to CRMP3, CRMP4, and CRMP5. See Example 2 and Figure 2. Therefore, this antibody does not substantially cross-react with other CRMP proteins.

[0021] Furthermore, antibody NI-504.3E7 was shown to bind to full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2) in ELISA assays. Here, antibody NI-504.3E7 binds CRMP2 and pCRMP2 to low nanomolar concentrations in the EC2 range. 50It was shown that it could be specifically recognized. See Example 4 and Table VII.

[0022] In contrast, most antibodies available to date bind exclusively to phosphorylated CRMP2. For example, the anti-pCRMP2 antibody 3F4 recognizes triply phosphorylated and biphosphorylated CRMP2, but not unphosphorylated or monophosphorylated CRMP2. See Uchida et al., Genes Cells 10 (2005), 165-79. Also, commercially available polyclonal anti-phospho-CRMP-2 (pThr) antibodies produced against synthetic peptides derived from the Thr509 phosphorylation site of human CRMP2. 509 The antibody (Sigma-Aldrich® SAB4504698, available from Merck KGaA (Darmstadt, Germany)) binds only to pCRMP2. The same is true for the antibody pCRMP-2(Thr514)#9397 from Cell Signaling Technology, Inc (Leiden, Netherlands). Furthermore, International Publication No. 2011 / 007209A1 describes an anti-CRMP2 antibody specific to pCRMP2 with phosphorylated tyrosine 479.

[0023] In addition to the original antibody NI-504.3E7, 40 variants of antibody NI-504.3E7 (NI-504.3E7_V1 to NI-504.3E7_V40) were generated by exchanging amino acids in the variable heavy (VH) chain region, variable light (VL) chain region, and / or complementarity-determining region (CDR). The nucleotide and amino acid sequences of the variable regions (VH, VL) of antibody NI-504.3E7, as well as their variant VH and VL chains and CDRs, are shown in Tables II and III. In particular, three variant VL chains containing two, four, or five amino acid exchanges, namely VL_3E7_var1, VL_3E7_var2, and VL_3E7_var3, were generated. See Table V and the alignment in Figure 1. Furthermore, there are 22 variant VH chains containing 2, 3, 4, 5, 6, 7, 8, or 10 amino acid exchanges, namely VH_3E7_var1, VH_3E7_var2, VH_3E7_var3, VH_3E7_var4, VH_3E7_var5, VH_3E7_var6, VH_3E7_var6a, VH_3E7_var6b, VH_3E7_var6c, and VH_3E7_var6 The following variants were produced: d, VH_3E7_var6ab, VH_3E7_var6ac, VH_3E7_var6ad, VH_3E7_var6bc, VH_3E7_var6bd, VH_3E7_var6cd, VH_3E7_var6bcd, VH_3E7_var6acd, VH_3E7_var6abd, VH_3E7_var6abc, VH_3E7_var7, and VH_3E7_var8. See the alignment in Table V and Figure 1. The assignment of which antibody variants (NI-504.3E7_V1 to NI-504.3E7_V40) contain which variant VH and VL chains is shown in Table IV.

[0024] As shown in Example 4 and Table VII, all mutants retained their ability to bind to CRMP2 and pCRMP2. Furthermore, the results of experiments performed using the original antibody NI-504.3E7 described above were confirmed in at least one exemplary mutant.

[0025] Epitope mapping revealed that, as shown in Example 3, antibody NI-504.3E7 binds to a linear epitope near the C-terminus of CRMP2. In particular, antibody NI-504.3E7 binds to CRMP2 peptides containing or consisting of the amino acid sequences TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63), and the minimal epitope was determined to contain the amino acid sequence 516-ASSAK-520 (SEQ ID NO: 64).

[0026] As described above, the antibody NI-504.3E7 reduces pCRMP2 levels in a concentration-dependent manner. See Example 5 and Figure 4. Therefore, although we do not intend to dwell on theory, the therapeutic effect of anti-CRMP2 antibodies may be due to their ability to bind to CRMP2 and conserve it, and / or instead neutralize CRMP2 and / or inhibit CRMP2 phosphorylation and / or capture phosphorylated CRMP2, resulting in a reduction in the amount of excess phosphorylated CRMP2 that is generally present in excess in the brains of AD patients and / or a reduction in the amount of phosphorylated CRMP2.

[0027] One explanation for this unique activity may be the epitope of antibody NI-504.3E7 (including 516-ASSAK-520, SEQ ID NO: 64), located at the C-terminus of CRMP2 and very close to the phosphorylation sites T514 and S522. This is supported by the fact that the corresponding anti-CRMP2 control antibody (NI-504.B) (which binds to a different epitope not close to the above phosphorylation sites) does not possess this property, i.e., does not reduce pCRMP2 levels in a concentration-dependent manner. See Example 5 and Figure 4.

[0028] As further illustrated in the examples, the cDNA sequence encoding the antibody NI-504.3E7 was originally cloned from human memory B cells. As mentioned above, in humans, both non-phosphorylated and phosphorylated CRMP2 exist. Furthermore, the tertiary structure of the C-terminal region (490-572) of CRMP2 is flexible and somewhat random, and undergoes post-translational modifications such as phosphorylation, altering its conformation. See Nakamura et al., Front.Cell Neurosci. 14(2020), 188. Therefore, antibodies originally produced in the human body, exposed to the natural environment and thus to the flexible and changing tertiary structure of the CRMP2 C-terminal region, may possess specific binding characteristics not found in antibodies produced against a particular antigen, such as the monoclonal anti-CRMP2 antibody 1B1 (sc-101348, Santa Cruz Biotechnology, Dallas, USA) and the polyclonal anti-CRMP2 antibody (ab36201) from abcam (Cambridge, UK) (both produced against synthetic CRMP2 C-terminal peptide), as well as the Sigma-Aldrich® polyclonal anti-CRMP2 antibody C2993 available from Merck KGaA (Darmstadt, Germany) (which was produced against unphosphorylated full-length CRMP2).

[0029] Indeed, as described by Mileusnic and Rose in Journal of Neurochemistry 118(2011), 616-625, doi:10.1111 / j.1471-4159.2011.07193.x, when administered to chicks, antibody ab36201 induced memory loss in relation to a passive avoidance task. Therefore, this antibody adversely affects cognitive function in chicks, in contrast to the antibody of the present invention which improves cognitive function in mice.

[0030] Furthermore, hyperphosphorylated tau is known to co-localize with phosphorylated CRMP2. See Takata et al., Am.J. Pathol 175 (2009), 17-24. In experiments conducted within the scope of the present invention, recombinant (p)tau and (p)CRMP2 actually co-aggregate in the ThioT assay, as shown here (see Example 8 and Figure 7), and as previously stated, antibody NI-503.3E7 also reduces amyloid plaque loading in a transgenic AD mouse model. Therefore, it is reasonable to expect that antibody NI-504.3E7 can inhibit the co-aggregation of p-tau and pCRMP2. In this context, unless otherwise specified, the term "NI-504.3E7" refers to the original antibody NI-504.3E7 and any variant thereof, representing equivalent CRMP2-specific binding molecules.

[0031] The extracellular neurite plaques composed of amyloid-beta (Aβ) protein and intracellular neurofibrillary tangles containing phosphorylated tau protein (both characteristic proteins of Alzheimer's disease (AD)), as well as the individual neurotoxicity of these proteins in AD, have been extensively studied. The coexistence of Aβ plaques and phosphorylated tau has been suggested to be related to a mechanism by which Aβ promotes the propagation of tau aggregation in neurite plaques. The interaction between Aβ and tau mediates cognitive impairment in AD patients. See Zhang et al., Int J Biol Sci. 17 (2021), 2181-2192. Furthermore, therapeutic agents developed to treat AD are often known to target both Aβ plaques and neurofibrillary tangles containing phosphorylated tau protein. For example, it has recently been shown that human anti-Aβ antibodies induce the neutralization and removal of neurotoxic oligomeric Aβ species from amyloid plaques and presynaptic axonal terminals in the brain, thereby protecting axons from amyloid damage and keeping non-phosphorylated microtubule-bound tau within its physiological axonal localization, thereby preventing the abnormal relocalization of tau from the axonal compartment to the cell body dendritic compartment in amyloid-affected neurons where tau is abnormally phosphorylated and prone to aggregation. Furthermore, extracellular species of pathological tau are removed by phagocytosis of microglia or macrophages, which is observed by a decrease in CSF levels of pathological phosphotau in aducanumab-treated patients. For example, see International Patent Publication No. 2021 / 081101A1 and Cummings et al. Alz.Res.Therapy 13(2021),98; Salloway and Cummings, Aducanumab, Amyloid Lowering, and Slowing of Alzheimer Disease, Neurology 97(2021),543-544.

[0032] As described above, CRMP2 exists in either a phosphorylated or unphosphorylated form, and phosphorylation controls neurite outgrowth. CRMP2 can be sequentially phosphorylated by GSK3β at S518, T514, and T509, but only after prior phosphorylation by Cdk5 at Ser522. The binding of antibody NI-504.3E7 to full-length phosphorylated CRMP2 has already been shown in Examples 2 and 4, and further experiments were performed to demonstrate that antibody NI-504.3E7 can bind to phosphorylated CRMP2 peptides. Experiments determining the binding specificity of antibody NI-504.3E7 using phosphorylated peptides showed that the affinity of antibody NI-504.3E7 to CRMP2 peptides phosphorylated at position T514 is approximately 1 / 300th of the affinity to unphosphorylated peptides and peptides phosphorylated at T509 and S522. This suggests that phosphorylation of T514 partially impairs the binding of antibody NI-504.3E7, possibly due to its proximity to the antibody's minimal epitope 516-ASSAK-520. See Example 6 and Figure 5.

[0033] The antibody NI-504.3E7 has also been shown to bind to recombinant CRMP2 full-length protein in humans, mice, and rats. It has also been shown that the antibody NI-504.3E7 is internalized in SH-SY5Y cells. See Example 12 and Figure 11. As shown in Example 9, in an ex vivo hippocampal section culture model, aggregated pCRMP2 and monomeric CRMP2 increase neuronal spine density, but the increase in spine density by CRMP2 / pCRMP2 is reversed by two different concentrations of NI-504.3E7. Such assays can be used as proof of antibody activity, particularly proof that the antibody interferes with CRMP2 signaling, which has not been described to date.

[0034] Accordingly, the present invention relates to a pharmaceutical composition comprising an antibody and an equivalent CRMP2-binding molecule, which may be a binding fragment or derivative of NI-504.3E7 having substantially the same binding specificity and activity as the antibody NI-504.3E7. In other words, the present invention relates to a pharmaceutical composition comprising an antibody and an equivalent CRMP2-binding molecule that (i) preferentially recognizes human CRMP2 over CRMP1, CRMP3, CRMP4 and CRMP5 (which can be tested in ELISA assays and Western blot analyses as described in Example 2), (ii) can reduce the level of pCRMP2 in a concentration-dependent manner when CRMP2 is subjected to a phosphorylation assay (which can be determined as described in Example 5), and / or (iii) can reverse the increase in neuronal spine density induced by CRMP2, as measured in an ex vivo hippocampal section culture model (which can be tested as described in Example 9). Accordingly, the present invention relates to a pharmaceutical composition comprising an antibody and an equivalent CRMP2 binding molecule, which may be a binding fragment or derivative of NI-504.3E7 having substantially the same binding specificity and activity as the antibody NI-504.3E7 with respect to feature (i), or feature (ii), or feature (iii), or feature (i) and (ii), or feature (ii) and (iii), or feature (i), (ii), and (iii).

[0035] The described characteristics can be readily determined by following the experiments and assays disclosed in the attached examples, in which case antibody NI-504.3E7 can be used as the reference antibody. Typically, such equivalent CRMP2-binding molecules will compete with the corresponding reference antibody for binding to CRMP2 and pCRMP2 in the same epitopes and peptides as described above, respectively. Preferably, such equivalent CRMP2-binding molecules have the same epitopes as the reference antibody.

[0036] As described above, 40 variants of the antibody NI-504.3E7 (NI-504.3E7_V1 to NI-504.3E7_V40) were generated by exchanging amino acids in the variable heavy (VH) chain region, the variable light (VL) chain region, and / or the complementarity-determining region (CDR).

[0037] Therefore, in one embodiment, the antibody of the present invention may be characterized by a complementarity-determining region (CDR) or hypervariable region of the variable heavy (VH) chain and variable light (VL) chain of antibody NI-504.3E7, which contains the amino acid sequences of SEQ ID NO: 2 (VH_3E7) and SEQ ID NO: 7 (VL_3E7) shown in Tables II and IV.

[0038] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V1, which contains the amino acid sequences of SEQ ID NO: 2 (VH_3E7) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0039] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V2, which contains the amino acid sequences of SEQ ID NO: 2 (VH_3E7) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0040] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V3, which contains the amino acid sequences of SEQ ID NO: 12 (VH_3E7_var1) and SEQ ID NO: 7 (VL_3E7) shown in Tables II and IV.

[0041] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V4, which contains the amino acid sequences of SEQ ID NO: 12 (VH_3E7_var1) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0042] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V5, which contains the amino acid sequences of SEQ ID NO: 12 (VH_3E7_var1) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0043] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V6, which contains the amino acid sequences of SEQ ID NO: 14 (VH_3E7_var2) and SEQ ID NO: 7 (VL_3E7) shown in Tables II and IV.

[0044] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V7, which contains the amino acid sequences of SEQ ID NO: 14 (VH_3E7_var2) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0045] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V8, which contains the amino acid sequences of SEQ ID NO: 14 (VH_3E7_var2) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0046] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V9, which contains the amino acid sequences of SEQ ID NO: 16 (VH_3E7_var3) and SEQ ID NO: 7 (VL_3E7) shown in Tables II and IV.

[0047] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V10, which contains the amino acid sequences of SEQ ID NO: 16 (VH_3E7_var3) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0048] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V11, which contains the amino acid sequences of SEQ ID NO: 16 (VH_3E7_var3) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0049] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V12, which contains the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 7 (VL_3E7) shown in Tables II and IV.

[0050] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V13, which contains the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0051] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V14, which contains the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0052] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V15, which contains the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 60 (VL_3E7_var3) shown in Tables II and IV.

[0053] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V16, which contains the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 7 (VL_3E7) shown in Tables II and IV.

[0054] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V17, which contains the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0055] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V18, which contains the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0056] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V19, which contains the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 60 (VL_3E7_var3) shown in Tables II and IV.

[0057] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V20, which contains the amino acid sequences of SEQ ID NO: 22 (VH_3E7_var6) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0058] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V21, which contains the amino acid sequences of SEQ ID NO: 24 (VH_3E7_var6a) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0059] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V22, which contains the amino acid sequences of SEQ ID NO: 26 (VH_3E7_var6b) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0060] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V23, which contains the amino acid sequences of SEQ ID NO: 28 (VH_3E7_var6c) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0061] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V24, which contains the amino acid sequences of SEQ ID NO: 30 (VH_3E7_var6d) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0062] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V25, which contains the amino acid sequences of SEQ ID NO: 32 (VH_3E7_var6ab) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0063] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V26, which contains the amino acid sequences of SEQ ID NO: 34 (VH_3E7_var6ac) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV. In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V27, which contains the amino acid sequences of SEQ ID NO: 36 (VH_3E7_var6ad) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0064] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V28, which contains the amino acid sequences of SEQ ID NO: 38 (VH_3E7_var6bc) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0065] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V29, which contains the amino acid sequences of SEQ ID NO: 40 (VH_3E7_var6bd) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0066] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V30, which contains the amino acid sequences of SEQ ID NO: 42 (VH_3E7_var6cd) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0067] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V31, which contains the amino acid sequences of SEQ ID NO: 44 (VH_3E7_var6bcd) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0068] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V32, which contains the amino acid sequences of SEQ ID NO: 46 (VH_3E7_var6acd) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0069] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V33, which contains the amino acid sequences of SEQ ID NO: 48 (VH_3E7_var6abd) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0070] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V34, which contains the amino acid sequences of SEQ ID NO: 50 (VH_3E7_var6abc) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0071] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V35, which contains the amino acid sequences of SEQ ID NO: 22 (VH_3E7_var6) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0072] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH and VL chains of antibody NI-504.3E7_V36, which contains the amino acid sequences of SEQ ID NO: 22 (VH_3E7_var6) and SEQ ID NO: 60 (VL_3E7_var3) shown in Tables II and IV.

[0073] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V37, which contains the amino acid sequences of SEQ ID NO: 52 (VH_3E7_var7) and SEQ ID NO: 56 (VL_3E7_var1) shown in Tables II and IV.

[0074] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V38, which contains the amino acid sequences of SEQ ID NO: 52 (VH_3E7_var7) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0075] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V39, which contains the amino acid sequences of SEQ ID NO: 52 (VH_3E7_var7) and SEQ ID NO: 60 (VL_3E7_var3) shown in Tables II and IV.

[0076] In one embodiment, the antibody of the present invention may be characterized by the CDR or hypervariable region of the VH chain and VL chain of antibody NI-504.3E7_V40, which contains the amino acid sequences of SEQ ID NO: 54 (VH_3E7_var8) and SEQ ID NO: 58 (VL_3E7_var2) shown in Tables II and IV.

[0077] The present invention is illustrated and described with reference to the human-derived antibody first obtained in the experiments described in the Examples performed in accordance with the present invention, but it should be understood that the present invention also includes antibodies and equivalent CRMP2-binding molecules, such as synthetic and bioengineered derivatives of antibody NI-504.3E7 (this means any engineered antibody or antibody-like CRMP2-binding molecule synthesized by chemical or recombinant techniques), which retain one or more of the functional properties of the present invention antibody, in particular preferentially recognizing human CRMP2 over CRMP1, CRMP3, CRMP4, and CRMP5, being able to dose-dependently reduce pCRMP2 levels when CRMP2 is subjected to a phosphorylation assay, and / or being able to reverse the increase in neuronal spine density induced by CRMP2, as measured in an ex vivo hippocampal section culture model. Therefore, for the sake of brevity, the present invention may be described with reference to one or more antibodies, but unless otherwise stated, the meaning of the term “antibody” is intended and includes its synthetic and bioengineered derivatives, as well as equivalent CRMP2-binding molecules.

[0078] As described above and demonstrated in the examples, the present invention has for the first time established CRMP2 and pCRMP2 as therapeutic targets in the treatment of neurodegenerative diseases. Accordingly, the present invention also relates to molecules that can target CRMP2 and pCRMP2, respectively, and can interfere with CRMP2 signaling, for use as pharmaceuticals. In particular, these molecules are intended for use in methods to prevent, slow the progression of, or treat neurodegenerative diseases, cognitive impairments, and / or to improve memory and learning abilities.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but exemplary methods and materials are described below. Any publications, patent applications, patents, and other documents referenced herein are incorporated by reference in their entirety. Materials, methods, and examples are illustrative and not intended to be limiting.

[0080] Further embodiments of the present invention will become apparent from the following description, examples, and claims. [Brief explanation of the drawing]

[0081] [Figure 1-1]Figure 1: Alignment of the amino acid sequences of the variable region of the anti-CRMP2 specific human antibody NI-504.3E7, i.e., the heavy chain (VH;A) and lambda light chain (VL;B), to 40 variants NI-504.3E7_V1~V40 containing amino acid substitutions in VH, VL, and / or CDR. The complementarity-determining region (CDR) is indicated by an underline. The region adjacent to the CDR is known as the framework region. The Kabat numbering scheme was used as shown in Table I below (see http: / / www.bioinf.org.uk / abs / ; Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interests" (1983)). Unless otherwise specified, references to the numbering of specific amino acid residue positions in the antibodies of the present invention or their CRMP2-binding fragments, variants, or derivatives follow the Kabat numbering system. However, this is theoretical and does not apply equally to all antibodies of the present invention. For example, depending on the position of the first CDR, subsequent CDRs may be shifted in either direction. Therefore, in the event of any accidental error or inconsistency in the representation of CDRs in Figure 1 and / or the sequence listing, a person skilled in the art will be well in a position to determine the correct Kabat CDR sequence based on the disclosures of this application, i.e., based on the variable heavy (VH) chain and variable light (VL) chain amino acid sequences of antibody NI-504.3E7 and its variants, which will be used to define the antibody and its CRMP2 binding fragment relating to this application. The variable heavy chain VH sequence (A) of antibody NI-504.3E7 shown in SEQ ID NO: 2 and the light chain VL sequence (B) of antibody NI-504.3E7 shown in SEQ ID NO: 7 are shown, and they are aligned to the VH and VL sequences of 40 variants of NI.504.3E7.As will be further explained herein, within the CDR and / or framework region, conservative amino acid substitutions are preferred, such as the exchange of positions of two amino acids, where the physicochemical properties of the original amino acids are considered individually or in conjunction with adjacent amino acids, as illustrated in Mirsky et al., Mol. Biol. Evol. 32 (2014) 806-819, page 813, Figure 6, particularly in the AB or LG model. [Figure 1-2] Figure 1 (continued) [Figure 1-3] Figure 1 (continued) [Figure 1-4] Figure 1 (continued) [Figure 1-5] Figure 1 (continued) [Figure 1-6] Figure 1 (continued) [Figure 2] Figure 2: The anti-CRMP2 antibody NI-504.3E7 of the present invention exhibits preferential binding to CRMP2 and weak binding to CRMP1. (A) The binding activity of NI-504.3E7 was evaluated for recombinant human CRMP proteins 1-5 by ELISA. The line represents the sigmoid curve fit. Data are shown as mean + SEM. (B) The binding specificity of NI-504.3E7 was evaluated for recombinant human CRMP proteins 1-5 by Western blotting. The numbers indicate molecular weight in kilodaltons (kDa). [Figure 3] Figure 3: (A) Determination of epitopes related to antibody NI-504.3E7 binding. Epitope mapping using an overlap peptide array representing the human CRMP2 protein showed that the anti-CRMP2 antibody NI-504.3E7 of the present invention binds to peptide 128 (SEQ ID NO: 62) and peptide 129 (SEQ ID NO: 63). (B) No binding was observed with the anti-human control (secondary antibody control). [Figure 4-1]Figure 4: The anti-CRMP2 antibody NI-504.3E7 of the present invention reduces pCRMP2 levels in a concentration-dependent manner. Phosphorylation levels at three selected phospho sites (T509 (A-E), T514 (F-J), S522 (K-O)) were analyzed in a phosphorylation assay after phosphorylation for 1 hour or 24 hours in the presence of different concentrations of CRMP2-specific antibody or human IgG control (isotype control antibody), and readouts were performed by Western blotting (A, F, K) (numbers indicate molecular weight expressed in kilodaltons (kDa)). Quantification of the Western blotting analysis was performed based on signal band intensity (B-E, G-J, L-O). Specifically, signal band intensity was measured and displayed either directly or normalized to the total CRMP2 protein level evaluated with the commercially available CRMP2 antibody C2993. [Figure 4-2] Figure 4 (continued) [Figure 4-3] Figure 4 (continued) [Figure 5] Figure 5: When measured by ELISA assay, the anti-CRMP2 antibody NI-504.3E7 of the present invention specifically binds to CRMP2 peptide and pCRMP2 peptide. The affinity of antibody NI-504.3E7 for CRMP2 peptide phosphorylated at position T514 is approximately 1 / 300th of the affinity for unphosphorylated peptide and peptide phosphorylated at positions T509 and S522. Phosphorylation of threonine 514 indicates partially impaired binding of antibody NI-504.3E7. Unphosphorylated CRMP2 protein and BSA were used as positive and negative controls, respectively. Lines represent sigmoid curve fits. The sequences of peptides bound by antibody NI-504.3E7 are shown. Data are presented as mean + SEM. [Figure 6]Figure 6: The anti-CRMP2 antibody NI-504.3E7 of the present invention exhibits orthologous protein binding to recombinant human, mouse, and rat CRMP2 proteins. Western blotting detected the binding specificity of NI-504.3E7 for recombinant human CRMP2 (SUMO-CRMP2) protein, mouse CRMP2 (CRMP2-His6) protein, and rat CRMP2 (CRMP2-His6) protein. No binding was observed with human IgG control (isotype control antibody) or anti-human control (secondary antibody control). The numbers indicate molecular weight in kilodaltons (kDa). [Figure 7] Figure 7: Aggregation of recombinant CRMP2 and recombinant pCRMP2 is increased in the presence of recombinant tau protein and recombinant p-tau protein. Thio-T positive signals (RFUs) indicate the level of aggregation of various proteins or protein combinations in the 240-hour agglutination assay. The medium was used as a negative control. Data are shown as mean + SEM. [Figure 8] Figure 8: The increase in spine density driven by CRMP2 / phosphoCRMP2 is reversed at two different concentrations by the anti-CRMP2 antibody NI-504.3E7 of the present invention. pCRMP2 aggregates and CRMP2 monomers (monomerized CRMP2) were evaluated for neuronal spine density in a hippocampal section culture model. Dendritic spine density was evaluated by threshold image analysis. Data are presented as mean + SEM. Statistical analysis: One-way ANOVA***p<0.001**p<0.01; analysis by Kruskal-Wallis post-hoc test compared to untreated control. [Figure 9] Figure 9: The anti-CRMP2 antibody NI-504.3E7 of the present invention detects neurons in the hippocampus of Alzheimer's disease patients. A) Clear neuronal staining is observed in Alzheimer's disease tissue with antibody NI-504.3E7, but no such staining is observed with the anti-human control. B) A high-magnification image of the illustrated region is shown as an enlarged image. [Figure 10]Figure 10: The anti-CRMP2 antibody NI-504.3E7 of the present invention reduces amyloid-beta plaques in female APPPS1 / TTBK1 transgenic mice. Threshold-based image analysis was performed on cortical and hippocampal amyloid-beta tissue staining. The number and size of amyloid-beta plaques were compared between NI-504.3E7-injected APPPS1 / TTBK1 transgenic mice and PBS-injected APPPS1 / TTBK1 transgenic mice. Mice administered with antibody NI-504.3E7 showed a 63% reduction in amyloid plaque load in the cortex and hippocampus (A, C) and a reduction in plaque size (B, D). The mean reduction in plaque size in female TTBK1 / APP mice administered with antibody NI-504.3E7 was 30% in the cortex (B) and 68% in the hippocampus (D). Data are shown as mean + SEM. Group size: n=7, Statistical analysis: Independent t-test ≤ 0.0001. [Figure 11] Figure 11: The anti-CRMP2 antibody NI-504.3E7 of the present invention binds to differentiated SH-SY5Y cells. A positive Cy3 signal was detected in cells stained with NI-504.3E7. SH-SY5Y cells stained with commercially available anti-CRMP2 antibody (Abcam 129082) and anti-β-III-tubulin antibody were used as positive controls. No signal was observed with human IgG control (isotype control antibody) or anti-human control (secondary antibody control). Nuclei were visualized using DAPI. Images were acquired using a confocal microscope (20x objective lens). Scale bar: 50 μm. [Figure 12] Figure 12: The anti-CRMP2 antibody NI-504.3E7 of the present invention improves long-term potentiation (LTP) in aged C57Bl / 6J mice. LTP analysis was performed in young mice and a control group (aged mice administered PBS). In aged mice, the LTP signal was reduced compared to young mice. After administration of the antibody to aged mice, the LTP signal increased again, thus demonstrating that antibody NI-504.3E7 improves LTP in aged mice. Data are presented as mean + SEM. Group size: n=8, statistical analysis: two-way ANOVA, *p<0.05, ***p<0.001 compared to the PBS group. [Figure 13] Figure 13: The anti-CRMP2 antibody NI-504.3E7 of the present invention improves cognitive function in aged C57Bl / 6J mice. When the discrimination index was assayed in young mice and a control group (aged mice administered PBS), the discrimination index decreased in aged mice compared to young mice. After antibody administration, the discrimination index increased, thus demonstrating that antibody NI-504.3E7 improves cognitive function in aged mice. Data are presented as mean + SEM. Group size: n=11~12, statistical analysis: ## p<0.01 compared to the young group (t-test), *** p<0.001 compared to the PBS group (Dunnett's test). [Modes for carrying out the invention]

[0082] Detailed description of the invention The present invention is characterized in the claims and relates to embodiments disclosed herein and illustrated in the following examples and drawings. In general, the present invention relates to pharmaceutical compositions comprising recombinant, human-derived, preferably monoclonal antibodies that bind to human collagen reaction-mediated protein 2 (CRMP2), and equivalent binding molecules such as their binding fragments and derivatives. In particular, the present invention relates to pharmaceutical compositions comprising anti-CRMP2 antibodies that have unique binding characteristics, namely that they can bind to full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2), and preferably to an epitope comprising amino acid sequence 516-ASSAK-520 (SEQ ID NO: 64).

[0083] To avoid misunderstanding, while expressions such as “in some embodiments,” “in a particular embodiment,” “in certain cases,” “in some cases,” “in a further embodiment,” and “in one embodiment” are used, it should be emphasized that each of the embodiments described therein should be read with the understanding that each of the features of those embodiments can be combined, and that this disclosure should be treated as if the combination of features of those embodiments were detailed as a single embodiment. The same applies to any combination of embodiments and features of the appended claims and embodiments illustrated in the examples, which are also intended to be combined with features from the corresponding embodiments disclosed herein. Herein, embodiments are characterized by dependencies for the sake of consistent and concise description, but in practice, combinations of embodiments and features that can be constructed by (multiple) dependencies should be understood as being literally disclosed, and not as selections from different options.

[0084] Unless otherwise stated, terms used herein are given the definitions found in the "Oxford Dictionary of Biochemistry and Molecular Biology" (Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0-19-850673-2; 2nd edition, published 2006, ISBN 0-19-852917-1 978-0-19852917-0).

[0085] The term "derivative" refers to synthetic and bioengineered derivatives of an antibody that retain the functional properties of both the parent human antibody and the human-derived antibody, respectively. This includes any engineered antibody and CRMP2-binding molecule synthesized by chemical or recombinant techniques.

[0086] Furthermore, when referring to the anti-CRMP2 antibody of the present invention, equivalent binding molecules such as its binding fragments and derivatives are also included in this definition.

[0087] An antibody is "human-derived" if it has the CDR region of an antibody obtained from a human, or if the CDR of the antibody contains one or more variant CDR regions of an antibody obtained from a human, while maintaining the binding specificity of the human antibody. Human-derived antibodies typically contain a human framework region, preferably the framework region of an antibody obtained from a human, or one or more variant human framework regions of an antibody obtained from a human. Therefore, human-derived antibodies typically contain human VL and VH domains, or variant human VL or VH domains. Human-derived antibodies typically contain a human constant region. A human is preferably a person without neurodegenerative diseases such as Alzheimer's disease or tauopathy. A human is preferably a healthy person. A human may be elderly. Elderly people are typically over 60, over 65, over 70, over 75, or over 80 years old. Antibodies obtained from humans can be isolated from antibody libraries, such as blood lymphocyte libraries collected from elderly individuals.

[0088] The variant CDR region of a human-derived antibody typically contains one or more amino acid substitutions, preferably one, two, three, or four, more preferably one or two, and most preferably one. In one preferred embodiment, only VL-CDR3 contains one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two, and most preferably one.

[0089] The variant framework region of a human-derived antibody typically contains one or more amino acid substitutions, preferably one, two, three, four, five, or six, more preferably one or two, and most preferably one amino acid substitution.

[0090] In one preferred embodiment, mutants VH and VL each contain one or more amino acid substitutions and / or deletions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and / or deletions, preferably VL chain mutants contain 1, 2, 3, 4, or 5, preferably 2, 4, or 5 amino acid substitutions and / or deletions, and / or VH chain mutants contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2, 3, 4, 5, 6, 7, 8, or 10 amino acid substitutions and / or deletions. Most preferably, mutants VH and VL each contain one or two amino acid substitutions and / or deletions.

[0091] Conservative amino acid substitutions, which consider the physicochemical properties of the original amino acid either alone or in conjunction with adjacent amino acids, are preferred. Amino acid substitutions and deletions do not substantially alter the binding affinity of the original antibody. Amino acid substitutions and deletions do not substantially alter the immunogenicity of the original antibody.

[0092] As described herein, mutant antibodies NI-504.3E7_V1 to NI-504.3E7_V40 were created based on the antibody NI-504.3E7. Therefore, when referring to the antibodies of the present invention, it includes not only the antibody NI-504.3E7, its binding fragments and derivatives, and equivalent binding molecules, as well as competing antibodies, but also the corresponding mutants. In particular, the results of experiments performed with the original antibody NI-504.3E7 have been confirmed with one or more of these mutants.

[0093] Accordingly, the present invention provides a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment that selectively binds to human collagen reaction-mediated protein 2 (CRMP2), wherein the antibody can bind to the C-terminus of CRMP2, preferably the antibody can bind to a CRMP2 peptide consisting of the amino acid sequence TPKTVTPASSAKTSP (SEQ ID NO: 62) or the amino acid sequence VTPASSAKTSPAKQQ (SEQ ID NO: 63), and / or the antibody requires the amino acid sequence 516-ASSAK-520 (SEQ ID NO: 64) in CRMP2 to bind to the C-terminus of CRMP2. The antibody or antigen-binding fragment typically binds to CRMP2 such that the phosphorylated CRMP2 (pCRMP2) level decreases in a concentration-dependent manner when CRMP2 is subjected to a phosphorylation assay. The phosphorylation assay can be performed in combination with Western blot analysis. This phosphorylation assay can detect levels of T509 pCRMP2, T514 pCRMP2, and / or S522 pCRMP2. Antibodies typically bind to recombinant CRMP2 full-length protein from mice and rats, as well as recombinant CRMP2 full-length protein from humans. Antibodies or antigen-binding fragments can typically reverse the increase in neuronal spine density induced by CRMP2, as determined in ex vivo hippocampal section culture models. Antibodies are preferably human antibodies or antibodies derived from humans.

[0094] The antibody selectively binds to collagen reaction-mediating protein 2 (CRMP2) and preferentially recognizes human CRMP2 over CRMP1, CRMP3, CRMP4, and CRMP5. Selective antibody binding can be measured using ELISA assays and Western blotting analysis, as described in Example 2.

[0095] The antibody or antigen-binding molecule of the present invention may be the following antibody or antigen-binding molecule against human CRMP2 and / or pCRMP2: - Human CRMP2 is recognized preferentially over CRMP1, CRMP3, CRMP4, and CRMP5; -When CRMP2 is subjected to a phosphorylation assay, the level of phosphorylated CRMP2 (pCRMP2) can be reduced in a concentration-dependent manner, and / or -When measured in an ex vivo hippocampal section culture model, it was possible to reverse the increase in neuronal spine density induced by CRMP2. The antibody or antigen-binding molecule inhibits the binding of an antibody containing a heavy chain variable (VH) region and a light chain variable (VL) region to human CRMP2, wherein the VH region includes complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region includes CDRs 1, 2, and 3, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 or 61. In addition, the binding of the antibody or antigen-binding molecule to human CRMP2 may be inhibited by an antibody containing the VH and VL CDR amino acid sequences (a) to (f). The antibody is preferably a human antibody or a human-derived antibody.

[0096] The antibody or antigen-binding molecule of the present invention may be the following antibody or antigen-binding molecule against human CRMP2 and / or pCRMP2: - Human CRMP2 is recognized preferentially over CRMP1, CRMP3, CRMP4, and CRMP5; -When CRMP2 is subjected to a phosphorylation assay, the phosphorylated CRMP2 (pCRMP2) level can be reduced in a concentration-dependent manner, and / or -When determined in an ex vivo hippocampal section culture model, it is possible to reverse the increase in neuronal spine density induced by CRMP2, The binding of the antibody or antigen-binding molecule to human CRMP2 is inhibited by an antibody whose variable regions include a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region includes complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region includes CDRs 1, 2, and 3, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 or 61. In addition, the antibody or antigen-binding molecule may inhibit the binding of antibodies containing VH and VL CDR amino acid sequences (a) to (f) to human CRMP2.

[0097] Inhibition of antibody binding to human CRMP2 can be determined using a simple immunoassay (e.g., electrochemiluminescence assay, competitive ELISA, solid-phase radioimmunoassay (SPRIA), or blockade Western blot) that demonstrates the ability of one antibody to block the binding of another antibody to a target antigen.

[0098] Furthermore, with respect to CRMP2 proteins and anti-CRMP2 antibodies, those skilled in the art can rely on their recombinant production, purification, modification, formulation into pharmaceutical compositions and therapeutic use in host cells, as well as general terminology and features in the art, when carrying out the claimed invention. See, for example, "Antibodies A Laboratory Manual" (2nd edition, 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA). This document also describes antibody purification and storage, engineering of antibodies including the use of degenerate oligonucleotides, 5'-RACE, phage display and mutagenesis, immunoblotting protocols and modern screening and labeling techniques.

[0099] As further illustrated in the examples, the antibodies of the present invention were initially isolated from human donors and are shown to specifically recognize at least human full-length CRMP2, and optionally mouse and rat full-length CRMP2 as well. See Example 7.

[0100] Since CRMP2 sequences are highly conserved across mammalian species, and the antibody NI-504.3E7 binds to human, mouse, and rat CRMP2, it is reasonable to expect that the antibody of the present invention will bind to mammalian CRMP2 in general. Therefore, in one embodiment, the antibody of the present invention can specifically bind to mammalian CRMP2, preferably recombinant full-length mammalian CRMP2. In a preferred embodiment, the antibody of the present invention can bind to recombinant human CRMP2, particularly recombinant human full-length CRMP2. In one embodiment, the antibody of the present invention can also bind to mouse and rat CRMP2, particularly recombinant full-length mouse and rat CRMP2.

[0101] Furthermore, since antibody NI-504.3E7 does not bind to E. coli cell lysates, it is confirmed that there is no cross-reactivity when recombinant CRMP2 is produced in E. coli.

[0102] As further demonstrated in the examples, the antibodies of the present invention not only specifically bind to full-length CRMP2 in humans, and optionally to mouse and rats, but also selectively bind to CRMP2. In particular, the antibodies of the present invention have been shown to bind to human full-length CRMP2 on Western blot, but not to human full-length CRMP1, CRMP3, CRMP4, and CRMP5 (see Example 2). Thus, in one embodiment, the antibodies of the present invention selectively bind to CRMP2 and preferably do not bind to CRMP1, CRMP3, CRMP4, and / or CRMP5, or show only weak binding. In one embodiment, the antibodies of the present invention do not bind to CRMP3, CRMP4, and CRMP5, but show weak binding to CRMP1.

[0103] "Antibodies that selectively or specifically bind to CRMP2," "antibodies that selectively or specifically recognize CRMP2," and "antibodies that are selective or specific to CRMP2" specifically, generally, and collectively mean antibodies that, as determined by ELISA as illustrated in Example 2 and shown in Figure 2A, bind to full-length recombinant or native CRMP2 (e.g., those present in brain tissue), and optionally to denatured CRMP2, preferably with at least one order of priority over CRMP1, CRMP3, CRMP4, and CRMP5, and antibodies that, as determined by Western blotting as illustrated in Example 2 and shown in Figure 2B, do not substantially cross-react with CRMP1, CRMP3, CRMP4, and CRMP5. As described above, the binding specificity of an antibody can be determined, for example, by a Western blotting or ELISA assay described in the examples herein, or by an equivalent assay.

[0104] As described above, CRMP2 is a phosphoprotein and can switch between a non-phosphorylated (active) form and a phosphorylated (inactive) form. As shown in Example 4 and Table VII, the antibodies of the present invention bind to full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2). Thus, in one embodiment, the antibodies of the present invention are capable of binding to full-length non-phosphorylated CRMP2 and pCRMP2 when determined by an ELISA assay.

[0105] Thus, unless otherwise indicated, when referring to an anti-CRMP2 antibody, a CRMP2-binding molecule and fragments or derivatives thereof, and an antibody of the present invention specific for CRMP2, it means that the antibody, CRMP2-binding molecule and fragments or derivatives thereof also bind to phosphorylated CRMP2 (pCRMP2). Thus, unless otherwise indicated, the terms "anti-CRMP2 antibody" and "CRMP2-binding molecule and fragments or derivatives thereof" and "CRMP2-specific antibody" include the corresponding antibodies and binding molecules and fragments or derivatives thereof that bind to pCRMP2.

[0106] Antibodies with high affinity for CRMP2 and / or pCRMP2, preferably for CRMP2 and pCRMP2, are of particular interest, and to obtain a measure of the binding affinity, the EC 50 of the antibody was determined in the ELISA assay performed in Example 4. The term "EC 50 " refers to the concentration of an antibody or an antigen-binding fragment thereof that induces a response that is 50% of the maximum response, i.e., the midpoint between the maximum response and the baseline, in the context of in vitro or in vivo assays using the antibody or its antigen-binding fragment.

[0107] As can be seen from Table VII, the binding affinity of antibody NI-504.3E7 has not only been determined, but also, as detailed below, has been determined from its 40 variants (NI-504.3E7_V1 to NI-504.3E7_V40) that have amino acid substitutions in the VH and / or VL chains, and / or within the CDR. In particular, antibody NI-504.3E7 binds recombinant human full-length CRMP2 and pCRMP2 at EC values ​​of 0.5 nM and 4.7 nM, respectively. 50 Therefore, it is specifically recognized. The values ​​for mutants NI-504.3E7_V1 to NI-504.3E7_V40 are listed in Table VII.

[0108] In one embodiment, the antibody of the present invention has an EC of CRMP2 of less than about 30 nM, preferably less than about 25 nM, preferably less than about 20 nM, preferably less than about 15 nM, preferably less than about 10 nM, preferably less than about 7.5 nM, preferably less than about 5 nM, preferably less than about 3 nM, and most preferably less than about 2 nM. 50 Recognizes pCRMP2 with an EC of less than about 120 nM, preferably less than about 110 nM, preferably less than about 100 nM, preferably less than about 90 nM, preferably less than about 80 nM, preferably less than about 70 nM, preferably less than about 60 nM, preferably less than about 50 nM, preferably less than about 40 nM, preferably less than about 30 nM, preferably less than about 25 nM, most preferably less than about 20 nM. 50 Recognized by the above EC. Preferably, the binding affinity for CRMP2 and pCRMP2 is in the same order of magnitude, or the binding affinity for pCRMP2 is one or two orders of magnitude lower than the binding affinity for CRMP2. Most preferably, the antibody of the present invention recognizes CRMP2 and pCRMP2 as described above. 50 Recognition is based on the value. Therefore, antibodies that do not specifically bind to CRMP2 / pCRMP2 require an EC of, for example, higher than approximately 150 nM, more preferably higher than approximately 400 nM. 50 The antibody binds to CRMP2 / pCRMP2, and most preferably EC 50It is not even possible to determine this. However, it also depends on the antibody format, for example, whether to use antibody fragments such as IgG1, IgG4, or Fab fragments, EC 50 The values ​​may deviate and may be higher than, for example, the values ​​described above and in the examples. Therefore, in this context, the term "approximately" means a value that may differ from the value determined for the reference antibody in the examples, where the difference is preferably less than one order of magnitude, most preferably within the same order of magnitude. For example, EC 50 The reference value can be ±10 nM.

[0109] The aforementioned characteristics, namely that antibody NI-504.3E7 exhibits preferential and selective binding to CRMP2 compared to CRMP1, CRMP3, CRMP4, and CRMP5, and that antibody NI-504.3E7 binds to both phosphorylated and unphosphorylated CRMP2, have also been confirmed by analyzing the binding affinity of the antibody to the corresponding CRMP and pCRMP peptides. In particular, antibody NI-504.3E7, when determined by ELISA, contains the CRMP2 peptide (H-EVSVTPKTVTPASSAKTSPAKQQC-NH2 (SEQ ID NO: 70)) and the pCRMP2 peptide (H-EVSV(pT)PKTV(pT)PASSAKT(pS)PAKQQC-NH2 (SEQ ID NO: 70)) which contains three phospho moieties T509, T514 and S522, as well as the corresponding peptide from (p)CRMP1 ((EVPA(pT)PKYA(pT)PAPSAKS(pS)PSKHQC-NH2 (SEQ ID NO: 69)) and the corresponding peptide from (p)CRMP4 (H-DLTT(pT)PKGG(pT)PAGSARG(pS)PTRP It preferentially binds to NC-NH2 (SEQ ID NO: 71). Furthermore, antibody NI-504.3E7 preferentially binds to the CRMP2 peptide (TVTPASSAKTSPAKQQAPPVRC-NH2 (SEQ ID NO: 67)) and the pCRMP2 peptide containing a single S522 phospho moiety (H-TVTPASSAKT(pS)PAKQQAPPVRC-NH2 (SEQ ID NO: 67)) over the corresponding peptide derived from (p)CRMP1 (H-YATPAPSAKS(pS)PSKHQPPPIRC-NH2 (SEQ ID NO: 66)) and the corresponding peptide derived from (p)CRMP4 (H-GGTPAGSARG(pS)PTRPNPPVRNC-NH2 (SEQ ID NO: 68)).

[0110] As shown in Example 6, the antibody NI-504.3E7 contains the CRMP2 peptide VCEVSVTPKTVTPASSAKTSPAKQQA (SEQ ID NO: 72) and different pCRMP2 peptides (VCEVSV(p)T, respectively) each containing one phospho moiety (T509, T514, or S522). 509PKTVTPASSAKTSPAKQQA (Sequence ID 72), VCEVSVTPKTV(p)T 514 PASSAKTSPAKQQA (Sequence ID 72), and VCEVSVTPKTVTPASSAKT(p)S 522 The antibody NI-504.3E7 specifically binds to PAKQQA (SEQ ID NO: 72) with different affinities. In particular, the affinity of antibody NI-504.3E7 to the CRMP2 peptide with phosphorylation at position T514 has been shown to be about 1 / 300th of that of the non-phosphorylated peptide and the peptide with phosphorylation at positions T509 and S522. Therefore, in one embodiment, the antibody of the present invention binds to both phosphorylated and non-phosphorylated CRMP2 peptides, where the phosphorylated peptide is preferably phosphorylated at T509, T514 and / or S522. Preferably, the binding of the antibody of the present invention to the CRMP2 peptide with phosphorylation at T514 (preferably the one described above) is less preferable than the binding to the non-phosphorylated CRMP2 peptide and the CRMP2 peptide with phosphorylation at positions T509 and S522, and preferably to the one described above.

[0111] In the phosphorylation assay described in Example 5, it was further demonstrated that the antibody of the present invention reduces the level of pCRMP2 in a concentration-dependent manner. In fact, this experiment was performed exemplary using the mutant NI-504.3E7_V20 to further confirm the fact that the mutant antibody retains the binding specificity and activity of the original antibody. As can be seen from Table VII, the binding affinity (EC) of NI-504.3E7_V20 to CRMP2 and pCRMP2 is shown. 50 The value (represented by the antibody level) is reduced compared to the original antibody. Therefore, since this variant has sufficient activity to reduce pCRMP2 levels in the phosphorylation assay, other variants should be able to do the same.

[0112] This activity was not observed with anti-CRMP2 control antibodies that bind to the different epitopes detailed above, nor with human IgG control antibodies. Therefore, this feature appears to be unique to the antibody of the present invention. In particular, Western blot images showed that when the antibody of the present invention and CRMP2 were subjected to phosphorylation assays for 1 hour and 24 hours under appropriate conditions, i.e., phosphorylation of T509, T514, and S522 in the CRMP2 protein, respectively, in the presence of glycogen synthase kinase-3 (GSK-3)β and cyclin-dependent kinase (Cdk) 5 / p35, the amounts of T509-phosphorylated CRMP2, T514-phosphorylated CRMP2, and S522-phosphorylated CRMP2 decreased compared to "no antibody control" and "human IgG antibody control." Furthermore, under most conditions, this decrease was dependent on the concentration of the antibody of the present invention. That is, the higher the amount of the antibody of the present invention, the greater the decrease in phosphorylated CRMP2.

[0113] Therefore, in one embodiment, when both the antibody and the CRMP2 protein are subjected to a phosphorylation assay, the antibody of the present invention can reduce the level of pCRMP2, preferably T509 pCRMP2, T514 pCRMP2, and / or S522 pCRMP2, in a concentration-dependent manner compared to a control. Preferably, the phosphorylation assay is performed for 1 hour or 24 hours under appropriate conditions in the presence of GSK-3β and Cdk 5 / p35, and the results are analyzed by Western blot analysis. Therefore, in a preferred embodiment, when the antibody and CRMP2 are subjected to a phosphorylation assay for 1 hour or 24 hours under appropriate conditions in the presence of glycogen synthase kinase-3 (GSK-3)β and Cdk 5 / p35, the antibody of the present invention can reduce the level of pCRMP2 in a concentration-dependent manner compared to a control, as determined by Western blot analysis, where preferably pCRMP2 is selected from T509 pCRMP2, T514 pCRMP2, and S522 pCRMP2. In a preferred embodiment, a larger amount of antibody leads to a greater reduction in the pCRMP2 level, while a smaller amount of antibody leads to a smaller reduction in the pCRMP2 level.

[0114] There are various appropriate controls, such as an "antibody-free control" where no antibody is added to the phosphorylation assay, a control antibody that does not recognize either CRMP2 or pCRMP2, such as a human IgG antibody control, or a CRMP2 antibody that is known not to reduce pCRMP2 levels in phosphorylation assays.

[0115] Overlap peptide array analysis revealed that antibody NI-504.3E7 binds to linear peptides containing the amino acid sequences TPKTVTPASSAKTSP (SEQ ID NO: 62) and VTPASSAKTSPAKQQ (SEQ ID NO: 63), which are part of the C-terminal domain of CRMP2, with VTPASSAKTSP (SEQ ID NO: 65) as the consensus sequence. See Example 3. The C-terminal domain spans amino acids 490 to 572. Therefore, in one embodiment, the antibody of the present invention binds to the C-terminal regions of CRMP2 and pCRMP2. In a particularly preferred embodiment, the antibody of the present invention binds to a peptide present in the C-terminal region of CRMP2 and pCRMP2, where the peptide is preferably TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63). Of course, the antibody also binds to peptides containing the aforementioned sequences.

[0116] Epitope mapping revealed that the antibody NI-504.3E7 binds to a linear peptide containing the consensus sequence ASSAK (SEQ ID NO: 64). Therefore, in one embodiment, the antibody of the present invention binds to an epitope containing or consisting of the amino acid sequence ASSAK (SEQ ID NO: 64).

[0117] As shown in Example 9, the antibody NI-504.3E7 can reverse the increase in spine density driven by CRMP2 / pCRMP2 at antibody concentrations of 7.5 μM and 0.7 μM. In particular, since CRMP2 is known to be involved in axonal growth, and experiments conducted within the scope of the present invention have shown that anti-CRMP2 antibodies act on spine density, preferably reducing it, this feature can be used as proof of activity for the antibody of the present invention, further indicating that this anti-CRMP2 antibody interferes with CRMP2 signaling in some way. Accordingly, the present invention also relates to a molecule that can interfere with CRMP2 signaling for use as a pharmaceutical, preferably for use in methods for the prevention, delay of progression, or treatment of neurodegenerative diseases, cognitive impairment, and / or for improving memory and learning abilities.

[0118] Furthermore, in one embodiment, the antibody of the present invention can reverse the increase in neuronal spine density induced by CRMP2 when determined in an ex vivo hippocampal section culture model, preferably at concentrations between 0.4 μM and 10 μM, preferably between 0.5 μM and 9 μM, preferably between 0.6 μM and 8 μM, and most preferably at 7.5 μM or 0.7 μM.

[0119] The increase in neuronal spine density is amplified by pCRMP2 aggregates or CRMP2 monomers.

[0120] In one embodiment, an anti-CRMP2 antibody and an equivalent CRMP2-binding molecule exhibit the immunohistochemical characteristics and in vivo activity of any one of the anti-CRMP2 antibodies illustrated in the following examples and drawings.

[0121] In particular, antibodies, antigen-binding fragments, and their derivatives have at least one of the following properties (i) to (iii): (i) Detection of neurons in the hippocampus of AD patients, (ii) Reduction of amyloid plaque load in female tautubulin kinase-1 (TTBK1) / amyloid precursor protein (APP) transgenic mice, and / or (iii) Improvement of cognitive function in aged mice.

[0122] More specifically, IHC analysis showed that the antibody NI-504.3E7 binds to hippocampal tissue and, in particular, detects neurons in samples from patients with tauopathy. Specifically, antibody NI-504.3E7 binds to hippocampal tissue from patients with AD. Furthermore, antibody NI-504.3E7 has been shown to bind to hippocampal tissue from patients with PSP, from patients with PiD, detect neurons in PiD hippocampal sections, detect neurons in FTD-PD hippocampal sections, and detect neurons in FTLD-TDP hippocampal tissue. Further studies have shown that antibody NI-504.3E7 reduces amyloid plaque load by 63% in the cortex and hippocampus, and reduces amyloid plaque size by 30% in the cortex and 68% in the hippocampus in a transgenic mouse model of Alzheimer's disease. In addition, in the cortex, a 50% reduction in the amount of plaque / area was observed in female TTBK1 / APP mice administered with the antibody NI-504.3E7 (area is defined as the entire cortex). Experiments described in Examples 13 and 14 further demonstrate that the antibody of the present invention improves LTP and cognitive function in aged mice when administered at doses of 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg. In fact, these experiments were performed exemplary using the mutant NI-504.3E7_V20 to further confirm the fact that the mutant antibody retains the activity of the original antibody, as described above with respect to the phosphorylation assay.

[0123] Accordingly, in one embodiment, the antibody of the present invention detects neurons in the hippocampus of patients with tauopathy, preferably AD, PSP, PiD, FTD-PD, and FTLD-TDP, most preferably patients with AD. In one embodiment, the antibody of the present invention reduces amyloid plaque load and / or mean amyloid plaque size and / or plaque / area amount in a transgenic mouse model of Alzheimer's disease, preferably in female tautubulin kinase-1 (TTBK1) / amyloid precursor protein (APP) transgenic mice. In one preferred embodiment, plaque load is reduced in the cortex and / or hippocampus by 5% to 95%, preferably 10% to 90%, preferably 15% to 85%, preferably 20% to 80%, preferably 25% to 75%, preferably 30% to 70%, preferably 35% to 65%, preferably 40% to 65%, preferably 45% to 65%, preferably 50% to 65%, preferably 55% to 65%, more preferably 60% to 65%, and most preferably 63%. In a further preferred embodiment, the average plaque size in the hippocampus is reduced by 5% to 95%, preferably 10% to 90%, preferably 15% to 85%, preferably 20% to 80%, preferably 25% to 75%, preferably 30% to 70%, preferably 35% to 70%, preferably 40% to 70%, preferably 45% to 70%, preferably 50% to 70%, preferably 55% to 70%, preferably 60% to 70%, more preferably 65% ​​to 70%, and most preferably 68%. In a further preferred embodiment, the average plaque size in the cortex is reduced by 5% to 60%, preferably 10% to 55%, preferably 15% to 50%, preferably 20% to 45%, preferably 25% to 40%, and most preferably 30%. In a further preferred embodiment, the amount of mottling / area is reduced by 20% to 70%, preferably 25% to 65%, preferably 30% to 60%, preferably 35% to 55%, preferably 40% to 50%, and most preferably 50%.In one embodiment, the antibody of the present invention improves cognitive function in aged mice when administered at doses preferably 0.1 mg / kg to 10 mg / kg, preferably 0.2 mg / kg to 10 mg / kg, preferably 0.3 mg / kg to 10 mg / kg, preferably 1 mg / kg to 10 mg / kg, most preferably 0.3 mg / kg, 1 mg / kg, 3 mg / kg, or 10 mg / kg, where the effect is most pronounced at doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg.

[0124] The present invention also relates to an antibody or antigen-binding molecule that generally competes with the antibody of the present invention for specific binding to human CRMP2 and / or pCRMP2, and retains one or more of the binding properties and functional characteristics described for the antibody of the present invention. Preferably, the competing antibody and antigen-binding molecule has at least one of the following properties (i) to (iii), preferably at least two of the following properties (i) to (iii), namely (i) and (ii), or (i) and (iii), or (ii) and (iii), most preferably all three of the following properties (i) to (iii): (i) Human CRMP2 is recognized preferentially over CRMP1, CRMP3, CRMP4, and CRMP5 (this can be tested by ELISA assay and Western blot analysis as described in Example 2); (ii) When CRMP2 is subjected to a phosphorylation assay, the level of pCRMP2 can be reduced in a concentration-dependent manner (this can be determined as described in Example 5); (iii) The increase in neuronal spine density induced by CRMP2 can be reversed when determined in an ex vivo hippocampal section culture model (this can be tested as described in Example 9).

[0125] In one embodiment, the present invention relates to a pharmaceutical composition comprising an antibody that recognizes the same epitope as antibody NI-504.3E7 and retains any one, preferably all, of the above binding characteristics (i) to (iii). Competitive antibodies can be identified by a simple immunoassay (e.g., electrochemiluminescence assay, competitive ELISA, solid-phase radioimmunoassay (SPRIA), or blocking Western blot) that demonstrates the ability of one antibody to block the binding of another antibody to a target antigen.

[0126] The present invention is exemplified by an anti-CRMP2 antibody and its antigen-binding fragment, characterized in that its variable region, i.e., the binding domain, contains a variable heavy (VH) chain and a variable light (VL) chain having the amino acid sequence shown in Figure 1. The corresponding nucleotide and amino acid sequences are shown in Table II below. In particular, Figure 1 shows the VH and VL chains of the antibody NI-504.3E7 and 40 of its variants (NI-504.3E7_V1 to NI-504.3E7_V40), where amino acid substitutions are performed in the VH chain, VL chain, and / or CDR. The CDR is underlined in the corresponding figure and table. The corresponding amino acid substitutions can be performed by conventional methods known in the art, such as PCR.

[0127] As is typical, the variable domain of each chain contains three hypervariable loops called complementarity-determining regions (CDRs, CDR-1, -2, and -3). The CDRs are separated by structurally conserved regions called framework regions (FR-1, -2, -3, -4) that display these loops on the surface of the variable domain by forming a “core” β-sheet structure. The length and composition of the CDR sequence are highly variable, especially in CDR3. The CDRs approximate the paratopes of antibodies that interact with antigens and thus contain antigen-binding residues. Therefore, it is common to define an antibody by its six CDRs. Exemplary sets of CDRs in the above amino acid sequences of the VH and VL chains are shown in Figure 1 and in Tables II and IV. However, as will be discussed below, those skilled in the art are well aware that, in addition to or instead of them, CDRs (especially in the case of CDR2 and CDR3) whose amino acid sequences differ from any one of those in Figure 1 by one, two, three, or even more amino acids may be used. As noted in the legend of Figure 1, those skilled in the art can easily identify CDRs according to the general principles summarized, for example, at www.bioinf.org.uk / abs. In this regard, the CDRs of the antibodies shown in Figure 1 are shown according to Kabat et al., but it is known to those skilled in the art that there are several commonly used definitions of CDRs. These are: (i) Kabat's definition based on sequence variability (which is the most commonly used), (ii) Chothia's definition based on the position of the structural loop region, (iii) AbM's definition as a compromise between the two, used by Oxford Molecular's AbM antibody modeling software, and (iv) Contact's definition based on the analysis of available complex crystal structures, which has recently been introduced. Since these are residues involved in interaction with the antigen, this definition is probably the most useful for mutagenesis to modify the affinity of the antibody.For a list of CDR residues in contact with each antibody, along with summary data for each CDR, please refer to, for example, www.bioinf.org.uk / abs (which also refers to antibody modeling software such as abYmod, available at abymod.abysis.org). Table I below shows the relationships between CDR positions defined by different concepts.

[0128] [Table 1]

[0129] For the definitions mentioned above, please also refer to Kontermann and Dubel, eds., "Antibody Engineering," Vol. 2, DOI 10.1007 / 978-3-642-01147-4_3, #Springer-Verlag Berlin Heidelberg 2010, in particular Chapter 3, "Protein Sequence and Structure Analysis of Antibody Variable Domains" (pp. 33-51), and Dondelinger et al., Front.Immunol.9(2018), 2278 (which specifically discusses the importance and significance of antibody numbering and antigen-binding surface / residue definitions). For example, see Figs. 4 and 6 by Dondelinger et al., which illustrate the differences in the classical CDR definitions by Kabat, Chothia (Chothia and Lesk, J.Mol.Biol.196(1987), 901-917), Contact (MacCallum et al., J.Mol.Biol.262(1996), 732-745), and IMGT (IMGT®, the international ImMunoGeneTics information system®, www.imgt.org). The AbM definition is a compromise between the two above, used by Oxford Molecular's AbM antibody modeling software.

[0130] [ka]

[0131] The diagram above illustrates alternative definitions of CDR-H1 (VH-CDR1). The numbering schemes of Kabat and Chothia are shown horizontally, and the definitions of CDR by Kabat, Chothia, AbM, and Contact are indicated by arrows above and below these two numbering schemes. Thus, based on the amino acid sequences of the VH and VL chains provided as corresponding variants of the two present invention antibodies NI-504.3E7, their epitopes, and the assays described in the examples, those skilled in the art can design and arrive at equivalent antibodies and similar CRMP2-binding molecules in which one or more of those CDRs defined by Kabat differ from the corresponding CDR of the parent present invention antibody, but substantially retain the CDR defined by Chothia. During experiments conducted within the scope of the present invention, it has even been demonstrated that certain amino acid substitutions do not substantially alter the binding affinity of the original antibody NI-504.3E7. Certain amino acid substitutions exist in the VH and / or VL chains of the variant antibodies, as shown in Figure 1 and, for example, Table V. Furthermore, amino acid substitutions are present even in VL-CDR3 in NI-504.3E7_V15, NI-504.3E7_V19, NI-504.3E7_V36, and NI-504.3E7_V39. As shown in Example 4 and Table VII, all mutants maintained their binding ability to CRMP2 and pCRMP2. In addition, the results of experiments performed using the original antibody NI-504.3E7, as described above, have been confirmed in one or more of the 40 mutants.

[0132] In one embodiment, the anti-CRMP2 antibody of the present invention, its antigen-binding fragment or derivative, or CRMP2-binding molecule comprises a variable heavy (VH) chain containing VH complementarity-determining regions (VH-CDRs) 1, 2, and 3 as defined by Kabat, and / or a variable light (VL) chain containing VL-CDRs 1, 2, and 3, where, NI-504.3E7 (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 3. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 4. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 5. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 8. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 shown in Figure 1 or Table III or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 9, and (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 shown in Figure 1 or Table III, or a variant thereof, wherein the variant contains the amino acid sequence resulting from a partial modification of SEQ ID NO: 10, or

[0133] (NI-504.3E7_V15, NI-504.3E7_V19, NI-504.3E7_V36, and NI-504.3E7_V39) (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 3. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 4. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 5. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 8. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 shown in Figure 1 or Table III or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 9, and (f) VL-CDR3 includes the amino acid sequence of SEQ ID NO: 61 (VL_3E7_var3) shown in Figure 1 or Table III, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 61.

[0134] In a preferred embodiment, the mutant CDR contains one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two, and most preferably one. In a preferred embodiment, only VL-CDR3 contains one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two, and most preferably one.

[0135] In another preferred embodiment, the mutant CDR contains an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 61, respectively. More preferably, VL-CDR3 alone contains a mutant amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequence shown in SEQ ID NO: 61. In addition to or instead of the above, the antibody or antigen-binding fragment of the present invention can be characterized as follows: NI-504.3E7 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 (VH_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 2, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7.

[0136] NI-504.3E7_V1 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 (VH_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 2, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0137] NI-504.3E7_V2 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 (VH_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 2, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0138] NI-504.3E7_V3 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 12 (VH_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 12, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7.

[0139] NI-504.3E7_V4 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 12 (VH_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 12, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0140] NI-504.3E7_V5 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 12 (VH_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 12, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0141] NI-504.3E7_V6 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 14 (VH_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 14, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7.

[0142] NI-504.3E7_V7 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 14 (VH_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 14, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0143] NI-504.3E7_V8 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 14 (VH_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 14, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0144] NI-504.3E7_V9 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 16 (VH_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 16, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7.

[0145] NI-504.3E7_V10 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 16 (VH_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 16, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0146] NI-504.3E7_V11 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 16 (VH_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 16, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0147] NI-504.3E7_V12 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7.

[0148] NI-504.3E7_V13 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0149] NI-504.3E7_V14 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0150] NI-504.3E7_V15 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60.

[0151] NI-504.3E7_V16 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7.

[0152] NI-504.3E7_V17 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0153] NI-504.3E7_V18 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0154] NI-504.3E7_V19 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60.

[0155] NI-504.3E7_V20 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 22 (VH_3E7_var6) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 22, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0156] NI-504.3E7_V21 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 24 (VH_3E7_var6a) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 24, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0157] NI-504.3E7_V22 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 26 (VH_3E7_var6b) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 26, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0158] NI-504.3E7_V23 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 28 (VH_3E7_var6c) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 28, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0159] NI-504.3E7_V24 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 30 (VH_3E7_var6d) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 30, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0160] NI-504.3E7_V25 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 32 (VH_3E7_var6ab) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 32, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0161] NI-504.3E7_V26 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 34 (VH_3E7_var6ac) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 34, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0162] NI-504.3E7_V27 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 36 (VH_3E7_var6ad) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 36, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58. NI-504.3E7_V28 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 38 (VH_3E7_var6bc) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 38, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0163] NI-504.3E7_V29 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 40 (VH_3E7_var6bd) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 40, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0164] NI-504.3E7_V30 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 42 (VH_3E7_var6cd) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 42, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0165] NI-504.3E7_V31 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 44 (VH_3E7_var6bcd) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 44, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0166] NI-504.3E7_V32 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 46 (VH_3E7_var6acd) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 46, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0167] NI-504.3E7_V33 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 48 (VH_3E7_var6abd) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 48, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0168] NI-504.3E7_V34 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 50 (VH_3E7_var6abc) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 50, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0169] NI-504.3E7_V35 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 22 (VH_3E7_var6) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 22, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0170] NI-504.3E7_V36 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 22 (VH_3E7_var6) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 22, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60.

[0171] NI-504.3E7_V37 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 52 (VH_3E7_var7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 52, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56.

[0172] NI-504.3E7_V38 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 52 (VH_3E7_var7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 52, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0173] NI-504.3E7_V39 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 52 (VH_3E7_var7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 52, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60.

[0174] NI-504.3E7_V40 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 54 (VH_3E7_var8) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 54, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0175] In a preferred embodiment, mutants VH and VL each contain one or more amino acid substitutions and / or deletions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and / or deletions, preferably VL chain mutants contain 1, 2, 3, 4, or 5, preferably 2, 4, or 5 amino acid substitutions and / or deletions, and / or VH chain mutants contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2, 3, 4, 5, 6, 7, 8, or 10 amino acid substitutions and / or deletions. Most preferably, mutants VH and VL each contain one or two amino acid substitutions and / or deletions. For example, mutant VL contains an amino acid deletion in which the N-terminal amino acid is deleted by post-translational modification, and in one embodiment, an amino acid deletion in which the first residue serine is deleted when a signal peptide is deleted.

[0176] In another preferred embodiment, the VH chain and VL chain variants described each contain an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99%, identical to the sequences shown in SEQ ID NOs: 2, 7, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60, respectively. In these embodiments, preferably, one or more of the CDRs as defined by Kabat are kept substantially immutable. However, under the simplified assumption that paratopes correspond to CDRs, Chothia's CDR definitions may be used in addition to or instead of the above, because they correlate very well with the structural loops present in the variable region. Therefore, in order to provide an anti-CRMP2 antibody equivalent to any one of the antibody NI-504.3E7 or variants V1-V40 of the present invention, preferably, at least one or two, most preferably one, of the one or more, preferably two or fewer, amino acid substitutions is made outside the CDR defined by Chothia and / or IMGT, if it is made in a CDR as defined by Kabat, and most preferably outside the overlap of the CDRs as defined by Kabat and Chothia.

[0177] For example, for amino acid substitutions within the amino acid sequences of the CDR, variable heavy chain, variable light chain, and framework, conservative amino acid substitutions are preferably performed according to the most frequently exchanged amino acids, as analyzed and described by, for example, Mirsky et al., Mol. Biol. Evol. 32 (2014), 806-819. See Figure 6 on page 813 of Mirsky et al.'s literature. In particular, S may be substituted with T in VH-CDR1; V may be substituted with E, T with S, and / or M with V in VH-CDR3; R may be substituted with K, R with E, and / or T in VL-CDR1; S may be substituted with A, and / or A with G in VL-CDR2; and P may be substituted with S in VL-CDR3. As described above, preferably, amino acid substitutions belonging to the same category are selected in either or preferably both of models LG and AB shown in Figure 6 of Mirsky et al. (2014), and the LG model is preferred in that it tends to maintain the properties of the amino acids. In this case, the amino acid substitutions are preferably selected such that the physicochemical properties of the original amino acids, i.e., hydrophobicity, polarity, or charge properties are substantially maintained, or, if two or more amino acid substitutions are made, they compensate for each other to give the physicochemical properties of the surface as a whole.

[0178] Of course, in addition to theoretical considerations, there are also experimental approaches to identify CDR variants without requiring a reasonable amount of time or excessive burden. For example, Tiller et al., Front Immunol. 8 (2017), 986 describes the easy affinity maturation of antibody variable domains using natural diversity mutagenesis. In fact, several years earlier, Rajpal et al. reported in PNAS 102 (2005), 8466-8471, a general method for significantly improving antibody affinity by using combinatorial libraries, illustrating their method with the anti-TNF-α antibody D2E7 (HUMIRA®) and identifying 38 substitutions at 21 CDR positions that result in higher affinity binding to TNF-α. More recently, Cannon et al., in PLOS Computational Biology, https: / / doi.org / 10.1371 / journal.pcbi.1006980 (May 1, 2019), described computational antibody affinity maturation based on experiments using de novo docking, modeling, and rational design in silico affinity maturation in combination with alanine scanning. This enabled fine-tuning of the protein-protein docking model, subsequently allowing the identification of two single point mutations that increased the affinity of the hybridoma-derived antibody AB1 for its antigen, mouse CCL20.

[0179] Therefore, each antibody is unique and may have different characteristics; nevertheless, once lead candidates are provided to a person skilled in the art, that person can, by considering the teachings of the present invention disclosed in this application and in light of previously developed computational design and experimental approaches, arrive at an equivalent anti-CRMP2 antibody that retains the desired characteristics of the antibody, such as those described with respect to the anti-CRMP2 antibody exemplified in the Examples and specifically defined in the Claims. In this regard, it is well understood that the mutant antibody substantially maintains the binding specificity of the parent antibody, for example, by binding to the peptide / epitope defined above, by binding to CRMP2 and pCRMP2, by selectively binding to CRMP2 and not specifically binding to CRMP1, CRMP3, CRMP4 and CRMP5, by reducing the level of pCRMP2 in a concentration-dependent manner in phosphorylation assays, by reversing the increase in neuronal spine density induced by CRMP2 when determined in an ex vivo hippocampal section culture model, and / or by competing with the parent antibody, i.e., antibody NI-504.3E7 and any one of the mutants NI-504.3E7_V1~NI-504.3E7_V40, for binding to their respective epitopes.

[0180] As is known in the art, variable heavy chain CDR3 (VH-CDR3) appears to primarily determine antigen specificity. See, for example, Xu and Davis, Immunity 13 (2000), 37-45. In this regard, while the diversity of heavy chain CDR3 drives specificity, it has been noted that VH-CDR1 and VH-CDR2 residues are broadly cross-reactive and readily modified by somatic high-frequency mutations. See Davis, Semin. Immunol. 16 (2004), 239-243. Therefore, in one embodiment, an antibody of the present invention having the immunological characteristics of a reference antibody and capable of competing for its binding to each epitope of CRMP2 comprises in its variable region a VH-CDR3 having at least the VH-CDR3 of the corresponding reference antibody, or a sequence that is at least 90% identical, preferably 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to the reference VH-CDR3. For example, a mutant antibody of a reference antibody may retain the VH-CDR3 of the reference (parent) antibody, but VH-CDR1 and / or VH-CDR2 may contain one or more amino acid substitutions. See above.

[0181] However, in addition to the theoretical and practical considerations presented in the literature, experiments conducted within the scope of the present invention have shown that the generated mutant antibodies (NI-504.3E7_V1 to NI-504.3E7_V40) retain the binding characteristics of the original antibody NI-504.3E7. For example, as shown in Table VII, all mutants still bind to phosphorylated and unphosphorylated CRMP2. Furthermore, the results of experiments conducted within the scope of the present invention have been confirmed for one or more of these mutants.

[0182] Therefore, in a more preferred embodiment, the amino acid substitution is present in the VL-CDR3, preferably where the VL-CDR3 comprises one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two amino acid substitutions, and most preferably one amino acid substitution. In a further preferred embodiment, the amino acid substitution is within the VL-CDR3, preferably at position 91 of the VL chain, and the amino acid substitution is most preferably D91E.

[0183] In another further preferred embodiment, the mutant CDR comprises an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 61, respectively.

[0184] Therefore, in one embodiment, (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 3. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 4. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 5. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 shown in Figure 1 or Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 8. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 shown in Figure 1 or Table III or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 9, and (f) VL-CDR3 includes the amino acid sequence of SEQ ID NO: 10 shown in Figure 1 or Table III, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 10. Regarding NI-504.3E7, which has the following characteristics: The mutant CDR comprises one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two, and most preferably one. In one preferred embodiment, the mutant CDR is VL-CDR3 and comprises one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two, and most preferably one. In a further preferred embodiment, the amino acid substitution is within VL-CDR3, preferably at position 91 of the VL chain, and the amino acid substitution is most preferably D91E.

[0185] In one embodiment, with respect to NI-504.3E7, the mutant CDR contains an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. In a preferred embodiment, the mutant CDR contains an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequence shown in SEQ ID NO: 10, respectively.

[0186] In another preferred embodiment, the VH chain and VL chain variants described each comprise one or more amino acid substitutions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, preferably the VL chain variant comprises 1, 2, 3, 4, or 5 amino acid substitutions, preferably 2, 4, or 5 amino acid substitutions, and / or the VH chain variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, preferably 2, 3, 4, 5, 6, 7, 8, or 10 amino acid substitutions. In one embodiment, the VH chain and VL chain variants described each comprise one or two amino acid substitutions.

[0187] In one embodiment, the listed VH chain variants include the amino acid substitutions T7S, Q19R, P28T, Y29F, V40S, V40A, I71T, M80T, R82Y, H84Q, and / or Y86S. In particular, in one preferred embodiment, the VH chain variant includes the following amino acid substitutions: (i) M80T and Y86S, (ii) V40S, M80T, and Y86S, (iii) I71T, M80T, and Y86S, (iv) V40S, I71T, M80T, and Y86S, (v)V40A, I71T, M80T, and Y86S, (vi) T7S, Q19R, V40A, I71T, M80T, R82Y, H84Q, and Y86S, (vii) Q19R, V40A, I71T, M80T, R82Y, H84Q, and Y86S, (viii) T7S, V40A, I71T, M80T, R82Y, H84Q, and Y86S, (ix) T7S, Q19R, V40A, I71T, M80T, H84Q, and Y86S, (x)T7S, Q19R, V40A, I71T, M80T, R82Y, and Y86S, (xi)V40A, I71T, M80T, R82Y, H84Q, and Y86S, (xii) Q19R, V40A, I71T, M80T, H84Q, and Y86S, (xiii) Q19R, V40A, I71T, M80T, R82Y, and Y86S, (xiv)T7S, V40A, I71T, M80T, H84Q, and Y86S, (xv)T7S, V40A, I71T, M80T, R82Y, and Y86S, (xvi)T7S, Q19R, V40A, I71T, M80T, and Y86S, (xvii)T7S, V40A, I71T, M80T, and Y86S, (xviii) Q19R, V40A, I71T, M80T, and Y86S, (ixx)V40A, I71T, M80T, R82Y, and Y86S, (xx)V40A, I71T, M80T, H84Q, and Y86S, (xxi)T7S, Q19R, V40S, I71T, M80T, R82Y, H84Q, and Y86S, or (xxii)T7S, Q19R, P28T, Y29F, V40A, I71T, M80T, R82Y, H84Q, and Y86S.

[0188] In one embodiment, the listed VL chain variants include the amino acid substitutions L37Q, L69T, N75S, L78Q, and / or D91E. In particular, in one preferred embodiment, the VL variant includes the following amino acid substitutions: (i) L69T and L78Q, (ii) L37Q, L69T, N75S, and L78Q, or (iii) L37Q, L69T, N75S, L78Q, and D91E.

[0189] As shown in Tables II and V, variants of the VH chain of antibody NI-504.3E7 (VH_3E7_var1, VH_3E7_var2, VH_3E7_var3, VH_3E7_var4, VH_3E7_var5, VH_3E7_var6, VH_3E7_var6a, VH_3E7_var6b, VH_3E7_var6c, VH_3E7_var6d, VH_3E7_var6ab, VH_3E7_var6ac, VH_3E7_var6ad, VH The following amino acid substitutions exist in variants of antibody NI-504.3E7 (VH_3E7_var6bc, VH_3E7_var6bd, VH_3E7_var6cd, VH_3E7_var6bcd, VH_3E7_var6acd, VH_3E7_var6abd, VH_3E7_var6abc, VH_3E7_var7, VH_3E7_var8) and in the VL chain (VL_3E7_var1, VL_3E7_var2, VL_3E7_var3). Preferred assignments for the variant VH and VL chains are shown in Table IV.

[0190] In one embodiment, the described VH chain and VL chain variants each include one or two amino acid substitutions, preferably one of those listed above. In another preferred embodiment, the described VH chain and VL chain variants each include an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99%, identical to the sequences shown in SEQ ID NOs: 2, 7, 12, and 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

[0191] With respect to antibody NI-504.3E7, the described VH chain and VL chain variants each contain an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 7, respectively.

[0192] Preferably, however, the antibody of the present invention contains, on one or both of its immunoglobulin chains, one, two or three CDRs of the variable regions shown in Figure 1 and Tables II and III, or one, two or three CDRs that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the CDRs of the variable regions shown in Figure 1 and Table III. In addition to or instead of the above, one or more FRs derived from the framework region (FR) are 80% identical to the corresponding FRs (these are located between the CDRs shown in Figure 1 and Table II), and preferably 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the FRs that are sequences between the underlined CDRs in Figure 1 and Table II. In some embodiments, there are one, two, three, or all four FRs (each at least 90%, 90-95%, and / or 95-99% identical to the FRs shown in Figure 1 and Table II).

[0193] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0194] [Table 3]

[0195] [Table 4]

[0196] [Table 5]

[0197] In a further additional or alternative embodiment of the present invention, anti-CRMP2 antibodies, their antigen-binding fragments, and synthetic or bioengineered variants can be optimized to have appropriate binding affinity and stability to the target. Thus, at least one amino acid in the CDR or variable region that is susceptible to modification selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, iso-aspartic acid formation, and / or unpaired cysteine ​​is replaced by a mutant amino acid lacking such modification, or at least one carbohydrate moiety is chemically or enzymatically removed or added to the antibody. See, for example, Liu et al., J. Pharm. Sci. 97 (2008), 2426-2447, Beck et al., Nat. Rev. Immunol. 10 (2010), 345-352, and Haberger et al., MAbs. 6 (2014), 327-339.

[0198] The immunoglobulin or the cDNA encoding it may be further modified. Therefore, in a further embodiment, the method of the present invention includes one of the steps of producing a chimeric antibody, a murine antibody, a single-chain antibody, a Fab fragment, a bispecific antibody, a fusion antibody, a labeled antibody, or an analogue thereof. Corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual" (CSH Press, Cold Spring Harbor (1988), 1st edition; 2nd edition by Edward A. Greenfield, Dana-Farber Cancer Institute (copyright) 2014, ISBN 978-1-936113-81-1). For example, Fab fragments and F(ab')2 fragments may be produced recombinantly or by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (for producing Fab fragments) or pepsin (for producing F(ab')2 fragments). The F(ab')2 fragment contains a variable region, a light chain constant region (CL), and a heavy chain CH1 domain. Such fragments are sufficient for use in immunodiagnostic procedures, for example, that involve coupling the immunospecific portion of an immunoglobulin with a detection reagent such as a radioisotope.

[0199] Accordingly, in one embodiment, the antibody of the present invention may be provided in a format selected from the group consisting of single-chain Fv fragments (scFv), F(ab') fragments, F(ab) fragments, and F(ab')2 fragments, Fd, Fv, single-chain antibodies, disulfide-stabilized Fv (dsFv), and nanobodies, and / or in a format as a chimeric mouse-human antibody or a mouse-derived antibody.

[0200] As described above, CRMP2 has been shown to co-localize with hyperphosphorylated tau and is therefore present in the corresponding intracellular neurofibrillary tangles. Thus, in one embodiment, it may be beneficial to use recombinant Fab (rFab) and scFv anti-tau antibodies that can more easily permeate the cell membrane.

[0201] Immunotherapeutic approaches using various antibody formats such as scFv, single-domain antibody fragments (VHH or sdAb), bispecific antibodies, intrabodies and nanobodies have shown therapeutic efficacy in several animal models of Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), Huntington's disease (HD), transmissible spongiform encephalopathy (TSE) and multiple sclerosis (MS). Recombinant antibody fragments have been demonstrated to neutralize toxic extracellular and intracellular misfolded proteins involved in the pathogenesis of neurodegenerative diseases and thus can be promising tools for developing antibody-based immunotherapeutics for these diseases. See the review by Manoutcharian et al., Curr Neuropharmacol 15(2017),779-788. Advantages recognized for using small Fab and scFv engineered antibody formats lacking effector functions include more efficient passage across the blood-brain barrier and minimizing the risk of triggering inflammatory side reactions. Additionally, scFv and single-domain antibodies can be expressed as a single gene and as intrabodies within mammalian cells, in addition to maintaining the binding specificity of full-length antibodies, and may alter the folding, interaction, modification, or intracellular localization of their respective targets. See, for example, Miller and Messer, Molecular Therapy 12(2005),394-401 for an overview. Further, alternative CRMP2-binding molecules such as designed ankyrin repeat proteins (DARPin) are provided. See amyloid Β peptide-specific DARPin described as a novel class of potential therapeutics for Alzheimer's disease in Hanenberg et al., J Biol Chem.289(2014),27080-27089. However, as exemplified in the examples, according to the present invention, IgG antibodies are preferably used.

[0202] The five major classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. These are distinguished by the type of heavy chain found in the molecule. IgG molecules have a heavy chain known as the gamma chain, IgM has a mu chain, IgA has an alpha chain, IgE has an epsilon chain, and IgD has a delta chain. For an overview, see, for example, Schroeder et al., Structure and function of immunoglobulins. J. Allergy Clin. Immunol. 125 (2010), S41 - S52. Further, there are different subclasses, IgA is further divided into subclasses IgA1 and IgA2, and IgG is further divided into subclasses IgG1, IgG2, IgG3, and IgG4. Additionally, there are two types of light chains, kappa (κ) and lambda (λ).

[0203] In principle, the antibodies of the present invention may be of any class and subclass and may contain any type of light chain, provided that, as shown for example in Table VI or VII and exemplified in the attached examples for the corresponding reference antibodies, the binding specificity for CRMP2 is maintained essentially unaffected. However, preferably intact IgG antibodies are used, in which case the antibody contains a constant domain. Thus, in one embodiment, the immunoglobulin heavy chain and / or light chain constant domain present in the antibody of the present invention is of the IgG type, IgM type, IgA type, IgD type or IgE type, preferably of the IgG type. In one embodiment, the immunoglobulin heavy chain and / or light chain constant domain present in the antibody of the present invention is of the IgA1, IgA2, IgG1, IgG2, IgG3 or IgG4 subclass, preferably of the IgG1, IgG2, IgG3 or IgG4 subclass, most preferably of the IgG2 subclass. In one embodiment, the light chain is a kappa (κ) or lambda (λ) light chain.

[0204] The constant domain may be native, i.e., originally cloned together with the variable domain, or it may be heterologous, for example, a mouse constant domain if animal testing is anticipated. In this case, the original isolated antibody was an IgG2 antibody. Here, the recombinant human-derived antibody of the present invention is preferably of the IgG4 type or IgG1 type, and preferably of the IgG1 type. Therefore, preferably, the constant domain is of human origin, of a different IgG subtype (e.g., IgG4 for IgG2, or IgG1 for IgG2) or of different allotypes and alleles, compared to the constant domain of the antibody as it naturally occurs in humans. The definition of "allotype" requires that antibody reagents for serological determination of the allotype are available. If the determination is made only at the sequence level, the polymorphism should be described as "allele." This does not prevent the establishment of an allele-allotype correspondence when the allele-allotype correspondence has been experimentally demonstrated, or when the individual sequence is identical to a sequence for which a correspondence with an allotype has been demonstrated.

[0205] In one preferred embodiment of the present invention, the constant domain is heterologous to the CDR and at least one of the VH and VL chains, for example, an immunoglobulin heavy chain constant domain and / or an immunoglobulin light chain constant domain (preferably of the IgG type). In addition to or instead of the above, the heterologous portion of the antibody may be a mammalian secreted signaling peptide. In other words, in one embodiment, the anti-CRMP2 antibody and CRMP2 binding fragment or derivative thereof of the present invention is a fusion protein comprising (i) a polypeptide sequence heterologous to the VH region and / or VL region or at least one CDR, and / or (ii) a non-natural variant of an immunoglobulin-derived polypeptide comprising a heavy chain constant domain comprising one or more amino acid deletions, substitutions and / or additions compared to the wild-type polypeptide. For example, the human constant domain of the recombinant human-derived antibody of the present invention may be a different IgG isotype or a different allotype from the constant domain of a parental antibody naturally produced by memory B cells, for example, to avoid or reduce immunogenicity that may result from alloimmunization. For an overview, see, for example, Jefferis and Lefranc, MAbs 1 (2009), 332-338. Therefore, in one embodiment, if the parent antibody is of the IgG1 subclass, the human constant domain of the recombinant human-derived antibody of the present invention is of the IgG2, IgG3, or IgG4 subclass. In one embodiment, if the parent antibody is of the IgG2 subclass, the human constant domain of the recombinant human-derived antibody of the present invention is of the IgG1, IgG3, or IgG4 subclass. In one embodiment, if the parent antibody is of the IgG3 subclass, the human constant domain of the recombinant human-derived antibody of the present invention is of the IgG1, IgG2, or IgG4 subclass. In one embodiment, if the parent antibody is of the IgG4 subclass, the human constant domain of the recombinant human-derived antibody of the present invention is of the IgG1, IgG2, or IgG3 subclass.In one preferred embodiment, the parent antibody is an IgG2 subclass, and therefore the human constant domain of the recombinant human-derived antibody of the present invention is an IgG1, IgG3, or IgG4 subclass, preferably an IgG1 or IgG4 subclass, most preferably an IgG1 subclass or isotype.

[0206] In an alternative or further embodiment, if the parent antibody contains a kappa (κ) light chain, the light chain of the recombinant human antibody of the present invention can be changed to a lambda (λ) light chain, or if the parent antibody contains a lambda (λ) light chain, the light chain of the recombinant human antibody of the present invention can be changed to a kappa (κ) light chain.

[0207] In addition to the four subclasses of IgG mentioned above, human heavy and light chain genes exhibit extensive structural polymorphisms, are closely linked, and are inherited as haplotypes. Allotype variants are immunogenic and may elicit an antibody response as a result of alloimmunity. Therefore, allotype switching can be of particular interest for providing non-immunogenic antibody therapeutics. To date, a wide range of allotypes (polymorphisms) have been known, but the focus is on serologically defined allotypes. Allotypes of the IgG protein are defined by the expression of a unique epitope recognized by a unique serum reagent. Allotypes expressed on the constant region of the IgG heavy chain are designated as Gm (genetic marker) with a subclass (e.g., G1m) and an allotype number (or letter) (e.g., G1m1 [or G1m(a)], G3m5 [or G3m(b1)]). Human immunoglobulin allotypes are listed in Table 1 of Jefferis and Lefrance, mAbs 1 (2009), 1-7 and in Fig. 1A of Irani et al., Molecular Immunology 67 (2015), 171-182, the contents of which are incorporated herein by reference. Accordingly, in one embodiment, the antibody of the present invention is one of the following allotypes, but is not limited to them: G1m1, G1m2, G1m3, G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, A2m3, Em1, Km1, Km2, and Km3. In one embodiment, if the parent antibody is one of the allotypes listed above, the recombinant human-derived antibody of the present invention is one of the above allotypes, excluding that of the parent antibody.

[0208] The four subclasses, IgG1, IgG2, IgG3, and IgG4, are highly conserved, differing in their constant regions, particularly the hinge and upper CH2 domain. These regions are involved in both binding to the IgG-Fc receptor (FcgR) and binding to C1q. As a result, the different subclasses have different effector functions, both in triggering FcgR-expressing cells to induce phagocytosis or antibody-dependent cell-mediated cytotoxicity, and in activating complement. The Fc region also contains a binding epitope for the neonatal Fc receptor (FcRn), which is responsible for prolonging half-life, placental transport, and bidirectional transport of IgG across the mucosal surface. However, FcRn is also expressed in myeloid cells and, along with classical FcgR and complement, is involved in both phagocytosis and antigen presentation. The effects of these characteristics, IgG polymorphisms, and post-translational modifications in the form of glycosylation on IgG function are described in Vidarsson et al., Front.Immunol.5(2014),520.doi:10.3389 / fimmu.2014.00520 and de Taeye et al., Antibodies 2019,8,30;doi:10.3390 / antib8020030. Preferably, the constant domains of the immunoglobulin heavy chain and / or light chain present in the antibody of the present invention are of the IgG type.

[0209] Therefore, in certain embodiments of the present invention, it is preferable that a particular IgG type, such as an IgG1 isotype and / or the constant region of the antibody, or the constant region of its antigen-binding fragment, variant, or derivative, is modified to give a desired biochemical characteristic. In particular, in one embodiment, the Fc portion of the antibody can be mutated using techniques known in the art to alter, i.e., decrease or increase, its immunoeffector function or increase its half-life. Therefore, in one embodiment, the Fc portion of the antibody is mutated to decrease its immunoeffector function, in another embodiment, the Fc portion of the antibody is mutated to increase its immunoeffector function, and in yet another embodiment, the antibody is mutated to increase its half-life.

[0210] For example, constant region modifications consistent with the present invention would relax complement binding, thereby reducing the serum half-life and nonspecific association of conjugated cytotoxins. Disulfide bond or oligosaccharide moiety modifications may be used with other constant region modifications that enable enhanced tissue-antigen interactions by increasing antigen specificity or antibody flexibility. Furthermore, mutations leading to enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent phagocytosis (ADCP) can also be added to the Fc region, by increasing FcγRIIIa binding and / or decreasing FcγRIIIb binding, and by increasing FcγRIIa binding and / or FcγRIIIa binding, respectively. For example, the GASDALIE Fc variant (G236A / S239D / A330L / I332E) exhibits increased affinity for FcγRIIIa. Another possibility is the enhancement of complement-dependent cytotoxicity (CDC) by increasing C1q binding and / or hexamerization.

[0211] In another embodiment, certain antibodies for use in the diagnostic and therapeutic methods described herein have a constant region, such as the IgG heavy chain constant region, that has been modified to eliminate glycosylation (referred to as non-glycosylated antibodies or "agly" antibodies in other sections herein). Such "agly" antibodies can be prepared by enzymatic preparation and by engineering manipulation of consensus glycosylation sites within the constant region. "Agly" antibodies are thought to have reduced effector function and therefore improved in vivo safety and stability profiles. Methods for generating non-glycosylated antibodies with desired effector function can be found, for example, in International Patent Publication No. 2005 / 018572A2 (this document is incorporated in its entirety by reference). Further approaches to reducing the effector function of antibodies involve reducing FcγR and C1q binding by mutations in the Fc region.

[0212] An overview is provided, for example, in the review article Wang et al., Protein Cell 9 (2018), 63-73, which includes Table 1 of examples of modifications to alter antibody effector function and antibody half-life. The mutations described in this article are incorporated herein by reference. The resulting physiological profiles, bioavailability, and other biochemical effects of the modifications (such as CRMP2 recognition and binding, in vivo distribution, and serum half-life) can be readily measured and quantified using well-known immunological techniques without excessive experimentation.

[0213] As mentioned above, in some cases, the inflammatory response should be avoided, and therefore the effector function of the antibody's constant domain may be weakened or completely eliminated. For example, when reduced effector function is desirable, IgG4 antibodies are potential candidates for immunotherapy. IgG4 Abs are dynamic molecules that can undergo a process known as Fab-arm exchange (FAE). This results in functional monovalent bispecific antibodies (bsAbs) with unknown specificity, and therefore potentially leads to reduced therapeutic efficacy. However, the stabilizing S228P mutation can prevent IgG4 FAE and thus restore the therapeutic usefulness of IgG4 antibodies. See Silva et al., J Biol Chem 290 (2015), 5462-5469. Furthermore, recombinant human IgG antibodies (hIgG) that completely lack binding to the Fcγ receptor (FcγR) and complement protein C1q, and therefore lose their immunoeffector function, are useful for a variety of therapeutic applications. The combination of Leu234Ala and Leu235Ala (commonly known as the LALA mutation) or the SPLE mutation has been shown to eliminate FcγRIIa binding and detectable binding to FcγRI, IIa, and IIIa for both IgG1 and IgG4, and the LALA-PG mutation has been found to be an improvement over the LALA mutation alone in that it inactivates Fc function in mouse and human IgG. For corresponding reviews, see, for example, Saunders, Front.Immunol.10(2019),1296.doi:10.3389 / fimmu.2019.01296 and Schlothauer et al., Protein Engineering, Design and Selection 29(2016),457-466.

[0214] Another early approach to reducing effector function is to mutate the glycosylation site of N297 with mutations such as N297A, N297Q, and N297G. The antibody half-life can be increased by introducing the following mutations: M252Y / S254T / T256E or M428L / N434S. See Wang et al., 2018.

[0215] As shown in Examples 13 and 14, administration of antibody NI-504-3E7 led to improved long-term potentiation (LTP) and cognitive function in aged mice. The same was observed with antibody NI-504.3E7 containing the LALA-PG mutation. Therefore, IgG1 effector function does not appear to be necessary or decisive for the observed biological effects of this antibody. This is consistent with the finding that antibody NI-504.3E7 can detect CRMP2 intracellularly (where effector function is not required), as demonstrated in Example 12 using fully differentiated SH-SY5Y cells. Therefore, in one embodiment, the antibody of the present invention has reduced Fc function or no Fc function. In one embodiment, the antibody of the present invention is an IgG4 class or IgG1 class having one of the above-mentioned mutations. In a preferred embodiment, the antibody of the present invention is an IgG4 class, preferably containing the S228P mutation. In another preferred embodiment, the antibody of the present invention is an antibody of an IgG1 subclass having an LALA mutation, most preferably the LALA-PG mutation.

[0216] The present invention also relates to antibody or antigen-binding fragments comprising heterologous polypeptides. For example, the antibody polypeptides of the present invention comprise, essentially consist of, or may consist of fusion proteins. A fusion protein is, for example, a chimeric molecule comprising an immunoglobulin CRMP2-binding domain having at least one target-binding site and at least one heterologous portion, i.e., a portion not naturally linked in nature. Their amino acid sequences are typically present in separate proteins that are combined in the fusion polypeptide, or are typically present in the same protein but may be in a new configuration in the fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis, or by creating and translating polynucleotides encoded so that the peptide regions are in a desired relationship.

[0217] When the term “heterogeneous” is applied to a polynucleotide or polypeptide, it means that the polynucleotide or polypeptide originates from an entity different from the rest of the entity being compared. For example, as used herein, a “heterogeneous polypeptide” fused to an antibody or its antigen-binding fragment, variant, or analog originates from a non-immunoglobulin polypeptide of the same species or an immunoglobulin polypeptide or non-immunoglobulin polypeptide of a different species.

[0218] As described above, chimeric molecules constitute fusion proteins. The antibodies of the present invention may be chimeric rodent-human antibodies, preferably chimeric mouse-human antibodies, which are particularly useful for diagnostic methods and studies in animals. In one embodiment, the antibody of the present invention is preferably a chimeric mouse-human antibody containing a mouse constant region, most preferably a mouse IgG2a constant region. The production of chimeric antibodies is described, for example, in International Patent Publication No. 89 / 09622.

[0219] The present invention also relates to one or more polynucleotides encoding the antibody of the present invention, its antigen-binding fragment or derivative, or its immunoglobulin VH and VL, wherein the polynucleotide is preferably cDNA.

[0220] In one preferred embodiment of the present invention, the polynucleotide comprises, essentially comprises, or consists of a nucleic acid having a polynucleotide sequence encoding the VH chain or VL chain of an anti-CRMP2 antibody, as shown in Table II. In this regard, it will be readily apparent to those skilled in the art that the polynucleotide encoding the light chain and / or heavy chain may be encoded by one or more polynucleotides. Thus, in one embodiment, the polynucleotide comprises, essentially comprises, or consists of a nucleic acid having polynucleotide sequences of the VH chain and VL chain of an anti-CRMP2 antibody, as shown in Table II.

[0221] In one embodiment of the present invention, a polynucleotide is ligated to a heterogeneous nucleic acid, such as an expression regulatory sequence including a promoter, a transcription and / or translation enhancer sequence, an internal ribosome binding site, or a nucleic acid encoding a peptide reader sequence for recombinant expression in a host.

[0222] Therefore, the present invention relates to polynucleotides encoding anti-CRMP2 antibodies, preferably in a preferred embodiment, human recombinant anti-CRMP2 antibodies or CRMP2-binding fragments, or derivatives thereof, wherein the polynucleotide is VH chains containing CDR1, 2, and 3 as defined by Kabat, and / or VL chains containing VL CDR1, 2, and 3. Code this, and here, (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 shown in Figure 1 and Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 3. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 shown in FIG. 1 and Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial modification of SEQ ID NO: 4. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 shown in FIG. 1 and Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial modification of SEQ ID NO: 5. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 shown in FIG. 1 and Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial modification of SEQ ID NO: 8. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 shown in FIG. 1 and Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial modification of SEQ ID NO: 9, and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 shown in FIG. 1 and Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial modification of SEQ ID NO: 10.

[0223] In one preferred embodiment, the mutant CDR contains one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two, most preferably one. In one preferred embodiment, only the VL-CDR contains one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two, most preferably one. In a further preferred embodiment, the amino acid substitution is within the VL-CDR3, preferably at position 91 of the VL chain, and the amino acid substitution is most preferably D91E. In another preferred embodiment, the mutant CDR contains an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequences shown in SEQ ID NOs. 3, SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 8, SEQ ID NOs. 9, and SEQ ID NOs. 10, respectively. In a preferred embodiment, the mutant CDR comprises an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequence shown in SEQ ID NO: 10.

[0224] The present invention further relates to polynucleotides encoding anti-CRMP2 antibodies, preferably in a preferred embodiment, human recombinant anti-CRMP2 antibodies or CRMP2-binding fragments, or derivatives thereof, wherein the polynucleotide is (i) VH chains containing CDR1, 2, and 3 as defined by Kabat, and / or VL chains containing VL CDR1, 2, and 3 Code this, and here, (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 shown in Figure 1 and Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 3. (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 shown in Figure 1 and Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 4. (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 shown in Figure 1 and Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 5. (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 shown in Figure 1 and Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 8. (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 shown in Figure 1 and Table III, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 9, and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 61 shown in Figure 1 and Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial modification of SEQ ID NO: 61.

[0225] In one preferred embodiment, the variant CDR comprises one or more amino acid substitutions, preferably one, two, three, or four, preferably one or two amino acid substitutions, most preferably one amino acid substitution. In another preferred embodiment, the variant comprises an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 61, respectively.

[0226] In a further or alternative embodiment, the present invention relates to a polynucleotide encoding an anti-CRMP2 antibody, preferably a human recombinant anti-CRMP2 antibody or CRMP2-binding fragment, or a derivative thereof, wherein the polynucleotide is This encodes the VH chain and / or VL chain, where, (a) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 2, and / or (b) VL includes the amino acid sequence shown in SEQ ID NO: 7 in Figure 1 or Table II, or a variant thereof, wherein the variant includes an amino acid sequence resulting from a partial modification of SEQ ID NO: 7.

[0227] In another preferred embodiment, the mutant VH and VL chains each contain one or more amino acid substitutions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, preferably the VL chain mutant contains 1, 2, 3, 4, or 5 amino acid substitutions, preferably 2, 4, or 5 amino acid substitutions, and / or the VH chain mutant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, preferably 2, 3, 4, 5, 6, 7, 8, or 10 amino acid substitutions. Most preferably, the mutant VH and VL each contain one or two amino acid substitutions.

[0228] In one embodiment, the listed VH chain variants include the amino acid substitutions T7S, Q19R, P28T, Y29F, V40S, V40A, I71T, M80T, R82Y, H84Q, and / or Y86S. In particular, in one preferred embodiment, the VH chain variant includes the following amino acid substitutions: (i) M80T and Y86S, (ii) V40S, M80T, and Y86S, (iii) I71T, M80T, and Y86S, (iv) V40S, I71T, M80T, and Y86S, (v)V40A, I71T, M80T, and Y86S, (vi) T7S, Q19R, V40A, I71T, M80T, R82Y, H84Q, and Y86S, (vii) Q19R, V40A, I71T, M80T, R82Y, H84Q, and Y86S, (viii) T7S, V40A, I71T, M80T, R82Y, H84Q, and Y86S, (ix) T7S, Q19R, V40A, I71T, M80T, H84Q, and Y86S, (x)T7S, Q19R, V40A, I71T, M80T, R82Y, and Y86S, (xi)V40A, I71T, M80T, R82Y, H84Q, and Y86S, (xii) Q19R, V40A, I71T, M80T, H84Q, and Y86S, (xiii) Q19R, V40A, I71T, M80T, R82Y, and Y86S, (xiv)T7S, V40A, I71T, M80T, H84Q, and Y86S, (xv)T7S, V40A, I71T, M80T, R82Y, and Y86S, (xvi)T7S, Q19R, V40A, I71T, M80T, and Y86S, (xvii)T7S, V40A, I71T, M80T, and Y86S, (xviii) Q19R, V40A, I71T, M80T, and Y86S, (ixx)V40A, I71T, M80T, R82Y, and Y86S, (xx)V40A, I71T, M80T, H84Q, and Y86S, (xxi)T7S, Q19R, V40S, I71T, M80T, R82Y, H84Q, and Y86S, or (xxii)T7S, Q19R, P28T, Y29F, V40A, I71T, M80T, R82Y, H84Q, and Y86S.

[0229] In one embodiment, the listed VL chain variants include the amino acid substitutions L37Q, L69T, N75S, L78Q, and / or D91E. In particular, in one preferred embodiment, the VL variant includes the following amino acid substitutions: (i) L69T and L78Q, (ii) L37Q, L69T, N75S, and L78Q, or (iii) L37Q, L69T, N75S, L78Q, and D91E.

[0230] As shown in Tables II and V, variants of the VH chain of antibody NI-504.3E7 (VH_3E7_var1, VH_3E7_var2, VH_3E7_var3, VH_3E7_var4, VH_3E7_var5, VH_3E7_var6, VH_3E7_var6a, VH_3E7_var6b, VH_3E7_var6c, VH_3E7_var6d, VH_3E7_var6ab, VH_3E7_var6ac, VH_3E7_var6ad, VH The following amino acid substitutions exist in variants of antibody NI-504.3E7 (VH_3E7_var6bc, VH_3E7_var6bd, VH_3E7_var6cd, VH_3E7_var6bcd, VH_3E7_var6acd, VH_3E7_var6abd, VH_3E7_var6abc, VH_3E7_var7, VH_3E7_var8) and in the VL chain (VL_3E7_var1, VL_3E7_var2, VL_3E7_var3). Preferred assignments for the variant VH and VL chains are shown in Table IV.

[0231] In another preferred embodiment, the VH chain and VL chain variants described respectively contain amino acid sequences that are at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical to the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 7, respectively.

[0232] In one embodiment, the present invention relates to a polynucleotide encoding an anti-CRMP2 antibody, preferably a human recombinant anti-CRMP2 antibody or CRMP2-binding fragment, or a derivative thereof, wherein the polynucleotide encodes a VH chain and / or VL chain, NI-504.3E7_V1 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 (VH_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 2, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V2 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 2 (VH_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 2, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V3 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 12 (VH_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 12, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7; NI-504.3E7_V4 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 12 (VH_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 12, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V5 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 12 (VH_3E7_var1) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 12, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V6 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 14 (VH_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 14, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7; NI-504.3E7_V7 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 14 (VH_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 14, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V8 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 14 (VH_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 14, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V9 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 16 (VH_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 16, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7; NI-504.3E7_V10 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 16 (VH_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 16, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V11 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 16 (VH_3E7_var3) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 16, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V12 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7; NI-504.3E7_V13 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V14 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V15 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 18 (VH_3E7_var4) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 18, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60; NI-504.3E7_V16 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 7 (VL_3E7) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 7; NI-504.3E7_V17 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V18 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V19 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 20 (VH_3E7_var5) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 20, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60; NI-504.3E7_V20 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 22 (VH_3E7_var6) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 22, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V21 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 24 (VH_3E7_var6a) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 24, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V22 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 26 (VH_3E7_var6b) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 26, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V23 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 28 (VH_3E7_var6c) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 28, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V24 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 30 (VH_3E7_var6d) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 30, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V25 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 32 (VH_3E7_var6ab) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 32, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V26 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 34 (VH_3E7_var6ac) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 34, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V27 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 36 (VH_3E7_var6ad) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 36, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V28 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 38 (VH_3E7_var6bc) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 38, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V29 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 40 (VH_3E7_var6bd) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 40, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V30 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 42 (VH_3E7_var6cd) shown in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 42, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V31 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 44 (VH_3E7_var6bcd) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 44, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V32 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 46 (VH_3E7_var6acd) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 46, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V33 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 48 (VH_3E7_var6abd) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 48, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V34 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 50 (VH_3E7_var6abc) in Figure 1 or Table II, or a variant thereof, wherein the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 50, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V35 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 22 (VH_3E7_var6) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 22, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V36 (i) The VH chain comprises the amino acid sequence shown in SEQ ID NO: 22 (VH_3E7_var6) in Figure 1 or Table II, or a variant thereof, where the variant comprises the amino acid sequence resulting from a partial modification of SEQ ID NO: 22, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60; NI-504.3E7_V37 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 52 (VH_3E7_var7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 52, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 56 (VL_3E7_var1) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 56; NI-504.3E7_V38 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 52 (VH_3E7_var7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 52, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58; NI-504.3E7_V39 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 52 (VH_3E7_var7) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 52, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 60 (VL_3E7_var3) in Figure 1 or Table II, or a variant thereof, wherein the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 60; NI-504.3E7_V40 (i) The VH chain includes the amino acid sequence shown in SEQ ID NO: 54 (VH_3E7_var8) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 54, and / or (ii) VL includes the amino acid sequence shown in SEQ ID NO: 58 (VL_3E7_var2) in Figure 1 or Table II, or a variant thereof, where the variant includes the amino acid sequence resulting from a partial modification of SEQ ID NO: 58.

[0233] In another preferred embodiment, the mutant VH and VL chains each contain one or more amino acid substitutions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, preferably the VL chain mutant contains 1, 2, 3, 4, or 5 amino acid substitutions, preferably 2, 4, or 5 amino acid substitutions, and / or the VH chain mutant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, preferably 2, 3, 4, 5, 6, 7, 8, or 10 amino acid substitutions. Most preferably, the mutant VH and VL each contain one or two amino acid substitutions.

[0234] In another preferred embodiment, the VH chain and VL chain variants described each contain an amino acid sequence that is at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% identical to the sequence shown in SEQ ID NOs: 2, 7, 12, and 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60.

[0235] In addition, the present invention relates to polynucleotides linked to heterologous nucleic acids, selected from the group consisting of the following: (a) A polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, comprising heavy chain variable regions (VH) containing CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 3, 4, and 5, respectively, wherein the VH binds to CRMP2 when paired with a light chain variable region (VL) containing the amino acid sequences shown in SEQ ID NOs. 7, 56, 58, or 60; (b) A polynucleotide encoding an immunoglobulin light chain or fragment thereof, comprising VLs containing CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, or SEQ ID NOs. 8, 9, and 61, respectively, wherein VLs bind to CRMP2 when paired with VHs containing amino acid sequences shown in SEQ ID NOs. 2, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, or 54; (c) (i) an immunoglobulin heavy chain or fragment thereof containing VH, which includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs: 3, 4, and 5, respectively, (ii) an immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, or SEQ ID NOs. 8, 9, and 61, respectively, Polynucleotides that code for (d) A polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, comprising VH containing the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, or SEQ ID NO: 54, wherein VH is a polynucleotide that binds to CRMP2 when paired with VL containing the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 58, or SEQ ID NO: 60; (e) A polynucleotide encoding an immunoglobulin light chain or fragment thereof, comprising a VL containing the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 58, or SEQ ID NO: 60, wherein the VL binds to CRMP2 when paired with a VH containing the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, or SEQ ID NO: 54; (f) Polynucleotides encoding an immunoglobulin heavy chain or fragment thereof containing VH having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, or SEQ ID NO: 54, and an immunoglobulin light chain or fragment thereof containing VL having the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 58, or SEQ ID NO: 60; (g) A polynucleotide according to any one of (a) to (f), wherein the CDR comprises one or more, preferably two or fewer, amino acid substitutions, and / or the variable region sequence is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, or SEQ ID NO: 54 or SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 58, or SEQ ID NO: 60.

[0236] The assignment of sequence numbers to the corresponding CDRs and VH and VL chains is shown in Tables III and II, and preferred combinations of VH and VL chains are shown in Table IV.

[0237] In one preferred embodiment, immunoglobulins, when paired as VH and VL, can bind to CRMP2 when measured by direct ELISA, capture ELISA, or an assay equivalent to the assay described in the Examples.

[0238] Furthermore, the present invention relates to one or more vectors comprising one or more of these polynucleotides, preferably wherein the vector is an expression vector, and the one or more polynucleotides are operably ligated to an expression control sequence.

[0239] Polynucleotides can be generated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, and PCR (see, for example, "Molecular Cloning: A Laboratory Manual" (4th edition): 3-volume set; Green and Sambrook (2012) ISBN 10:1936113422 / ISBN 13:9781936113422 Cold Spring Harbor Laboratory Press; updated edition (2014) ISBN 978-1-936113-42-2 and Ausubel et al., "Current Protocols in Molecular Biology" John Wiley & Sons, NY (1998) and updated editions. Both of these are incorporated herein by reference in their entirety) and, if necessary, can be manipulated to produce antibodies with different amino acid sequences, for example, to create amino acid substitutions, deletions, and / or insertions.

[0240] Once a polynucleotide encoding the antibody molecule of the present invention, or the heavy or light chain of the antibody, or a portion thereof (preferably containing the variable domain of the heavy or light chain), is obtained, a vector for producing the antibody molecule can be generated by recombinant DNA technology using techniques well known in the art. Accordingly, a method for preparing a protein by expressing a polynucleotide containing the nucleotide sequence encoding an antibody is described herein. An expression vector containing the antibody-coding sequence and appropriate transcription and translation control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention provides a replicable vector containing a nucleotide sequence encoding the antibody molecule of the present invention, or its heavy or light chain, or the variable domain of the heavy or light chain, operably linked to a promoter. Such a vector may contain a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, International Publication No. 86 / 05807A1 and International Publication No. 89 / 01036A1 and U.S. Patent No. 5,122,464). Furthermore, the variable domain of an antibody can be cloned into such a vector in order to express the entire heavy chain or the entire light chain.

[0241] The terms “vector” or “expression vector” are used herein to mean a vector used in accordance with the present invention as a vehicle for introducing a desired gene into a host cell and expressing it. Generally, a vector suitable for the present invention would include a selectable marker, a suitable restriction site to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells. The marker may confer protrophotrophy to a trophic host, or biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be introduced into the same cell by direct ligation to the DNA sequence to be expressed, or by co-transformation. Further elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splice signals, and transcription promoters, enhancers, and termination signals. To express a double-chain antibody, a single vector or multiple vectors encoding both the heavy and light chains may be co-expressed in host cells to express the entire immunoglobulin molecule, as detailed below.

[0242] Host cells may be co-transfected with two expression vectors of the present invention: a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. These two vectors may contain identical selectable markers enabling equal expression of the heavy chain polypeptide and the light chain polypeptide. Alternatively, a single vector encoding both the heavy chain polypeptide and the light chain polypeptide may be used. In such cases, it is advantageous to place the light chain before the heavy chain to avoid an excess of toxic free heavy chain. See Proudfoot, Nature 322 (1986), 52 and Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy and light chains may include cDNA or genomic DNA. Antibodies for use in the methods described herein are produced by transferring the expression vectors into host cells using conventional techniques and then culturing the transfected cells using conventional techniques. Thus, the present invention also relates to host cells comprising one or more polynucleotides or one or more vectors of the present invention.

[0243] As used herein, “host cell” refers to a cell containing a vector encoding at least one heterologous gene constructed using recombinant DNA techniques. In describing the process for isolating antibodies from a recombinant host, the terms “cell” and “cell culture” are used interchangeably to indicate the source of the antibody unless otherwise explicitly specified. In other words, the recovery of polypeptides from “cells” may mean recovery from whole cells after centrifugation, or recovery from a cell culture containing both culture medium and suspended cells.

[0244] Antibodies used in laboratory research / diagnosis can be expressed in any suitable host, such as mammalian cells, bacterial cells, yeast, plant cells, or insect cells. However, currently, almost all therapeutic antibodies are still produced in mammalian cell lines to reduce the risk of immunogenicity due to altered non-human glycosylation patterns. However, recent developments in glycosylation-engineered yeast, insect cell lines, and transgenic plants offer the prospect of obtaining antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments, including bispecific antibodies that are not glycosylated at all, are being successfully produced in bacteria and are progressing to clinical trials. The first therapeutic antibody products from non-mammalian sources can be expected in the coming years. A review of current antibody production systems that can be applied to prepare the human recombinant anti-CRMP2 antibody or its CRMP2-binding fragment or derivative of the present invention, including their usefulness for various applications, is published in Frenzel et al., Front Immunol. 4 (2013), 217 (published online on July 29, 2013, doi:10.3389 / fimmu.2013.00217). Transient expression of human antibodies in mammalian cells is described in Vazquez-Lombardi et al., Nature protocols, 13 (2018), 99-117 and Hunter et al., Optimization of protein expression in mammalian cells. Current Protocols in Protein Science 95 (2019), e77. doi:10.1002 / cpps.77.

[0245] Once the antibody molecules of the present invention are recombinantly expressed, the entire antibody, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified by standard procedures in the art, including, for example, chromatography (e.g., ion-exchange column chromatography, affinity column chromatography, particularly by affinity for specific antigens after protein A, and size fractionation column chromatography), centrifugation, solubility differential precipitation, e.g., ammonium sulfate precipitation, or any other standard technique for purifying proteins. See, for example, Scopes "Protein Purification" (Springer Verlag, NY (1982)) and "Antibodies A Laboratory Manual" (2nd edition, 2014, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA). Thus, the present invention also relates to a method for preparing an anti-CRMP2 antibody and / or its fragment or its immunoglobulin chain, comprising the following steps: (a) A step of culturing host cells defined above, containing the polynucleotide or vector defined above, under conditions that enable the expression of an anti-CRMP2 antibody, its CRMP2-binding fragment, or immunoglobulin chain, and (b) A step of isolating an anti-CRMP2 antibody, its CRMP2-binding fragment, or immunoglobulin chain from the culture.

[0246] Furthermore, the present invention also relates to anti-CRMP2 antibodies, CRMP2-binding fragments, derivatives thereof, and immunoglobulin chains encoded by the polynucleotides of the present invention described above and obtainable by recombinant production methods thereof. See above.

[0247] The antibodies of the present invention can also be used in diagnostic approaches. Since the antibodies of the present invention bind to pCRMP2 and CRMP2, total CRMP2 can be detected. Accordingly, in one embodiment, the present invention relates to a diagnostic method comprising determining the total level of CRMP2 (CRMP2 and pCRMP2) in a sample from a subject to be diagnosed using at least one antibody of the present invention or its CRMP2-binding fragment, wherein the increased level of total CRMP2 compared to a control (e.g., a sample from a healthy subject or a sample from a subject diagnosed with a disease associated with elevated total CRMP2 levels) indicates the corresponding disease in the subject.

[0248] Subjects diagnosed with such a disease are administered the anti-CRMP2 antibody of the present invention. Alternatively, if a subject is diagnosed according to the method of the present invention, and the information is communicated directly or indirectly to the subject or a physician or medical facility, and the subject is diagnosed with a disease associated with CRMP2 abnormality, the subject is treated with an agent that can improve, treat, or reduce the progression of at least one symptom of the disease in the subject. Preferably, the agent is an anti-CRMP2 antibody, most preferably the antibody of the present invention.

[0249] The subjects being diagnosed may be asymptomatic or preclinical in their condition. In one embodiment, the subjects being diagnosed and the control subjects are age-matched. The samples being analyzed may be any bodily fluid suspected to contain CRMP2 / pCRMP2, such as blood, CSF, or tissue.

[0250] The level of CRMP2 / pCRMP2 can be assessed by any suitable method known in the art, including, for example, analyzing CRMP2 / pCRMP2 by one or more techniques selected from Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectrometry (MS), matrix-assisted laser desorption / ionization-time-of-flight MS (MALDI-TOF), surface-enhanced laser desorption / ionization-time-of-flight MS (SELDI-TOF), high-performance liquid chromatography (HPLC), high-performance protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS / MS), laser densitometry, and IHC.

[0251] Furthermore, anti-CRMP2 antibodies or their CRMP2-binding fragments or derivatives can be used for in vivo imaging of CRMP2 / pCRMP2. In one embodiment, the in vivo imaging of CRMP2 / pCRMP2 includes positron emission tomography (PET), single-photon emission tomography (SPECT), near-infrared (NIR) optical imaging, or magnetic resonance imaging (MRI).

[0252] In certain embodiments, the antibody polypeptide includes an amino acid sequence or one or more portions not typically associated with the antibody. Therefore, the present invention further encompasses the antibody of the present invention, or its antigen-binding fragment, variant, or derivative, conjugated to a diagnostic or therapeutic agent. The antibody can be used diagnostically to monitor the onset or progression of a disease associated with abnormal levels / locations of pCRMP2 / CRMP2, for example, to demonstrate the presence of a disease associated with abnormal levels / locations of pCRMP2 / CRMP2 as described above, to indicate the risk of contracting a disease associated with abnormal levels / locations of pCRMP2 / CRMP2, or, for example, as part of a clinical trial procedure to determine the effectiveness of a given therapeutic and / or prophylactic regimen. Detection can be facilitated by coupling the antibody or its antigen-binding fragment, variant, or derivative to a detectable substance. Exemplary modifications are described in more detail below. For example, the antibody or its CRMP2-binding fragment of the present invention, such as a single-stranded Fv antibody fragment, may contain a flexible linker sequence or be modified to add a functional moiety or a detectable label (e.g., PEG, drug, toxin, or label, e.g., fluorescence, (chemical / bio)luminescence, radioactivity, enzyme, nuclear magnetism, heavy metal, tag, flag, etc.). For example, for general techniques see "Antibodies A Laboratory Manual," 2nd edition, 2014, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York, USA); for advances in fluorescent labeling strategies for dynamic cell imaging see Dean and Palmer, Nat. Chem. Biol. 10 (2014), 512-523; and for enzyme-based labeling strategies for antibody-drug conjugates and antibody mimetic products see Falck and Muller, Antibodies 7 (2018), 4; doi:10.3390 / antib7010004.

[0253] Furthermore, the anti-CRMP2 antibody of the present invention or its CRMP2-binding fragment or derivative may contain organ-specific targeting entities, particularly brain-targeting entities, and / or be contained in or conjugated to a vehicle such as an exosome or nanoparticle for delivery to the brain.

[0254] As is well known in this field, the blood-brain barrier (BBB) ​​limits the efficacy of drugs against central nervous system (CNS) diseases. For example, monoclonal antibodies do not efficiently cross the BBB, reaching a maximum of 0.11% one hour after injection (Banks et al. (2002), Peptides, 23, 2223-2226). The BBB is a specialized structural, physiological, and biochemical barrier that acts as the first boundary between the mutable blood environment and the extracellular fluid of the CNS. The BBB controls homeostasis of the nervous system by strictly controlling the movement of small or macromolecules from the blood to the brain. This allows only the selective transport of molecules essential for brain function. More specifically, over 98% of small molecule drugs and nearly 100% of large molecule drugs are excluded from drug delivery to the brain (Redzic (2011) Fluids Barriers CNS 8,3, Pardridge (2005) NeuroRx, 2,3-14). Therefore, polypeptides and antibodies, their antigen-binding fragments, and their variants or derivatives can each be modified to be permeable to the blood-brain barrier (BBB).

[0255] For example, the antibody and binding fragment can be fused to a cell-permeable peptide (CPP) (which is a short cationic and / or amphiphilic peptide that is eligible as a brain-targeting entity and is typically capable of transporting an associated molecular cargo (e.g., peptides, proteins, antibodies, etc.) across the cell membrane). However, anionic CCPs have also been reported. An example is given in Sharma et al. (2016) Int. J. Mol. Sci. 17, 806, and a method for fusing antibodies with CPPs is described, for example, in Gaston et al. (2019) Sci. Rep. 9, 18688 doi:10.1038 / s41598-019-55091-0. Furthermore, polyamine modification has been shown to dramatically increase the permeability of ia antibodies across the BBB (Poduslo and Curran (1996) J. Neurochem. 66, 1599-1609). The most studied method for delivering macromolecules to the brain is receptor-mediated transcytosis (RMT), with the main RMT receptors studied being the transferrin receptor (TfR) and the insulin receptor (IR). Therefore, RMT receptors are also brain-targeted entities. For example, bispecific antibodies are emerging as a promising scaffold for delivering therapeutic antibodies to the brain by engineering antibodies to incorporate one arm with specificity to the BBB RMT receptor (which drives antibody transfer across the BBB) and the other arm to CNS therapeutics. Essentially, bispecific antibodies can be created by fusing antibody fragments, such as Fab, scFv, or single-domain antibodies, to the N-terminus or C-terminus of a conventional IgG molecule, or by heterodimerization strategies such as the "knobs-into-holes" technology developed by Genentech. See Neves et al. (2016) Trends Biotech. 34, 36-48 for further details. Therefore, the anti-CRMP2 antibody of the present invention can be a bispecific antibody that binds to CRMP2 and the BBB RMT receptor.

[0256] Another approach involves using lipid nanoparticles / nanoexosomes to deliver the antibodies or binding fragments of the present invention across the blood-brain barrier (BBB) ​​or to the brain or kidneys. For example, nanoliposomes double-decorated with an anti-CRMP2 monoclonal antibody and an anti-RMT antibody, such as an anti-TfR monoclonal antibody, using biotin streptavidin conjugation can be used to improve delivery across the blood-brain barrier. The principle is outlined in Markoutsa et al. (2012) Eur.J.Pharm.Biopharm.81,49-56, and the principle of delivery to the heart or kidneys is outlined in Huang et al., Front.Bioeng.Biotechnol.9(2021),DOI:10.3389 / fbioe.2021.683247 and Yang et al., Adv.Drug Deliv.Rev.160(2020),1-18.

[0257] Furthermore, as summarized in Tosi et al. (2013) (Curr. Med. Chem. 20, 2212-25), biodegradable nanoparticles formulated from poly(D,L-lactide-co-glycolide) (PLGA) have been extensively studied for sustained and targeted delivery of various drugs. Accordingly, the antibodies and binding fragments of the present invention are conjugated to nanoparticles and nanoexosomes, respectively.

[0258] Anti-CRMP2 antibodies or their CRMP2-binding fragments, synthetic derivatives, or bioengineered derivatives, particularly recombinant antibodies of human origin (which may be fusion proteins and / or labeled, as described above), are available for a variety of applications according to standard techniques known in the field. See, for example, "Antibodies: A Laboratory Manual," 2nd edition, 2014, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York, USA). Recent advances in the design, manufacture, and formulation of therapeutic antibodies are described in Sifniotis et al., Antibodies 2019, 8(2), 36; https: / / doi.org / 10.3390 / antib8020036. This document also discusses the development of computational approaches for the strategic design of functionally modified antibodies.

[0259] The present invention relates to compositions comprising the CRMP2-binding molecules of the present invention described above, such as antibodies or their CRMP2-binding fragments, variants, or bioengineered derivatives, or the polynucleotides, vectors, or cells of the present invention as defined above. In one embodiment, the composition of the present invention is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0260] The present invention also provides pharmaceutical and diagnostic compositions, respectively, in the form of packs or kits, each containing one or more containers filled with the above components, such as the anti-CRMP2 antibody of the present invention, its CRMP2-binding fragment, biotechnological derivatives or variants, polynucleotides, vectors, or cells. Such containers may be associated with a notice in the form prescribed by the administrative agency that regulates the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting the agency's approval for manufacture, use, or sale for human administration. In addition to or instead of the above, the kit includes reagents and / or instructions for use in a suitable immuno-based diagnostic assay. The compositions of the present invention, e.g., the kits, are, naturally, particularly suited to risk assessment, diagnosis, prevention, and treatment of diseases or disorders involving the presence of CRMP2, and are in particular applicable to the treatment of diseases generally associated with CRMP2, as discussed above.

[0261] The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art. See, for example, "Remington: The Science and Practice of Pharmacy" (2000), published by the University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. Compositions containing such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to a subject in an appropriate dose. Administration of a suitable composition can be achieved by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, topical or intradermal administration, or by spinal or brain delivery. Aerosol formulations, such as nasal spray formulations, contain a purified aqueous solution or other solution of the active ingredient together with a preservative and an isotonic agent. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa.

[0262] The dosage regimen is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any given patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, overall health, and other medications being administered concurrently. See, for example, Bai et al., Clinical Pharmacokinetics, 51(2012), 119-135.

[0263] As described in detail above, the antibody of the present invention has been shown to improve cognitive function in aged mice and reduce amyloid-beta plaque load in the mouse cortex and hippocampus. Therefore, the present invention provides for the first time a therapeutically useful anti-CRMP2 antibody. Thus, in one embodiment, the present invention relates to the antibody or its antigen-binding fragment, derivative, or variant for use as a pharmaceutical.

[0264] Experiments demonstrating improved cognitive function and reduced amyloid-beta load in aged mice were conducted in an AD disease mouse model. Furthermore, pCRMP2 is known to co-localize with p-tau in neurofibrillary tangles, and experiments conducted within the scope of the present invention confirmed that pCRMP2 co-aggregates with p-tau. In addition, staining of hippocampal tissue with the antibody of the present invention was observed in patients with various tauopathies. Thus, it is reasonable to expect that the antibody of the present invention and its corresponding CRMP2-binding fragment will be useful not only for the treatment of AD but also for the treatment of further neurodegenerative diseases, particularly further diseases related to tau and / or Aβ pathology, more specifically diseases related to cognitive impairment, loss of memory and learning ability, and neurodegenerative diseases in general.

[0265] The antibodies of the present invention can reduce or eliminate at least one symptom of neurodegenerative disease in a subject. The symptoms may be hyperphosphorylation of CRMP2, accumulation of pCRMP2, particularly in neurofibrillary tangles, CRMP2 depletion, co-aggregation of pCRMP2 and p-tau, and / or accumulation of amyloid-beta plaques in the subject's brain.

[0266] Neurodegenerative diseases are chronic diseases characterized by the progressive loss of neurons in the brain and spinal cord, with the most prominent and common group being dementia-related tauopathy. Neurodegenerative tauopathy has common pathological lesions consisting of intracellular aggregates of abnormal filaments composed primarily of pathologically hyperphosphorylated tau in neurons and / or glial cells. The clinical features of tauopathy are heterogeneous and are characterized by dementia and / or motor syndromes. The progressive accumulation of fibrous tau inclusions, combined with other deposits, such as β-amyloid in AD, can cause neuronal and glial degeneration. Because the clinical manifestations of tauopathies are heterogeneous, a potentially inexhaustible list of tauopathic diseases, including AD, amyotrophic lateral sclerosis and Parkinson's dementia syndrome (ALS-PDC), argyrophilic granule dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease (CJD), Boxer dementia, diffuse neurofibrillary tangles with calcification, FTD, FTDP-17, frontotemporal lobar degeneration (FLD), Haller-Holden-Spats disease, PiD, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, tangle-only dementia, and multiple infarct dementia, may be provided. For a general overview, see, for example, Lee et al. (2001) Annu. Rev. Neurosci. 24, 1121-1159 (this document classifies and organizes the unique members of tauopathy in Table 1) or Sergeant et al. (2005), Bioch. Biophy. Acta 1739, 179-97 (this document has a list in Figure 2).

[0267] In one embodiment, the present invention relates to a pharmaceutical composition comprising an antibody or a corresponding CRMP2-binding fragment for use in the treatment of diseases of the nervous system, preferably tau and / or Aβ conditions, particularly diseases associated with cognitive impairment, diseases associated with loss of memory and learning ability, and neurodegenerative diseases in general. In a preferred embodiment, the present invention is not limited to AD, ALS-PDC, AGD, British-type amyloid angiopathy, cerebral amyloid angiopathy, CJD, Boxer dementia, diffuse neurofibrillary tangles with calcification, FTD, FTDP-17, FLD, Haller-Holden-Spats disease, PiD, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, neurofibrillary tangle dementia, polyinfarct dementia, Lewy body disease, mild cognitive impairment, cognitive impairment, Lewy body dementia (DLB), Parkinson's disease, ALS, spinal cord injury, etc. The present invention relates to a pharmaceutical composition comprising an antibody or a corresponding CRMP2-binding fragment for use in the treatment of disorders of the nervous system, including traumatic brain injury, Huntington's disease, multiple sclerosis, corticobasal degeneration (CBD), stroke, cerebrovascular disease, neurological disorders, CNS disorders, neuropathic pain, chronic pain, lower back pain, schizophrenia, depression, anxiety disorders, bipolar disorder, autism spectrum disorder, attention deficit / hyperactivity disorder, learning disabilities, motor disorders, obsessive-compulsive disorder, personality disorders, sleep disorders, delirium, developmental disorders, intellectual disabilities, post-traumatic stress disorder, and familial Danish dementia (FDD). In a more preferred embodiment, the present invention relates to a pharmaceutical composition comprising an antibody or a corresponding CRMP2-binding fragment for use in the treatment of tauopathy, most preferably AD, PSP, PiD, FTD, and FLD, for use in the treatment of AD, preferably AD, PSP, PiD, FTD, and FLD, preferably AD, PSD, AGD, British type amyloid angiopathy, cerebral amyloid angiopathy, CJD, Boxer dementia, diffuse neurofibrillary tangles with calcification, FTD, FTDP-17, FLD, Haller-Holden-Spats disease, PiD, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, neurofibrillary tangle dementia, polyinfarct dementia, and FDD.

[0268] Accordingly, the present invention also relates to a method for treating diseases associated with tau and / or Aβ pathology, particularly diseases associated with cognitive impairment, loss of memory and learning ability, and neurodegenerative diseases in general, comprising the step of administering a therapeutically effective amount of any one of the CRMP2-binding molecules, particularly a human-derived antibody of the present invention, to a subject in need thereof. The present invention provides a method for treating a neurodegenerative disease of a subject by administering a therapeutically effective amount of any one of the anti-CRMP2-binding molecules of the present invention, wherein the administration of the anti-CRMP2 antibody improves, treats, or reduces the progression of at least one symptom of the neurodegenerative disease of the subject. Preferably, the method is used for the treatment of neurodegenerative diseases, preferably tauopathies including the above diseases, preferably AD, PSP, PiD, FTD and FLD, most preferably AD.

[0269] In addition, in one embodiment, the present invention relates to a peptide having an epitope of (p)CRMP2 that is specifically recognized by an antibody NI-504.3F4, preferably in which one or more amino acids are substituted, deleted, and / or added, by any antibody of the present invention, wherein the peptide is recognized by any antibody of the present invention. In a preferred embodiment, the peptide of the present invention consists of 10 to 25 consecutive amino acids of CRMP2, comprising the amino acid sequence ASSAK (SEQ ID NO: 64), and optionally, the peptide further comprises a heterologous amino acid sequence or functional moiety. Such heterologous amino acid sequence or functional moiety is defined above. Most preferably, the peptide is that shown in SEQ ID NO: 62 or 63.

[0270] In one embodiment of the present invention, such peptides may be used for antibody screening, for example, screening for CRMP2-binding molecules equivalent to the antibody NI-504.3E7, and / or screening for antibodies that compete with the antibody of the present invention, or such peptides may be used as immunogens to produce antibodies for diagnostic use, for example, in mice or rabbits. The peptides of the present invention can be formulated into arrays, kits, and compositions, respectively, as described above. In connection therewith, the present invention also relates to the above-described kits further comprising the peptides of the present invention. In one embodiment, the present invention is [1] A pharmaceutical composition comprising a human monoclonal antibody or its antigen-binding fragment that selectively binds to human collagen reaction-mediating protein 2 (CRMP2), and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding fragment binds to an epitope comprising the amino acid sequence ASSAK (SEQ ID NO: 64), [2] The pharmaceutical composition according to [1], in which the antibody can be bound to the CRMP2 peptide consisting of the amino acid sequence TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63), [3] The pharmaceutical composition according to [1], in which the antibody can bind to full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2), [4] The pharmaceutical composition according to [3], wherein the antibody can reduce the level of pCRMP2 in a concentration-dependent manner when the antibody and CRMP2 are subjected to a phosphorylation assay, and / or can bind to human, mouse, and rat recombinant CRMP2 full-length protein. [5] The pharmaceutical composition according to [3], wherein the antibody binds to full-length human CRMP2 with an EC50 of approximately ≤10.0 nM and / or to pCRMP2 with an EC50 of approximately ≤100.0 nM, as determined by ELISA. [6] A pharmaceutical composition comprising a human monoclonal antibody or its antigen-binding fragment that selectively binds to human collagen reaction-mediated protein 2 (CRMP2) and whose variable region includes a heavy chain variable (VH) region and a light chain variable (VL) region, and a pharmaceutically acceptable carrier, wherein the VH region includes complementarity-determining regions (CDRs) 1, 2 and 3, and the VL region includes CDRs 1, 2 and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 61, or Here, one or more of the CDRs may contain one or more amino acid substitutions, a pharmaceutical composition, [7] The antibody or antigen-binding fragment is in its variable region (0) Sequence IDs 2 and 7, (1) Sequence IDs 2 and 56, (2) Sequence ID 2 and Sequence ID 58, (3) Sequence ID 12 and Sequence ID 7, (4) Sequence IDs 12 and 56, (5) Sequence IDs 12 and 58, (6) Sequence ID 14 and Sequence ID 7, (7) Sequence IDs 14 and 56, (8) Sequence IDs 14 and 58, (9) Sequence ID 16 and Sequence ID 7, (10) Sequence IDs 16 and 56, (11) Sequence IDs 16 and 58, (12) Sequence ID 18 and Sequence ID 7, (13) Sequence IDs 18 and 56, (14) Sequence IDs 18 and 58, (15) Sequence IDs 18 and 60, (16) Sequence IDs 20 and 7, (17) Sequence IDs 20 and 56, (18) Sequence IDs 20 and 58, (19) Sequence IDs 20 and 60, (20) Sequence IDs 22 and 58, (21) Sequence IDs 24 and 58, (22) Sequence IDs 26 and 58, (23) Sequence IDs 28 and 58, (24) Sequence IDs 30 and 58, (25) Sequence IDs 32 and 58, (26) Sequence IDs 34 and 58, (27) Sequence IDs 36 and 58, (28) Sequence IDs 38 and 58, (29) Sequence IDs 40 and 58, (30) Sequence IDs 42 and 58, (31) Sequence IDs 44 and 58, (32) Sequence IDs 46 and 58, (33) Sequence IDs 48 and 58, (34) Sequence IDs 50 and 58, (35) Sequence IDs 22 and 56, (36) Sequence IDs 22 and 60, (37) Sequence IDs 52 and 56, (38) Sequence IDs 52 and 58, (39) Sequence IDs 52 and 60, and (40) Sequence IDs 54 and 58 It includes amino acid sequences of the VH region and VL region selected from, or A pair of VH amino acid sequences and VL amino acid sequences, wherein VH and / or VL may contain one or more amino acid substitutions and / or amino acid deletions, [6] the pharmaceutical composition, [8] The pharmaceutical composition according to [7], comprising a pair of VH and / or VL which are at least 90% identical to one of the amino acid sequences (0) to (40), [9] A pharmaceutical composition comprising a human monoclonal antibody or its antigen-binding fragment that selectively binds to human collagen reaction-mediated protein 2 (CRMP2) and whose variable region includes a heavy chain variable (VH) region and a light chain variable (VL) region, and a pharmaceutically acceptable carrier, wherein the VH region includes complementarity-determining regions (CDRs) 1, 2 and 3, and the VL region includes CDRs 1, 2 and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 is a pharmaceutical composition containing the amino acid sequence of SEQ ID NO: 10.

[10] The antibody or antigen-binding fragment comprises the amino acid sequences of the VH region and the VL region in its variable region. The VH region contains the amino acid sequence of SEQ ID NO: 22, and The pharmaceutical composition according to [9], wherein the VL region comprises the amino acid sequence of SEQ ID NO: 58.

[11] A pharmaceutical composition according to any one of [1] to

[10] that can reverse the increase in neuronal spine density induced by CRMP2 when determined in an ex vivo hippocampal section culture model.

[12] The pharmaceutical composition according to

[11] , wherein CRMP2 is a pCRMP2 aggregate or a CEMP2 monomer.

[13] Antibodies or antigen-binding fragments (a) Includes the steady-state region; (b) comprising a heterologous polypeptide, wherein the antibody or antigen-binding fragment may be a chimeric mouse-human antibody; and / or (c) A pharmaceutical composition according to any one of the items [1] to

[10] , selected from the group consisting of single-chain Fv fragments (scFv), F(ab') fragments, F(ab) fragments, and F(ab')2 fragments, Fd, Fv, single-chain antibodies, disulfide-stabilized Fv (dsFv), and nanobodies.

[14] The pharmaceutical composition according to

[13] , wherein the constant region is of the IgG type.

[15] The pharmaceutical composition according to

[13] , wherein the constant region is an IgG1 class or an IgG1 isotype.

[16] The pharmaceutical composition according to

[13] , wherein the heterologous polypeptide is the mouse IgG2a constant region.

[17] A pharmaceutical composition comprising an antibody or antigen-binding molecule that competes with the antibody described in any one of [1] to

[10] for specific binding to human CRMP2 and / or pCRMP2, and a pharmaceutically acceptable carrier, wherein the antibody is - Human CRMP2 is recognized preferentially over CRMP1, CRMP3, CRMP4, and CRMP5; -When CRMP2 is subjected to a phosphorylation assay, the phosphorylated CRMP2 (pCRMP2) level can be reduced in a concentration-dependent manner, and / or - When determined in an ex vivo hippocampal section culture model, it is possible to reverse the increase in neuronal spine density induced by CRMP2. Pharmaceutical compositions,

[18] A pharmaceutical composition according to any one of [1] to

[10] , wherein an antibody, antigen-binding fragment, or immunoglobulin VH or VL is encoded by one or more polynucleotides.

[19] The pharmaceutical composition according to

[18] , wherein the polynucleotide is cDNA and / or is operably linked to a heterologous nucleic acid.

[20] The pharmaceutical composition described in

[19] , wherein the heterogeneous nucleic acid is an expression control sequence.

[21] A pharmaceutical composition according to any one of [1] to

[20] for the prevention, delay of progression, and / or treatment of a disease of the nervous system.

[22] Disorders of the nervous system include Alzheimer's disease (AD), amyotrophic lateral sclerosis and Parkinson's syndrome (ALS-PDC), argyrophilic granule dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease (CJD), Boxer dementia, diffuse neurofibrillary tangles with calcification, frontotemporal dementia (FTD), FTDP-17, frontotemporal lobar degeneration (FLD), Haller-Holden-Spats disease, Pick's disease (PiD), prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), neurofibrillary tangle dementia, polyinfarct dementia, and Lev - A pharmaceutical composition according to

[21] , which is a disease including corpuscle disease, mild cognitive impairment, cognitive impairment, Lewy body dementia (DLB), Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal cord injury, traumatic brain injury, Huntington's disease, multiple sclerosis, corticobasal degeneration (CBD), stroke, cerebrovascular disease, neurological disorders, CNS disorders, neuropathic pain, chronic pain, lower back pain, schizophrenia, depression, anxiety disorders, bipolar disorder, autism spectrum disorder, attention deficit / hyperactivity disorder, learning disability, motor disorder, obsessive-compulsive disorder, personality disorders, sleep disorders, delirium, developmental disorders, intellectual disability, post-traumatic stress disorder, and familial Danish dementia (FDD).

[23] The pharmaceutical composition according to

[21] , wherein the neurological disorder is Alzheimer's disease (AD), including mild, moderate, or severe AD, or mild cognitive impairment.

[24] A pharmacopoeia according to

[21] , wherein the disorder of the nervous system is a disorder associated with tau and / or Aβ pathology, in particular a disorder associated with cognitive impairment, loss of memory and learning ability.

[25] The pharmaceutical composition according to

[24] , wherein the disease associated with tau and / or Aβ pathology is Alzheimer's disease (AD), including mild, moderate, and severe AD.

[26] The pharmaceutical composition according to

[21] , wherein the disease of the nervous system is a neurodegenerative disease.

[27] The pharmaceutical composition according to

[26] , wherein the neurodegenerative disease is tauopathy.

[28] The pharmaceutical composition according to

[27] , wherein tauopathy is AD, ALS-PDC, AGD, British type amyloid angiopathy, cerebral amyloid angiopathy, CJD, Boxer dementia, diffuse neurofibrillary tangles with calcification, FTD, FTDP-17, FLD, Haller-Holden-Spats disease, PiD, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, neurofibrillary tangle dementia, polyinfarct dementia, and FDD.

[29] The pharmaceutical composition according to

[27] , wherein tauopathy is Alzheimer's disease (AD), including mild, moderate, and severe AD, and / or dementia symptoms in Alzheimer's disease (AD).

[30] The pharmaceutical composition described in

[21] , wherein the disorder of the nervous system is cognitive impairment.

[31] A pharmaceutical composition according to any one of [1] to

[20] for improving memory and / or learning ability.

[32] A prophylactic, progression-inhibiting, and / or therapeutic agent for a neurological disorder comprising the antibody or antigen-binding fragment described in [1] as an active ingredient, wherein the prophylactic and / or therapeutic agent is administered in combination with at least one agent selected from the group consisting of donepezil hydrochloride, galantamine hydrobromide, huperzine A, idebenone, lecacecarnine hydrochloride, memantine hydrochloride, memantine hydrochloride / donepezil hydrochloride, porcine brain protein-derived proteolytic peptide fraction, rivastigmine tartrate, tacrine hydrochloride, aducanumab, lecanemab, and donanemab,

[32] [1] A prophylactic, prolongation-inhibiting, and / or therapeutic agent for diseases of the nervous system, comprising the antibody or antigen-binding fragment described above.

[33] Antibodies or antigen-binding fragments described in [1] used for the prevention, delaying of progression, and / or treatment of diseases of the nervous system.

[34] A human monoclonal antibody or antigen-binding fragment that selectively binds to human collagen reaction-mediating protein 2 (CRMP2) and comprises a heavy chain variable (VH) region and a light chain variable (VL) region in its variable region, wherein the VH region comprises complementarity-determining regions (CDRs) 1, 2 and 3, and the VL region comprises CDRs 1, 2 and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 61, or Here, one or more of the CDRs may contain one or more amino acid substitutions, and are used for the manufacture of pharmaceuticals for the prevention, delay of progression, and / or treatment of diseases of the nervous system.

[35]

[34] The use described above, wherein the antibody or antigen-binding fragment is in its variable region (0) Sequence IDs 2 and 7, (1) Sequence IDs 2 and 56, (2) Sequence ID 2 and Sequence ID 58, (3) Sequence ID 12 and Sequence ID 7, (4) Sequence IDs 12 and 56, (5) Sequence IDs 12 and 58, (6) Sequence ID 14 and Sequence ID 7, (7) Sequence IDs 14 and 56, (8) Sequence IDs 14 and 58, (9) Sequence ID 16 and Sequence ID 7, (10) Sequence IDs 16 and 56, (11) Sequence IDs 16 and 58, (12) Sequence ID 18 and Sequence ID 7, (13) Sequence IDs 18 and 56, (14) Sequence IDs 18 and 58, (15) Sequence IDs 18 and 60, (16) Sequence IDs 20 and 7, (17) Sequence IDs 20 and 56, (18) Sequence IDs 20 and 58, (19) Sequence IDs 20 and 60, (20) Sequence IDs 22 and 58, (21) Sequence IDs 24 and 58, (22) Sequence IDs 26 and 58, (23) Sequence IDs 28 and 58, (24) Sequence IDs 30 and 58, (25) Sequence IDs 32 and 58, (26) Sequence IDs 34 and 58, (27) Sequence IDs 36 and 58, (28) Sequence IDs 38 and 58, (29) Sequence IDs 40 and 58, (30) Sequence IDs 42 and 58, (31) Sequence IDs 44 and 58, (32) Sequence IDs 46 and 58, (33) Sequence IDs 48 and 58, (34) Sequence IDs 50 and 58, (35) Sequence IDs 22 and 56, (36) Sequence IDs 22 and 60, (37) Sequence IDs 52 and 56, (38) Sequence IDs 52 and 58, (39) Sequence IDs 52 and 60, and (40) Sequence IDs 54 and 58 It includes amino acid sequences of the VH region and VL region selected from, or A pair of VH amino acid sequences and VL amino acid sequences, wherein VH and / or VL may contain one or more amino acid substitutions and / or amino acid deletions. Human-derived monoclonal antibodies or their antigen-binding fragments, for use,

[36] Use according to

[35] , comprising a pair in which VH and / or VL are at least 90% identical to any one amino acid sequence of (0) to (40),

[37] Disorders of the nervous system include Alzheimer's disease (AD), amyotrophic lateral sclerosis and Parkinson's syndrome (ALS-PDC), argyrophilic granule dementia (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease (CJD), Boxer dementia, diffuse neurofibrillary tangles with calcification, frontotemporal dementia (FTD), FTDP-17, frontotemporal lobar degeneration (FLD), Haller-Holden-Spats disease, Pick's disease (PiD), prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy (PSP), neurofibrillary tangle dementia, polyinfarct dementia, and Lewy body dementia. The use described in any one of paragraphs

[34] to

[36] includes, , mild cognitive impairment, cognitive impairment, Lewy body dementia (DLB), Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal cord injury, traumatic brain injury, Huntington's disease, multiple sclerosis, corticobasal degeneration (CBD), stroke, cerebrovascular disease, neurological disorders, CNS disorders, neuropathic pain, chronic pain, lower back pain, schizophrenia, depression, anxiety disorders, bipolar disorder, autism spectrum disorder, attention deficit / hyperactivity disorder, learning disabilities, motor disorders, obsessive-compulsive disorder, personality disorders, sleep disorders, delirium, developmental disorders, intellectual disabilities, post-traumatic stress disorder, and familial Danish dementia (FDD).

[38] The use described in

[37] if the neurological disorder is Alzheimer's disease (AD) or mild cognitive impairment,

[39] Disorders of the nervous system are disorders associated with tau and / or Aβ pathology, particularly disorders with cognitive impairment, disorders associated with loss of memory and learning ability, as described in any one of paragraphs

[34] to

[36] ,

[40] The use described in

[39] if the disease associated with tau and / or Aβ pathology is Alzheimer's disease (AD),

[41] The use described in any one of paragraphs

[34] to

[36] , where the disorder of the nervous system is a neurodegenerative disease.

[42] If the neurodegenerative disease is a tauopathy,

[41] use,

[43] Tauopathy is a disease that includes AD, ALS-PDC, AGD, British type amyloid angiopathy, cerebral amyloid angiopathy, CJD, Boxer dementia, diffuse neurofibrillary tangles with calcification, FTD, FTDP-17, FLD, Haller-Holden-Spats disease, PiD, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, neurofibrillary tangle dementia, polyinfarct dementia, and FDD, as described in

[42] .

[44] Tauopathy is a dementia symptom in Alzheimer's disease (AD) and / or Alzheimer's disease, as described in

[43] .

[45] If a disorder of the nervous system is a cognitive impairment, use as described in

[34] -

[36] ,

[46] A human monoclonal antibody or antigen-binding fragment that selectively binds to human collagen reaction-mediated protein 2 (CRMP2) and comprises a heavy chain variable (VH) region and a light chain variable (VL) region in its variable region, wherein the VH region comprises complementarity-determining regions (CDRs) 1, 2 and 3, and the VL region comprises CDRs 1, 2 and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 61, or Here, one or more of the CDRs may contain one or more amino acid substitutions, and are used for the manufacture of a pharmaceutical product for improving memory and / or learning ability of an antibody or antigen-binding fragment.

[47]

[46] The use described above, wherein the antibody or antigen-binding fragment is in its variable region (0) Sequence IDs 2 and 7, (1) Sequence IDs 2 and 56, (2) Sequence ID 2 and Sequence ID 58, (3) Sequence ID 12 and Sequence ID 7, (4) Sequence IDs 12 and 56, (5) Sequence IDs 12 and 58, (6) Sequence ID 14 and Sequence ID 7, (7) Sequence IDs 14 and 56, (8) Sequence IDs 14 and 58, (9) Sequence ID 16 and Sequence ID 7, (10) Sequence IDs 16 and 56, (11) Sequence IDs 16 and 58, (12) Sequence ID 18 and Sequence ID 7, (13) Sequence IDs 18 and 56, (14) Sequence IDs 18 and 58, (15) Sequence IDs 18 and 60, (16) Sequence IDs 20 and 7, (17) Sequence IDs 20 and 56, (18) Sequence IDs 20 and 58, (19) Sequence IDs 20 and 60, (20) Sequence IDs 22 and 58, (21) Sequence IDs 24 and 58, (22) Sequence IDs 26 and 58, (23) Sequence IDs 28 and 58, (24) Sequence IDs 30 and 58, (25) Sequence IDs 32 and 58, (26) Sequence IDs 34 and 58, (27) Sequence IDs 36 and 58, (28) Sequence IDs 38 and 58, (29) Sequence IDs 40 and 58, (30) Sequence IDs 42 and 58, (31) Sequence IDs 44 and 58, (32) Sequence IDs 46 and 58, (33) Sequence IDs 48 and 58, (34) Sequence IDs 50 and 58, (35) Sequence IDs 22 and 56, (36) Sequence IDs 22 and 60, (37) Sequence IDs 52 and 56, (38) Sequence IDs 52 and 58, (39) Sequence IDs 52 and 60, and (40) Sequence IDs 54 and 58 It includes amino acid sequences of the VH region and VL region selected from, or A pair of VH amino acid sequences and VL amino acid sequences, wherein VH and / or VL may contain one or more amino acid substitutions and / or amino acid deletions. An antibody or its antigen-binding fragment, used,

[48] ​​Use as described in

[47] , comprising a pair in which VH and / or VL are at least 90% identical to any one amino acid sequence of (0) to (40),

[49] A human monoclonal antibody or antigen-binding fragment that selectively binds to human collagen reaction-mediating protein 2 (CRMP2) and comprises a heavy chain variable (VH) region and a light chain variable (VL) region in its variable region, wherein the VH region comprises complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region comprises CDRs 1, 2, and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 is sequence number 10 The use of antibodies or antigen-binding fragments for the manufacture of pharmaceuticals for the prevention, delay of progression, and / or treatment of diseases of the nervous system.

[50] The antibody or antigen-binding fragment comprises the amino acid sequences of the VH region and the VL region in its variable region. The VH region contains the amino acid sequence of SEQ ID NO: 22, and The use described in

[49] , in which the VL region contains the amino acid sequence of SEQ ID NO: 58,

[51] The use described in

[49] or

[50] , if the disorder of the nervous system is Alzheimer's disease (AD) or mild cognitive impairment,

[52] The use described in

[49] or

[50] , where the disorder of the nervous system is a neurodegenerative disease.

[53] The use described in

[52] for neurodegenerative diseases, tauopathies,

[54] The use described in

[53] , where tauopathy is Alzheimer's disease (AD) and / or a dementia symptom in Alzheimer's disease (AD),

[55] A human monoclonal antibody or antigen-binding fragment that selectively binds to human collagen reaction-mediated protein 2 (CRMP2) and comprises a heavy chain variable (VH) region and a light chain variable (VL) region in its variable region, wherein the VH region comprises complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region comprises CDRs 1, 2, and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c)VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 is sequence number 10 The use of antibodies or antigen-binding fragments for the manufacture of pharmaceuticals to improve memory and / or learning ability,

[56] The antibody or antigen-binding fragment comprises the amino acid sequences of the VH region and the VL region in its variable region. The VH region contains the amino acid sequence of SEQ ID NO: 22, and The use described in

[55] , wherein the VL region comprises the amino acid sequence of SEQ ID NO: 58. Regarding.

[0271] Throughout this specification, several documents are cited. The contents of all cited documents (including references cited throughout this application, including the background art section, published patents, published patent applications, and manufacturer specifications, instructions, etc.) are expressly incorporated herein by reference. However, no cited document is acknowledged to be actual prior art with respect to the present invention.

[0272] A more complete understanding can be gained by referring to the following specific examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. [Examples]

[0273] The following shows the results of experiments conducted using the original antibody NI-504.3E7. However, these results were confirmed using one or more variants, i.e., one or more of NI-504.3E7_V1 to NI-504.3E7_V40.

[0274] Example 1: Isolation, identification, cloning, and recombinant expression of anti-CRMP2 antibody As an attempt to identify and isolate potential CRMP and CRMP2-specific antibodies that may have protective effects in aged individuals, we applied our proprietary Reverse Translational Medicine® (RTM) technology platform (Neurimmune AG, 8952 Schlieren / Zurich, Switzerland) to obtain cDNA sequences encoding recombinant human CRMP2 and pCRMP2-binding human antibodies from anonymized blood lymphocyte libraries collected from healthy aged subjects. Positive hits were counter-screened to exclude clones that cross-reacted with unrelated proteins. Selected CRMP2-reactive B cell clones were subjected to cDNA cloning of IgG heavy chain and light chain variable region sequences (see Table II for selected antibodies) and subcloned into expression vectors encoding human IgG1 constant domain sequences. Expression constructs encoding IgG1 heavy chain and light chain were transiently expressed in the ExpiCHO cell line and purified by protein A affinity chromatography. The results of the first screening are summarized in Table VI.

[0275] [Table 6]

[0276] Example 2: NI-504.3E7 selectively binds to human CRMP2. The antibody NI-504.3E7 was tested for its binding specificity to full-length CRMP1, CRMP2, CRMP3, CRMP4, and CRMP5 by direct ELISA and Western blotting.

[0277] ELISA assay For direct ELISA, 96-well microplates (Costar) were coated with 5 μg / mL of recombinant human CRMP1-5 proteins (CRMP1(Origene), CRMP2(Origene), CRMP3(Origene), CRMP4(Origene), and CRMP5(Origene)) in PBS (Gibco) and incubated overnight at 4°C.

[0278] The plates were blocked in blocking buffer (2% BSA (Sigma-Aldrich)) in PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP399; containing 0.1% Tween® 20 (Sigma-Aldrich)) for 1 hour at room temperature. After three washes with PBS-T using a plate washer (Bio Tek Microplate Staker 3), the plates were incubated with serial dilutions of antibody NI-504.3E7 (30 μL / well) at room temperature for 1 hour, followed by three more washes with PBS-T. The conjugated antibody NI-504.3E7 was detected at room temperature for 1 hour using HRP-conjugated anti-human Fc secondary antibody (Jackson ImmunoResearch, catalog 709-036-098), followed by three washes with PBS-T and chromogenic substrate TMB (Clinical) for absorbance measurement at 450 nm using a plate reader (Thermo Fisher Scientific). The conversion was performed using Science. Maximum half-value concentration EC 50 To estimate the reaction, logarithmically transformed absorbance values ​​were fitted using log(agonist) vs. reaction-variable slope (4-parameter) curve fitting in GraphPad Prism version 9.3, applying the formula Y = minimum + (maximum - minimum) / (1 + 10^((LogEC50 - X) * Hill gradient)). The data are displayed as mean + SEM.

[0279] Western blot For Western blot analysis, 0.2 μg each of recombinant human CRMP1 (Origene), CRMP2 (Origene), CRMP3 (Origene), CRMP4 (Origene), and CRMP5 (Origene) were denatured in LDS sample buffer (NuPAGE) + 4% β-mercaptoethanol (Sigma-Aldrich) at 95°C for 5 minutes. The samples were loaded onto 4-12% Bis-Tris protein gels (NuPAGE), and the gels were run in 1×MOPS SDS electrophoresis buffer (NuPAGE) at 200V for 45 minutes. The proteins were transferred to PVDF membranes (NuPAGE) for 7 minutes at 20V using iBlot. The membrane was blocked at room temperature for 1 hour using PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP399; containing 0.1% Tween® 20 (Sigma-Aldrich)) containing 2% BSA (Sigma-Aldrich). Antibody NI-504.3E7 was incubated at room temperature for 1 hour at 5 μg / mL in PBS-T containing 2% BSA. After three washes with PBS-T, the membrane was incubated at room temperature for 1 hour with HRP-conjugated anti-human-Fc secondary antibody (Jackson ImmunoResearch, catalog 709-036-098), washed three more times with PBS-T, and incubated with ECL (Amersham) for 2 minutes. The chemiluminescence signal was detected using Image Quant LAS 4000 (Amersham).

[0280] result In ELISA assays, antibody NI-504.3E7 showed preferential binding to CRMP2 compared to CRMP1, CRMP3, CRMP4, and CRMP5. In particular, antibody NI-504.3E7 showed high affinity for CRMP2, i.e., 4 nM EC2. 50 It has been shown that they are joined together, and CRMP1 has EC 50Only a weak binding of 69 nM was observed. See Figure 2A. Furthermore, even weaker binding was observed for CRMP3, CRMP4, and CRMP5. The same results were obtained by Western blot analysis. In particular, antibody NI-504.3E7 showed strong binding to CRMP2, only very weak binding to CRMP1, and no binding to CRMP3, CRMP4, and CRMP5. See Figure 2B. Thus, antibody NI-504.3E7 selectively binds to CRMP2.

[0281] Example 3: NI-504.3E7 binds to the linear epitope at the C-terminus of CRMP2. To evaluate the binding epitope of antibody NI-504.3E7, epitope mapping was performed using a cellulose membrane array with overlap peptides covering the full length of the CRMP2 protein (15 amino acid length) and 11 amino acid overlaps. The membrane was prepared by JPT Membrane Technologies (Germany). The membrane was blocked with 1×RotiBlock (Roth) at room temperature for 1 hour, and antibody NI-504.3E7 was incubated overnight at 4°C at 0.04 μg / mL in 1×RotiBlock. After three washes with 1×TBS-T (TBS 20×, Biocave Medical, catalog TWB945M; containing 0.1% Tween® 20 (Sigma-Aldrich)), the membrane was incubated with HRP-conjugated anti-human H+L secondary antibody (Jackson ImmunoResearch, catalog 709-036-149) at room temperature for 1 hour and washed three times with TBS-T. The antibody NI-504.3E7 bound to the membrane was detected using ECL (Amersham) for 2 minutes, and the chemiluminescence signal was detected using Image Quant LAS 4000 (Amersham).

[0282] As shown in Figure 3, by using an overlap peptide array, it was found that antibody NI-504.3E7 binds to two linear peptides 128 and 129 (sequence numbers 62 and 63) that have the consensus sequence VTPASSAKTSP (sequence number 65). Furthermore, the minimal epitope of antibody NI-504.3E7 contains the amino acid sequence 516-ASSAK-520 (sequence number 64).

[0283] Example 4: NI-504.3E7 binds to non-phosphorylated CRMP2 and phosphorylated CRMP2. ELISA assays were performed to determine the binding specificity of antibody NI-504.3E7 and 40 variants against CRMP2 and pCRMP2. The variants contain one or more amino acid substitutions in the VH chain, VL chain, and / or CDR. ELISA assays were performed using CRMP2 and pCRMP2 as described in Example 2. EC assays were performed for antibody NI-504.3E7 and antibody variants NI-504.3E7_V1~V40. 50 The values ​​are listed in Table VII.

[0284] [Table 7]

[0285] As shown in Table VII, the antibody NI-504.3E7 contains recombinant human full-length CRMP2 and pCRMP2 at EC2 concentrations of 0.5 nM and 4.7 nM, respectively. 50 Therefore, it is specifically recognized. As can be seen from this, all the mutants still bind to CRMP2 and pCRMP2, and thus it is confirmed that one or more amino acid substitutions do not substantially change the antibody's binding affinity.

[0286] Example 5: NI-504.3E7 reduces pCRMP2 levels in a concentration-dependent manner. To determine whether the phosphorylation of CRMP2 changes in the presence of the antibody or isotype control of the present invention when its concentration is increased, a phosphorylation assay was performed in combination with Western blotting analysis. To further confirm the fact that the mutant antibody retains the binding specificity and activity of the original antibody, this assay was also performed with antibody NI-504.3E7_V20 as an example. In particular, this experimental setup was performed to observe the levels of T509 pCRMP2 (Figures 4A-4E), T514 pCRMP2 (Figures 4F-4J), and S522 pCRMP2 (Figures 4K-4O) when CRMP2 was subjected to the phosphorylation assay with either antibody NI-504.3E7_V20 or an anti-CRMP2 control antibody (NI-504.B) or an isotype control antibody.

[0287] CRMP2 protein (Alexotech) was incubated for 1 hour in the presence of CRMP2 antibodies (NI-504.3E7 and NI-504.B) at different concentrations (one-third, equimolar, or 3x compared to CRMP2) and one human IgG control (isotype control antibody). CRMP2 phosphorylation was performed at 30°C for 1 hour or 24 hours using a phosphorylation buffer containing a phosphorylation mix (50 mM Tris-HCl (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 1.5 mM DTT (Sigma-Aldrich)), 0.25 μg GSK3β (SignalChem), 0.25 μg CDK5 / p35 (SignalChem), and 0.7 mM ATP (SignalChem). The level of CRMP2 phosphorylation was analyzed by Western blotting. 200 ng of phosphorylated CRMP2 protein was denatured in LDS sample buffer + 4% β-mercaptoethanol (NuPAGE) at 95°C for 10 minutes. The sample was loaded onto a 4-12% Bis-Tris protein gel (NuPAGE), and the gel was run in 1× MES SDS electrophoresis buffer (NuPAGE) at 200V for 45 minutes. The protein was transferred to a nitrocellulose membrane (NuPAGE) for 7 minutes at 20V using iBlot. The membrane was blocked overnight at 4°C using PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP399; containing 0.1% Tween® 20 (Sigma-Aldrich)) containing 2% BSA (Sigma-Aldrich). The following day, the membranes were incubated at room temperature for 1 hour with anti-CRMP2 antibody C2993 (Sigma-Aldrich), pCRMP2 (Thr-514) antibody (Cell Signaling, catalog 9397S), pCRMP2 (Thr-509) antibody (Thermo Fisher Scientific, catalog PA5-37551), or pCRMP2 (Ser-522) antibody (ECM Bioscience, catalog CP2191), and then washed three times with PBS-T.The membrane was incubated with HRP-conjugated goat anti-rabbit IgG(H+L) (Jackson ImmunoResearch, catalog 111-035-045) at room temperature for 1 hour. After washing three times with PBS-T, the membrane was incubated with ECL (Amersham) for 1 minute, and the chemiluminescence signal was detected using Image Quant LAS 4000 (Amersham). Signal band density analysis was performed using the Java®-based image processing program ImageJ.

[0288] To normalize the pCRMP2 levels, samples were analyzed for total CRMP2 levels using antibody C2993. The results showed that total CRMP2 levels were equivalent across all samples and the untreated loading control (200 ng / well) from Alexotech. Graphs C, E, H, J, M, and O in Figure 4 are normalized to their respective total CRMP2 levels.

[0289] As can be seen in the figure, antibody NI-504.3E7_V20 reduces T509 pCRMP2 levels in a concentration-dependent manner after 1 hour or 24 hours of phosphorylation. Furthermore, antibody NI-504.3E7_V20 reduces T514 pCRMP2 levels after 1 hour or 24 hours of phosphorylation compared to antibody NI-504.B and isotype controls. Antibody NI-504.3E7_V20 also reduces S522 pCRMP2 levels after 1 hour of phosphorylation compared to antibody NI-504.B and isotype controls. These results indicate that antibody NI-504.3E7_V20 reduces pCRMP2 levels in a concentration-dependent manner. Further repetition of this study confirmed the above results and also demonstrated a concentration-dependent reduction in S522 pCRMP2 levels after 24 hours of phosphorylation.

[0290] To ensure that antibody binding affinity was not reduced or altered by the phosphorylation procedure up to 24 hours, separate phosphorylation experiments were performed in the absence of the CRMP2 protein. Antibodies NI-504.3E7_V20 and NI-504.B, as well as isotype control antibodies, were incubated for 24 hours under standard phosphorylation conditions. Binding affinity was evaluated directly by CRMP2 ELISA. In conclusion, all antibodies showed comparable binding before and after phosphorylation, and their CRMP2 binding was not affected by the phosphorylation procedure.

[0291] Example 6: NI-504.3E7 specifically binds to CRMP2 peptide and pCRMP2 peptide. To evaluate whether the antibody NI-504.3E7 binds to CRMP2 in its phosphorylated state, four different peptides (Bachem) covering the antibody's epitopes, either without phosphorylation sites or with phosphorylation at specific sites (T509, T514, S522), were tested in ELISA format. The sequences of these peptides are as follows: Peptide 4152960:VCEVSVTPKTVTPASSAKTSPAKQQA (SEQ ID NO: 72) Peptide 4152961:VCEVSV(p)T 509 PKTVTPASSAKTSPAKQQA (Sequence ID 72) Peptide 4152962:VCEVSVTPKTV(p)T 514 PASSAKTSPAKQQA (Sequence ID 72) Peptide 4152963:VCEVSVTPKTVTPASSAKT(p)S 522 PAKQQA (Sequence ID 72)

[0292] For direct ELISA, 96-well microplates (Costar) were coated with 30 μL / well of each peptide at 5 μg / mL in PBS and incubated overnight at 4°C. The ELISA plates were blocked at room temperature for 1 hour with PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP399; containing 0.1% Tween® 20 (Sigma-Aldrich)) containing 2% BSA (Sigma-Aldrich), incubated at room temperature for 1 hour with 30 μL / well serial dilutions of antibody NI-504.3E7, and then washed three times with PBS-T. The conjugated antibodies were detected using HRP-conjugated anti-human Fc secondary antibody (Jackson ImmunoResearch, catalog 709-036-098) and chromogenic substrate TMB (Clinical Science) for absorbance measurement at 450 nm using a plate reader (Thermo Fisher Scientific). Maximum half-limit EC 50 To estimate the reaction, logarithmically transformed absorbance values ​​were fitted using log(agonist) vs. reaction-variable slope (4-parameter) curve fitting in GraphPad Prism version 9.3, applying the formula Y = minimum + (maximum - minimum) / (1 + 10^((LogEC50 - X) * Hill gradient)). The data are displayed as mean + SEM.

[0293] As shown in Figure 5, the affinity of antibody NI-504.3E7 for the CRMP2 peptide with phosphorylation at position T514 (peptide 4152962) is approximately 1 / 300th of that for the non-phosphorylated peptide and for the peptide with phosphorylation at positions T509 (peptide 4152961) and S522 (peptide 4152963). The phosphorylation at T514 appears to partially impair the binding of antibody NI-504.3E7, likely due to it being an antibody epitope.

[0294] Example 7: NI-504.3E7 can bind to recombinant CRMP2 full-length protein in humans, mice, and rats. To determine the binding of the antibody NI-504.3E7 to different species of CRMP2, 0.5 μg of recombinant human SUMO-CRMP2 (Boston Biochem), recombinant mouse CRMP2-His6 (MyBioSource, catalog MBS1185591), and recombinant rat CRMP2-His6 (MyBioSource, catalog MBS961395) were denatured in LDS sample buffer + 4% β-mercaptoethanol (NuPAGE) at 70°C for 10 minutes. The samples were loaded onto 4-12% Bis-Tris protein gel (NuPAGE), and the gels were run in 1× MES SDS electrophoresis buffer (NuPAGE) at 200 V for 60 minutes. The proteins were transferred to a nitrocellulose membrane (NuPAGE) for 7 minutes at 20 V using iBlot. The membrane was blocked for 1 hour at room temperature in 1×TBS-T (TBS 20×, Biocave Medical, catalog TWB945M; containing 0.1% Tween® 20 (Sigma-Aldrich)) containing 5% milk, and the antibody NI-504.3E7 was incubated overnight at 4°C at 5 μg / mL in TBS-T containing 1% milk. After three washes with TBS-T, the membrane was incubated with HRP-conjugated anti-human H+L secondary antibody (Jackson ImmunoResearch, catalog 709-036-149) at room temperature for 1 hour and washed three times with TBS-T. The membrane was incubated with ECL (Amersham) for 2 minutes, and the chemiluminescence signal was detected using Image Quant LAS 4000 (Amersham).

[0295] As shown in Figure 6, antibody NI-504.3E7 specifically binds to all analyzed CRMP2 species, namely human, mouse, and rat CRMP2, compared to isotype IgG1 and human IgG1 control. Therefore, antibody NI-504.3E7 can bind to recombinant CRMP2 full-length protein in humans, mice, and rats.

[0296] Example 8: Co-aggregation of recombinant (p)tau and (p)CRMP2 in the ThioT assay Based on Takata et al., Am J. Pathol. 175 (2009), 17-24 (in this paper, hyperphosphorylated tau co-localizes with phosphorylated CRMP2, as shown in Figure 1), a thioflavin-T (ThioT, Sigma-Aldrich) assay was performed to detect whether recombinant (p)tau and (p)CRMP2 proteins not only co-localize but also co-aggregate. ThioT is known to bind to a variety of fibrils, despite their completely different amino acid sequences, which strongly suggests that ThioT recognizes common structural features among fibrils. The ThioT assay measures the change in fluorescence intensity of ThioT when it binds to fibrils. It does not bind to the corresponding monomeric structure. Here, CRMP2, pCRMP2, tau + pCRMP2, and ptau + pCRMP2 were subjected to the ThioT assay, and the fluorescence signal was measured over 240 hours (exposure time was 5 minutes). Tau protein was obtained from rPeptide (Watkinsville, USA). Human CRMP2 was a custom-made protein obtained from Boston Biochem, Inc. (Cambridge, USA), containing a SUMO tag at the N-terminus and expressed in E. coli. In particular, amyloid fibril formation in composite samples of CRMP2 (CRMP2, 6.4 μM, Boston Biochem), phosphorylated CRMP2 (pCRMP2, 6.4 μM), tau (tau, 4.35 μM, tau-441 full-length protein (rPeptide, catalog T-1001)) and phosphorylated CRMP2 (pCRMP2, 6.4 μM), or phosphorylated tau (p-tau, 4.35 μM) and phosphorylated CRMP2 (pCRMP2, 6.4 μM) was measured by increasing ThioT fluorescence. 50 μM ThioT was used for CRMP2 and pCRMP2 samples, and 50 μM ThioT and 50 μM heparin were used for combined samples (tau + pCRMP2, ptau + pCRMP2). Agglutination assays were performed in plate format at room temperature with shaking at 300 rpm. Before each measurement, the plate was shaken at 600 rpm for 5 seconds, and fluorescence was measured at an emission wavelength of 490 nm after excitation at 456 nm. Data are shown as the mean of dual measurements taken at the time of display + SEM.

[0297] As can be seen in Figure 7, only weak ThioT binding was observed to CRMP2 (as determined by ThioT fluorescence), indicating that CRMP2 does not aggregate. During the incubation of pCRMP2 and during the incubation of pCRMP2 with tau, slightly higher ThioT binding was observed, and the ThioT fluorescence signal increased, indicating the formation of more aggregates. When pCRMP2 was incubated with ptau, the highest signal was obtained, indicating that pCRMP2 and ptau co-aggregate.

[0298] Example 9: Antibody NI-504.3E7 reverses CRMP2-induced spine density increase in a hippocampal section culture model. The effect of the antibody NI-504.3E7 on neuronal spine density driven by CRMP2 / pCRMP2, i.e., pCRMP2 aggregates and CRMP2 monomers, was analyzed in an ex vivo hippocampal section culture (HSC) model. Organotype hippocampal sections were prepared from 5-day-old C57BL / 6J wild-type mouse pups (N=8). The pups were decapitated, the brain was removed, the two cerebral hemispheres were separated, the midbrain was removed, and both hippocampi were extracted. The hippocampi were sectioned into 400 μm thick sections using a tissue chopper. The experimental procedure followed the publication Suendermann et al., Methods Mol Biol (2012), 277-293. Each condition was tested in duplicate. A total of 2 × 5 sections per condition were transferred to Multidish 6-well plates (Nunclon®) containing culture medium and Millicell cell culture inserts (Millipore, catalog ICM0RG50). Hippocampal sections were cultured, treated with each combination of target protein and antibody NI-504.3E7, infected, and fixed. Hippocampal sections were prepared on day 0 and cultured for 10 days with medium changes every 2-3 days. They were then treated with pCRMP2 aggregates or CRMP2 monomers, either combined with antibody NI-504.3E7 or without the antibody, at the indicated concentrations. After medium changes and infection, antibody NI-504.3E7 was diluted in NB medium containing N1 supplement for 4 days on day 10 and for an additional 2 days on day 14. Monomeric yeast CRMP2 was diluted in NB medium containing N1 supplement, while pCRMP2 aggregates were obtained after recombinant CRMP2 phosphorylation for 1 day at 37°C in a phosphorylation buffer containing Tris-HCl (Sigma-Aldrich), MgCl2 (Sigma-Aldrich), DTT (Sigma-Aldrich), GSK3β (SignalChem), CDK5 / p35 (SignalChem), and ATP (SignalChem). Aggregation of phosphorylated CRMP2 was performed at room temperature for 10 days at a concentration of 1 mg / mL in 5 mM Tris-HCl (pH 7.4) containing 5 mM heparin.

[0299] On day 14, section cultures were treated with GFP Sindbis virus (Sindobis expression system manual, Invitrogen catalog K750-01) applied by a droplet method using 1 μL / section of hSyn1-EGFP AAV9 construct diluted 1:3 from the supernatant. On day 16, hippocampal sections were fixed at +4°C for 2 hours with fixation solution (phosphate-buffered saline (Gibco), 25% paraformaldehyde (Sigma-Aldrich, catalog 158127), 25% sucrose (Sigma-Aldrich)), then mounted on glass slides, embedded in confocal matrix, and imaged using a confocal laser scanning microscope (Zeiss LSM 510). Semi-automated threshold analysis of spine density was performed by determining the number of spines per 1 μm of dendritic length. A total of 29–58 images were analyzed for each treatment condition, and 11–52 spines were detected on the dendritic segment in each image. Data are displayed as mean + SEM. Analysis by one-way ANOVA: ***p<0.001**p<0.01; Kruskal-Wallis test post-hoc vs. untreated.

[0300] As shown in Figure 8, neuronal spine density was increased by pCRMP2 aggregates or CRMP2 monomers compared to the untreated / aggregated buffer pool. This increase in spine density driven by CRMP2 / pCRMP2 was reversed by antibody NI-504.3E7 at antibody concentrations of 7.5 μM and 0.7 μM. Therefore, antibody NI-504.3E7 can reverse the increase in spine density induced by aggregated pCRMP2 and monomeric CRMP2 in hippocampal section culture models.

[0301] Example 10: NI-504.3E7 detects neurons in AD hippocampal sections. The binding of the antibody NI-504.3E7 to tissue sections was analyzed by immunohistochemistry (IHC). For the binding analysis of the antibody NI-504.3E7 in human Alzheimer's disease (AD) tissue, deparaffinized and rehydrated FFPE brain sections from human AD donors were boiled for 15 minutes in citrate buffer (20 mM citrate monohydrate, Sigma-Aldrich, catalog C7129 and 80 mM sodium citrate tribasic dihydrate, Sigma-Aldrich, catalog 71405), washed with water, and endogenous peroxidase was blocked by incubating the sections with methanol (Sigma-Aldrich) containing 3% hydrogen peroxide, followed by further washing with water. The sections were blocked at room temperature for 1 hour in blocking buffer (PBS (Gibco) containing 5% goat serum (Vector Laboratories); 5% horse serum (Vector Laboratories); and 4% bovine serum albumin (Sigma-Aldrich)). Antibody NI-504.3E7 was diluted to a concentration of 6.67 nM in PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP399; containing 0.1% Tween® 20 (Sigma-Aldrich)) containing 2% BSA, incubated overnight at 4°C on the sections, and then washed three times with water. Detection was performed using Biotin-SP AffiniPure goat anti-human IgG(H+L) (Jackson ImmunoResearch, catalog 109-065-088) at room temperature for 1 hour. Then, nuclear staining with diaminobenzidine (DAB, Thermo Fisher Scientific, catalog 1856090) and hematoxylin (Roth, catalog T865) was performed using the Vectastain ABC kit (Vector Laboratories) at room temperature for 20 minutes.

[0302] As shown in Figure 9, the antibody NI-504.3E7 detects neurons in the hippocampus of AD patients. Similar observations were made in hippocampal tissue from patients with PSP, PiD, FTD-PiD, and FTLD-TDP. The results are summarized in Table VIII below.

[0303] [Table 8]

[0304] Example 11: NI-504.3E7 reduces amyloid-β plaques in a transgenic mouse model of Alzheimer's disease. The in vivo efficacy of antibody NI-504.3E7 was analyzed in a tau-tubulin kinase-1 (TTBK1) / amyloid precursor protein (APP) dual transgenic mouse model of Alzheimer's disease. Specifically, the in vivo efficacy of antibody NI-504.3E7 was evaluated in female APPPS1 / TTBK1 transgenic mice (APPPS1#034829-JAX, Jackson Laboratories; TTBK1#B6 / 129-TTBK1-Tg Line 141, Tsuneya Ikezu, University of Nebraska) by weekly intravenous injection of 30 mg / kg over a 9-month period (from 3 months to 12 months of age). To enable repeated injections into the mouse model, the antibody for chimeric mouse IgG2a was formulated in PBS and used for injection. The volume of antibody and vehicle (PBS) was adjusted according to the animal's body weight. At the end of the in-life phase, mice were perfused transcardially with ice-cold PBS, the mouse brains were post-fixed with paraffin, and sagittal sections were prepared at 3 μm thickness.

[0305] For amyloid plaque analysis, deparaffinized, rehydrated FFPE mouse brain sections, including the cortex and hippocampus, were boiled in citrate buffer for 15 minutes and washed with water. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide solution (Sigma-Aldrich), followed by further washing with water. Sections were blocked in PBS containing 5% goat serum (Vector Laboratories), 5% horse serum (Vector Laboratories), and 4% bovine serum albumin (Sigma-Aldrich). Next, sections were incubated with 6.67 nM anti-β-amyloid antibody 6E10 (Biolegend), then washed with water, and detected with a 2.3 ug / ml secondary antibody Biotin-SP AffiniPure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, catalog 115-065-003) combined with nuclear staining using the Vectastain ABC kit (Vector Laboratories), diaminobenzidine (Thermo Fisher Scientific, catalog 1856090), and hematoxylin (Roth, catalog T865). For image analysis, three 3 μm thick sections located 60 μm apart were used for detection. The number and size of plaques in the depicted brain regions were evaluated by threshold determination using the Java®-based image processing program ImageJ. Amyloid-β plaque load represents the percentage of area covered by plaques stained with each antibody. Plaque size represents the average plaque size of all plaques in the cortex or hippocampus. Data are presented as mean + SEM. Group size: n=7, statistical analysis: unpaired t-test ≤ 0.0001.

[0306] As shown in Figures 10A and 10C, mice administered with the antibody NI-504.3E7 showed a 63% reduction in amyloid plaque load in the cortex and hippocampus. Furthermore, not only was the amyloid plaque load reduced, but the size of plaques in the cortex and hippocampus was also reduced (Figures 10B and 10D). In addition, in the cortex, a 50% reduction in plaque / area amount was observed in female TTBK1 / APP mice administered with the antibody NI-504.3E7 (area is defined as the entire cortex). The reduction in mean plaque size in female TTBK1 / APP mice administered with the antibody NI-504.3E7 was 30% in the cortex (Figure 10B) and 68% in the hippocampus (Figure 10D).

[0307] Example 12: NI-504.3E7 can bind to CRMP2 in fully differentiated SH-SY5Y cells. To further analyze the mechanism of action of antibody NI-504.3E7, its binding to differentiated cells was observed. For this analysis, SH-SY5Y cells were maintained in MEM medium (Thermo Fisher Scientific, catalog 41090028) supplemented with 1% MEM NEAA (Gibco), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 1% penicillin-streptomycin (Gibco), and 10% thermoinactivated FBS (Gibco). Differentiation of SH-SY5Y cells was performed in two stages, with 10 μM total trans retinoic acid (Sigma-Aldrich, catalog R2625) added to the medium during the 7-day pre-differentiation period. The medium was changed every 2-3 days. Predifferentiated cells were seeded at a density of 50,000 cells / well on poly-D-lysine coated 8-well 15μ slides (Ibidi, catalog 80825) and cultured for 10 days to fully differentiate in MEM medium (Thermo Fisher Scientific, catalog 41090028) supplemented with 1% MEM NEAA (Gibco), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 1% penicillin-streptomycin (Gibco), 50 ng / ml BDNF (Sigma-Aldrich, catalog SRP3014), 10 ng / ml NGF (RnD Systems), and 24 nM cholecalciferol (Sigma-Aldrich, catalog C9756). The medium was changed every 3-4 days. Fully differentiated cells were washed with PBS and fixed with 4% PFA (Sigma-Aldrich) for 20 minutes at room temperature. After two washes with PBS, the cells were permeabilized and blocked for 1 hour in PBS containing 10% FBS and 0.2% Triton®-X-100 (Sigma-Aldrich). Primary antibody incubation was performed overnight at 4°C using 50 nM human IgG control (isotype control antibody), 50 nM NI-504.3E7, 1 μg / ml anti-β-III-tubulin (Abcam, catalog ab78078), or 1% anti-CRMP2 (Abcam, catalog ab129082), diluted in blocking buffer (PBS containing 10% FBS).After two washes with PBS, the cells were stained in blocking buffer at room temperature for 1 hour with 2.7 μg / ml of the following secondary antibodies: anti-mouse Cy3 (Jackson ImmunoResearch, catalog 715-165-150), anti-rabbit Cy3 (Jackson ImmunoResearch, catalog 711-165-152), or anti-human Cy3 (Jackson ImmunoResearch, catalog 709-165-149). The nuclei were stained with DAPI (Thermo Fisher Scientific) in PBS for 5 minutes, and imaging was performed using inverted confocal microscopy (20x / dry objective lens, Leica DMI6000 AFC, model SP8).

[0308] As shown in Figure 11, the antibody NI-504.3E7 bound to differentiated SH-SY5Y cells, and no signal was observed with human IgG control or anti-human control. These results demonstrate the involvement of the target in the cell, namely CRMP2.

[0309] Example 13: NI-504.3E7 improves long-term potentiation (LTP) in aged mice. In addition to its ability to reduce amyloid plaque load in the transgenic mouse model of Alzheimer's disease shown in Example 11, the NI-504.3E7 antibody family's ability to improve long-term potentiation (LTP) was confirmed with one of its variants. In particular, similar to the phosphorylation assay described in Example 5, this assay was also performed exemplary using antibody NI-504.3E7_V20 to further confirm the fact that the variant antibody retains the activity of the original antibody.

[0310] The efficacy of the antibody NI-504.3E7_V20 was analyzed in aged C57Bl / 6J male mice (90 weeks old). The antibody for chimeric mouse IgG2a was formulated in PBS and administered by injection. The volume of antibody and vehicle (PBS) was adjusted according to the animal's body weight, and intraperitoneal injection was administered once a week for 5 weeks. Different doses of antibody, namely 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg, were administered. At the end of the in-life phase, the mice were anesthetized with 5% isoflurane and decapitated. The brain was dissected from the skull and immediately immersed for a total of 3–4 minutes in ice-cold, freshly prepared artificial cerebrospinal fluid (aCSF) (95% O2, 5% CO2) (pH 7.4) containing 124 mM NaCl, 3.75 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 26.5 mM NaHCO3, 1.25 mM NaH2PO4, and 10 mM glucose, while continuously supplied with oxygen. Acute sections (350 μm thick) were prepared using a vibratome (VT 1000S; Leica Microsystems, Bannockburn, Illinois). The sections were incubated in standard aCSF (124 mM NaCl, 3.75 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 26.5 mM NaHCO3, 1.25 mM NaH2PO4, 10 mM glucose) at room temperature for at least 1 hour before being recorded. For electrophysiological recording, a single section was placed in the recording chamber (room temperature) and immersed. For the remaining experiment, gas-treated aCSF (95% O2, 5% CO2; pH 7.4) was added at a constant rate (2 mL·min). -1The cells were continuously perfused with aCSF. Extracellular excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 zona radiata using a glass micropipette filled with aCSF. fEPSPs were induced by electrical stimulation of the Schaffer collateral-comiculation pathway at 0.1 Hz (i.e., a single pulse every 10 seconds) using a glass stimulating electrode (filamented borosilicate capillary glass, standard wall; OD: 1.5 mm; ID: 0.86 mm; length: 75 mm; Harvard Apparatus reference no. W3 30-0060) placed in the zona radiata. Stable baseline fEPSPs were then recorded for 10 minutes by stimulating at 30% of the maximum field amplitude (a single pulse every 10 seconds, i.e., 0.1 Hz). After 10 minutes of stabilization, LTP was assessed by delivering conditioning stimuli consisting of one train of 100 Hz stimulation. Following conditioning stimuli, a one-hour test period was recorded in which responses were again induced by single stimuli at the same intensity every 10 seconds (0.1 Hz). The signals were amplified using an Axopatch 200B amplifier (Molecular Devices, Union City, California) digitized with a Digidata 1322A interface (Axon Instruments, Molecular Devices, USA) and sampled at 10 kHz. Recordings were acquired using Clampex (Molecular Devices) and analyzed with Clampfit (Molecular Devices). LTP was calculated as a percentage of the baseline fEPSP gradient recorded over the 10 minutes prior to LTP induction. The mean gradient response from 40 to 60 minutes after LTP induction was used for analysis. Data are presented as mean + SEM. Group size: n=8, statistical analysis: two-way ANOVA, for the PBS group. * p<0.05, *** p<0.001.

[0311] As shown in Figure 12, LTP was reduced in aged mice compared to juvenile mice. See PBS control. However, after administering the antibody to aged mice, LTP increased compared to the LTP of PBS control mice, and especially after administration of 3 mg / kg, 10 mg / kg, and 30 mg / kg of the antibody, it nearly reached the LTP scores of juvenile mice. Therefore, the antibody NI-504.3E7_V20 was shown to improve LTP in aged mice as early as a dose of 3 mg / kg.

[0312] Example 14: NI-504.3E7 improves cognitive function in aged mice. In addition to its ability to reduce amyloid plaque load in the transgenic mouse model of Alzheimer's disease shown in Example 11 and its ability to improve LTP in aged mice shown in Example 13, the ability of the NI-504.3E7 antibody family to improve cognitive function in aged mice was confirmed by one of its variants. In particular, similar to the phosphorylation assay described in Example 5 and the LTP assay described in Example 13, this assay was also performed exemplary using the antibody NI-504.3E7_V20 to further confirm the fact that the variant antibody retains the activity of the original antibody.

[0313] The effect of the antibody NI-504.3E7_V20 on cognitive function was analyzed in aged C57Bl / 6J male mice. Mice aged 7 weeks (young) and 73 weeks (aged) were used in the experiment. The antibody and vehicle (PBS) were administered intraperitoneally once a week for 5 weeks. Cognitive function was assessed using a novel object recognition (NOR) test. During acclimatization on day 1, each mouse was placed in a test chamber, allowed to move freely for 10 minutes, and then returned to its home cage. Acclimatization was repeated twice, at least 4 hours apart. In the acquisition trial on day 2, two objects of the same color and shape were placed in the test chamber, and each mouse was allowed to move freely for 10 minutes. In the evaluation trial on day 3, one of the objects used in the acquisition trial (a known object) was replaced with a novel object of different color and shape, and each mouse was allowed to move freely for 10 minutes, with the process recorded using a video camera. The evaluation trial began 24 hours after the acquisition trial. Based on video footage recorded during the evaluation trials, the time spent searching for each object was measured using a stopwatch. The discrimination index was calculated using the following formula: (Time spent searching for new objects - Time spent searching for known objects) / (Time spent searching for new objects + Time spent searching for known objects). Data are presented as mean + SEM. Group size: n=11~12, statistical analysis: ## Compared to the younger group, p<0.01 (t-test). *** Compared to the PBS group, p<0.001 (Dunnett test).

[0314] As shown in Figure 13, the discrimination index was reduced in aged mice compared to young mice. See PBS control. However, after administration of the antibody at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, the discrimination index significantly increased. Thus, the antibody NI-504.3E7_V20 improved the discrimination index in aged mice as early as 1 mg / kg. These results demonstrate that the antibody NI-504.3E7_V20 improves cognitive function in aged mice.

Claims

1. A pharmaceutical composition comprising a human-derived monoclonal antibody or its antigen-binding fragment that selectively binds to human collagen reaction-mediated protein 2 (CRMP2), and a pharmaceutically acceptable carrier, wherein the antibody or its antigen-binding fragment binds to an epitope containing the amino acid sequence ASSAK (SEQ ID NO: 64).

2. The pharmaceutical composition according to claim 1, wherein the antibody can be bound to a CRMP2 peptide consisting of the amino acid sequence TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63).

3. The pharmaceutical composition according to claim 1, wherein the antibody can bind to full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2).

4. The pharmaceutical composition according to claim 3, wherein the antibody can reduce the level of pCRMP2 in a concentration-dependent manner when the antibody and CRMP2 are subjected to a phosphorylation assay, and / or can bind to human, mouse, and rat recombinant CRMP2 full-length protein.

5. The pharmaceutical composition according to claim 3, wherein the antibody binds to full-length human CRMP2 with an EC50 of approximately ≤10.0 nM and / or to pCRMP2 with an EC50 of approximately ≤100.0 nM, as determined by ELISA.

6. A pharmaceutical composition comprising a human-derived monoclonal antibody or its antigen-binding fragment, which selectively binds to human collagen reaction-mediated protein 2 (CRMP2) and whose variable region includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, and a pharmaceutically acceptable carrier, wherein the VH region includes complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region includes CDRs 1, 2, and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c) VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 61, or Herein, one or more of the CDRs may contain one or more amino acid substitutions in the pharmaceutical composition.

7. The antibody or antigen-binding fragment is in its variable region. (0) Sequence IDs 2 and 7, (1) Sequence IDs 2 and 56, (2) Sequence IDs 2 and 58, (3) Sequence ID 12 and Sequence ID 7, (4) Sequence IDs 12 and 56, (5) Sequence IDs 12 and 58, (6) Sequence ID 14 and Sequence ID 7, (7) Sequence IDs 14 and 56, (8) Sequence IDs 14 and 58, (9) Sequence ID 16 and Sequence ID 7, (10) Sequence IDs 16 and 56, (11) Sequence IDs 16 and 58, (12) Sequence ID 18 and Sequence ID 7, (13) Sequence ID 18 and Sequence ID 56, (14) Sequence ID 18 and Sequence ID 58, (15) Sequence ID 18 and Sequence ID 60, (16) Sequence ID 20 and Sequence ID 7, (17) Sequence IDs 20 and 56, (18) Sequence IDs 20 and 58, (19) Sequence IDs 20 and 60, (20) Sequence IDs 22 and 58, (21) Sequence IDs 24 and 58, (22) Sequence IDs 26 and 58, (23) Sequence IDs 28 and 58, (24) Sequence IDs 30 and 58, (25) Sequence IDs 32 and 58, (26) Sequence IDs 34 and 58, (27) Sequence IDs 36 and 58, (28) Sequence IDs 38 and 58, (29) Sequence IDs 40 and 58, (30) Sequence IDs 42 and 58, (31) Sequence IDs 44 and 58, (32) Sequence IDs 46 and 58, (33) Sequence IDs 48 and 58, (34) Sequence IDs 50 and 58, (35) Sequence IDs 22 and 56, (36) Sequence IDs 22 and 60, (37) Sequence IDs 52 and 56, (38) Sequence IDs 52 and 58, (39) Sequence IDs 52 and 60, and (40) Sequence IDs 54 and 58 It includes amino acid sequences of the VH region and VL region selected from, or The pharmaceutical composition according to claim 6, comprising a pair of VH amino acid sequences and VL amino acid sequences, wherein VH and / or VL may contain one or more amino acid substitutions and / or amino acid deletions.

8. The pharmaceutical composition according to claim 7, comprising a pair of VH and / or VL that are at least 90% identical to any one amino acid sequence from (0) to (40).

9. A pharmaceutical composition comprising a human-derived monoclonal antibody or its antigen-binding fragment, which selectively binds to human collagen reaction-mediated protein 2 (CRMP2) and whose variable region includes a heavy-chain variable (VH) region and a light-chain variable (VL) region, and a pharmaceutically acceptable carrier, wherein the VH region includes complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region includes CDRs 1, 2, and 3, where, (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3, (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4, (c) VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5, (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8, (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and (f) VL-CDR3 is a pharmaceutical composition containing the amino acid sequence of SEQ ID NO:

10.

10. An antibody or antigen-binding fragment contains amino acid sequences of a VH region and a VL region in its variable region. The VH region contains the amino acid sequence of SEQ ID NO: 22, and The pharmaceutical composition according to claim 9, wherein the VL region contains the amino acid sequence of SEQ ID NO:

58.

11. A pharmaceutical composition according to any one of claims 1 to 10, which can reverse the increase in neuronal spine density induced by CRMP2 when determined in an ex vivo hippocampal section culture model.

12. The pharmaceutical composition according to claim 11, wherein CRMP2 is a pCRMP2 aggregate or a CEMP2 monomer.

13. Antibodies or antigen-binding fragments (a) Includes the steady-state region; (b) comprising a heterologous polypeptide, wherein the antibody or antigen-binding fragment may be a chimeric mouse-human antibody; and / or (c) Single-chain Fv fragment (scFv), F(ab') fragment, F(ab) fragment, F(ab') ２ Selected from the group consisting of fragments, Fd, Fv, single-chain antibodies, disulfide-stabilized Fv (dsFv), and nanobodies, A pharmaceutical composition according to any one of claims 1 to 10.

14. The pharmaceutical composition according to claim 13, wherein the steady-state region is of the IgG type.

15. The pharmaceutical composition according to claim 13, wherein the steady-state region is of the IgG1 class or IgG1 isotype.

16. The pharmaceutical composition according to claim 13, wherein the heterologous polypeptide is the mouse IgG2a constant region.

17. A pharmaceutical composition comprising an antibody or antigen-binding molecule that competes with the antibody described in any one of claims 1 to 10 for specific binding to human CRMP2 and / or pCRMP2, and a pharmaceutically acceptable carrier, wherein the antibody is - Human CRMP2 is recognized preferentially over CRMP1, CRMP3, CRMP4, and CRMP5; - When CRMP2 is subjected to a phosphorylation assay, the phosphorylated CRMP2 (pCRMP2) level can be reduced in a concentration-dependent manner, and / or - When determined in an ex vivo hippocampal section culture model, it is possible to reverse the increase in neuronal spine density induced by CRMP2. Pharmaceutical composition.

18. The pharmaceutical composition according to any one of claims 1 to 10, wherein an antibody, an antigen-binding fragment, or immunoglobulin VH or VL is encoded by one or more polynucleotides.

19. The pharmaceutical composition according to claim 18, wherein the polynucleotide is cDNA and / or is operably linked to a heterologous nucleic acid.

20. The pharmaceutical composition according to claim 19, wherein the heterogeneous nucleic acid is an expression control sequence.

21. A pharmaceutical composition according to any one of claims 1 to 20 for the prevention, delay of progression, and / or treatment of a disorder of the nervous system.

22. The pharmaceutical composition according to claim 21, wherein the neurological disorder is Alzheimer's disease (AD), including mild, moderate, and severe AD, or mild cognitive impairment.

23. An agent for the prevention, inhibition of progression, and / or treatment of a disease of the nervous system, comprising the antibody or antigen-binding fragment described in claim 1.

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