Human-derived anti-brain failure response regulatory protein 2 (CRMP2) antibody

Abstract

Brain failure response regulatory protein 2 (CRMP2) is established as a therapeutic target for neurodegenerative disorders. Novel human anti-CRMP2 antibodies and fragments, derivatives and variants thereof are provided. Polynucleotides, vectors, host cells, and kits associated with the CRMP2 specific antibodies are also provided. The antibodies, immunoglobulin chains, and binding fragments, derivatives, and variants thereof are useful in pharmaceutical compositions for targeted therapy against CRMP2.

Description

Technical Field

[0001] This invention generally relates to novel human anti-brain aging response regulatory protein 2 (CRMP2) antibodies and equivalent antigen-binding molecules, and their uses, in particular, their use as medicaments for the treatment of neurodegenerative diseases. Background Technology

[0002] The pathogenesis of neurodegenerative diseases is highly complex and diverse. Neurodegenerative diseases are chronic conditions characterized by the progressive loss of neurons in the brain and spinal cord. The most prominent and prevalent category is dementia-related tau protein diseases, 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 material into insoluble amyloid plaques observed in Alzheimer's disease (AD), and the hyperphosphorylation of tau and the formation of intracellular neurofibrillary tangles observed in AD, PiD, PSP, and FTD, as well as neuronal death and a variety of related processes including neuroinflammation, synaptic and circuit dysfunction, mitochondrial and bioenergetic disorders. One of the earliest neuropathological changes in AD is neuronal degeneration, which may be caused by the deposition of β-amyloid plaques and neurofibrillary tangles in the brain. Neurodegeneration, including neuronal dysfunction and loss of functional synapses, can lead to cognitive and memory impairments, 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 profound and chronic effects of Alzheimer's disease (AD), current treatments fail to achieve satisfactory therapeutic effects or halt disease progression. Currently, only seven drugs are FDA-approved for AD treatment: donepezil, rivastigmine, galantamine, tacrine, memantine, aducanumab, and lecanemab. The first four are acetylcholinesterase inhibitors (AChEIs), while memantine is an N-methyl-D-aspartate receptor (NMDAR) antagonist, and aducanumab and lecanemab are human anti-Aβ antibodies targeting aggregated forms of Aβ. US and European guidelines list AChEIs as a first-line drug therapy for mild to moderate Alzheimer's disease (AD). However, AChEIs have shown only moderate efficacy for cognitive deficits and non-significant efficacy for functional abilities in mild to moderate AD. Memantine has shown very limited efficacy for cognitive symptoms, with no functional improvement. Various novel treatments for AD have been investigated, including pharmacological and non-pharmacological interventions, including anti-Aβ therapy, anti-Tau therapy, anti-neuroinflammatory therapy, neuroprotective agents, and brain stimulation.

[0004] Therefore, new drugs are still needed in the treatment of neurodegenerative diseases such as tau proteinosis, especially AD. Invention Overview

[0006] According to the present invention, the solution to the problem defined above is provided by embodiments characterized in the claims, disclosed in the specification, and further illustrated in the following examples and figures. Therefore, the present invention relates to anti-CRMP2 antibodies, CRMP2 binding fragments, and equivalent CRMP2 binding molecules, which preferably exhibit substantially the same binding characteristics and biological properties as shown in the appended examples and figures as the lead antibody named NI-504.3E7.

[0007] As described in the background section, since current drug therapies for the treatment of AD (the most common neurodegenerative disease) have only shown variable or insignificant success, the inventors have sought alternative target proteins that may play a role in the pathogenesis and / or progression of the disease, wherein targeting one or more forms of the target protein (e.g., specific conformations, denatured vs. naturally folded, monomers vs. polymers, phosphorylated vs. non-phosphorylated, etc.) leads to disease-improving effects.

[0008] Ultimately, after diligent work on various hypotheses and research on different types of disease models, the inventors devised a novel treatment strategy and selected brain aging response regulatory protein 2 (CRMP2) as a novel therapeutic target for neurodegenerative diseases such as Alzheimer's disease (AD).

[0009] CRMPs (Responsive Brain Response Proteins) represent a family of cytoplasmic proteins (CRMP1–5) that are highly expressed in the developing brain. CRMPs act as signaling molecules involved in regulating microtubule polymerization and stabilization, actin bridging and endocytosis, leading to neuronal differentiation; see Brustovetsky et al., Cells 10 (2021), 2781. CRMP2 is a cytoplasmic protein involved in axonal guidance and neurite growth via the serotonin 3A pathway. CRMP2 maintains high expression levels in adults compared to other members of the CRMP family. CRMP2 is a phosphoprotein; while it binds to tubulin heterodimers and promotes microtubule assembly and stabilization, phosphorylation of CRMP2 negatively regulates its ability to bind and stabilize tubulin. Therefore, phosphorylation affects the affinity of CRMP2 for assembled microtubules and tubulin dimers, which subsequently leads to axonal elongation or retraction. In Brustovetsky et al., Cells 10 (2021), 2781, the association between increased CRMP2 phosphorylation and decreased CRMP2 binding to activator-associated protein 1 (Drp1), Miro 2, and kinin 1 light chain (KLC1) is explained in more detail. The switching between inactive (phosphorylated) and active (non-phosphorylated) CRMP2 is a persistent physiological adaptive mechanism to prevent aberrant neuronal budding. Overall, a balance exists between active and inactive CRMP2. Furthermore, CRMP2 has been reported to be crucial for neurogenesis and associated with many neurodegenerative diseases, and is therefore considered a specific marker for AD; see, for example, Soutar et al., Curr Alzheimer Res 6 (2009), 269–278.

[0010] However, CRMP2 has not yet been considered a potential target for the inhibition of pathological processes in the brain, possibly because, in this sense, the pathological form of CRMP2 is unknown to known target proteins in neurodegeneration, such as Aβ and hyperphosphorylated Tau and their aggregates.

[0011] Therefore, the results of in vivo experiments conducted by the inventors are even more surprising, demonstrating that targeting CRMP2 via anti-CRMP2 antibodies resulted in significant therapeutic effects in various animal models of neurodegenerative diseases, one of which is illustrated in the examples. In particular, the inventors can demonstrate that the anti-CRMP2 antibody (designated NI-504.3E7) reduced the burden 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 burden in AD transgenic mouse models, targeting CRMP2 has been shown to improve long-term enhancement and cognitive function in aged mice; see Examples 13 and 14.

[0012] Characterization of antibody NI-504.3E7 in biochemical and in vitro cell assays revealed that this antibody selectively binds to human CRMP2 relative to the other four CRMP members (Examples 2 and 3). Figure 2 It can bind with high affinity to full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2) (Example 4), and reduce the level of pCRMP2 in a concentration-dependent manner (Example 5 and 6). Figure 4 This reverses the increase in dendritic spine density induced by pCRMP2 aggregates and monomeric CRMP, respectively; see Example 9 and... Figure 8 .

[0013] Given these properties of the antibody, the inventors hypothesize, but do not intend to be bound by theory, that the adverse effects of CRMP / CRMP2 in the brain caused by changes in the abundance and phosphorylation state of CRMP2 can be reversed by interfering with the levels of pCRMP2 and CRMP2, as well as the ratio between pCRMP2 and CRMP2, respectively.

[0014] For example, CRMP2 phosphorylation status has been reported to be altered in various neuropathologies, including Huntington's disease (HD) and Alzheimer's disease (AD). In particular, high levels of phosphorylated CRMP2 have been reported in the brains of individuals with AD associated with neurofibrillary tangles, suggesting that the accumulation of phosphorylated CRMP2 may be an early event in AD. It has been shown that not only are the levels of phosphorylated CRMP2 increased, but CRMP2 is also hyperphosphorylated, meaning that the relative level of CRMP2 phosphorylation is actually increased in brain regions associated with AD pathology; see Cole et al., J. Neurochem. 103 (2007), 1132-44.

[0015] Based on experimental evidence, the inventors believe, but are not intended to be bound by theory, that the reduction in CRMP2 and the increase in (hyper)phosphorylated CRMP2 can not only be markers of the neurodegenerative diseases described to date, but can actually actively participate in the onset and manifestation of the disease.

[0016] In this context, further experiments performed according to the invention have revealed that the exemplary antibody NI-504.3E7 is capable of detecting CRMP2 in cells, as demonstrated in Example 12 using fully differentiated SH-SY5Y cells.

[0017] In addition, to rule out the possibility that the observed biological activity depends on a specific amino acid sequence in the variable region of antibody NI-504.3E7, several variants with alterations in the frame region and one of the CDRs have been constructed and tested. As shown in Table VII, although some amino acid alterations affect the absolute EC50 of antibodies against CRMP2 and pCRMP2... 50 The values ​​are different, but for both CRMP and pCRMP2, all values ​​remain in the nanomolar range.

[0018] Therefore, in general, this invention relates to CRMP2 as a therapeutic target in the treatment of neurodegenerative diseases, and to any molecule capable of interfering with CRMP2 signaling, which is equivalent and similar in kind to that demonstrated against antibody NI-504.3E7, for use as a medicine, preferably for the prevention, delay, or treatment of neurodegenerative or cognitive disorders and / or for improving memory and learning abilities. Preferably, such a molecule is a CRMP2-binding molecule exhibiting the binding characteristics and biological activity demonstrated against antibody NI-504.3E7 as shown in the biochemical and in vitro cellular assays. Such a molecule can be screened and obtained from a compound library using the assays described in the examples, or can be computer-designed or derived from antibody NI-504.3E7.

[0019] However, due to their lack of immunogenicity and their use for proof of concept, human or human-derived antibodies are particularly preferred.

[0020] As illustrated in the examples, human monoclonal anti-CRMP2 antibodies have been cloned and identified during the complex antibody discovery process; see Example 1. Those antibodies were tested for their binding specificity and affinity. As can be derived from Table VI in Example 1, even some cloned antibodies showed EC50 values ​​of less than 20 nM in ELISA assays. 50 The values ​​show specific binding to the CRMP2 peptide, but it is noteworthy that only three antibodies showed EC50 values ​​of less than 20 nM in ELISA assays. 50The antibodies specifically bind to the CRMP2 protein. These three antibodies, namely NI-504.3E7, NI-504.10, and NI-504.12, further demonstrated binding signals in human brain tissue in immunohistochemical (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. Among these antibodies, only NI-504.3E7 showed selective binding to CRMP2, i.e., selective binding to both phosphorylated and non-phosphorylated CRMP2, while the other two antibodies also recognize CRMP1 and CRMP4.

[0021] More specifically, as determined in the ELISA assay, antibody NI-504.3E7 has been shown to have high affinity (i.e., at 4 nM EC50) 50 The full-length CRMP2 was bound, with only weak binding to CRMP1 observed, i.e., at an EC50 of 69 nM. 50 As determined in ELISA assays, antibody NI-504.3E7 binds very weakly to CRMP3, CRMP4, and CRMP5, and has no ECGs. 50 The values ​​can be determined. Similar results have been obtained by Western blot analysis, which showed that antibody NI-504.3E7 exhibited strong binding to full-length CRMP2, weak binding to CRMP1, and no binding to CRMP3, CRMP4, and CRMP5; see Example 2 and Figure 2 Therefore, the antibody does not cross-react with other CRMP proteins.

[0022] Furthermore, antibody NI-504.3E7 has been shown to bind to both full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2) in ELISA assays, with antibody NI-504.3E7 exhibiting EC50 values ​​in the low nanomolar range. 50 Specific recognition of CRMP2 and pCRMP2; see Example 4 and Table VII.

[0023] Conversely, most antibodies available to date bind only to phosphorylated CRMP2. For example, anti-pCRMP2 antibody 3F4 recognizes both triphosphorylated and diphosphorylated CRMP2, but not unphosphorylated or monophosphorylated CRMP2; see Uchida et al., Genes Cells 10 (2005), 165-79. Furthermore, commercially available polyclonal anti-phosphoryl-CRMP-2 (pThr... 509The antibody (SAB4504698 from Sigma-Aldrich (registered trademark), available at Merck KGaA, Darmstadt, Germany) is produced against a synthetic peptide derived from human CRMP2 around the Thr509 phosphorylation site and binds only to pCRMP2. This also applies to the antibody pCRMP-2 (Thr514)#9397 from Cell Signaling Technology, Inc., Leiden, The Netherlands. Furthermore, an anti-CRMP2 antibody specific for pCRMP2 phosphorylated at tyrosine 479 is described in WO 2011 / 007209 A1.

[0024] 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) have been 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). 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 described in Tables II and III. Specifically, three variant VL chains have been generated, namely VL_3E7_var1, VL_3E7_var2, and VL_3E7_var3, where the variants contain two, four, or five amino acid exchanges; see Tables V and III. Figure 1 The comparison was performed. In addition, 22 variant VH chains have been generated: 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_3 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, where variants contain two, three, four, five, six, seven, eight, or ten amino acid exchanges; see Table V and Figure 1 The comparisons were performed on the antibody variants (NI-504.3E7_V1 to NI-504.3E7_V40) that contained the variant VH and VL chains shown in Table IV.

[0025] As shown in Example 4 and Table VII, all variants retain their ability to bind CRMP2 and pCRMP2. Furthermore, the results of experiments performed as described above using the original antibody NI-504.3E7 have been confirmed with at least one exemplary variant.

[0026] Epitope mapping has been performed, as shown in Example 3, revealing that antibody NI-504.3E7 binds to a linear epitope near the C-terminus of CRMP2. Specifically, antibody NI-504.3E7 binds to the CRMP2 peptide, which comprises or consists of the amino acid sequences TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63), and the minimal epitope has been identified as comprising the amino acid sequence 516-ASSAK-520 (SEQ ID NO: 64).

[0027] As described above, antibody NI-504.3E7 reduces pCRMP2 levels in a concentration-dependent manner; see Example 5 and Figure 4 Therefore, not intended to be theoretically constrained, the therapeutic effect of anti-CRMP2 antibodies may be due to their ability to bind to and preserve CRMP2 and / or in turn neutralize CRMP2 and / or inhibit CRMP2 phosphorylation and / or capture phosphorylated CRMP2, resulting in an overall reduction in the amount of hyperphosphorylated CRMP2 and / or a reduction in the amount of phosphorylated CRMP2 that is excessively present in the brains of AD patients.

[0028] One explanation for this unique activity might be the epitope of antibody NI-504.3E7 (containing 516-ASSAK-520, SEQ ID NO: 64), which is 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 near the said phosphorylation sites) does not possess this property, i.e., it does not reduce pCRMP2 levels in a concentration-dependent manner. See Example 5 and Figure 4 .

[0029] As further illustrated in the examples, the cDNA sequence encoding antibody NI-504.3E7 was originally cloned from human memory B cells. As mentioned above, in humans, both phosphorylated and non-phosphorylated forms of CRMP2 exist. Furthermore, the ternary structure of the C-terminal region (490-572) of CRMP2 is flexible and somewhat random, altering its conformation upon post-translational modifications such as phosphorylation; see Nakamura et al., Front. Cell Neurosci. 14 (2020), 188. Therefore, antibodies initially produced in the human body (which are exposed to the natural environment and thus to the flexible and variable ternary structure of the CRMP2 C-terminal region) may have specific binding properties not found in antibodies produced against antigens, such as monoclonal anti-CRMP2 antibody 1B1 (sc-101348, Santa Cruz Biotechnology, Dallas, USA) and polyclonal anti-CRMP2 antibody (ab36201) from abcam, Cambridge, UK (both of which are produced against the synthetic CRMP2 C-terminal peptide), as well as polyclonal anti-CRMP2 antibody C2993 from Sigma-Aldrich (registered trademark), available at Merck KGaA, Darmstadt, Germany (which is produced against the unphosphorylated full-length CRMP2).

[0030] In fact, as described in Mileusnic and Rose, Journal of Neurochemistry 118 (2011), 616-635, doi: 10.1111 / j.1471-4159.2011.07193.x, antibody ab36201, when administered to young chicks, induces amnesia in passive avoidance tasks. Therefore, compared to the antibody of the present invention that improves cognitive function in mice, this antibody has a negative impact on the cognitive function of chicks.

[0031] It is also known that hyperphosphorylated Tau co-localizes with phosphorylated CRMP2; see Takata et al., Am. J. Pathol 175 (2009), 17-24. Experiments conducted within the scope of this invention have now demonstrated that recombinant (p)Tau and (p)CRMP2 do indeed co-aggregate in ThioT assays (see Example 8 and...). Figure 7Furthermore, as previously mentioned, antibody NI-503.3E7 has been shown to reduce amyloid plaque load in transgenic AD mouse models. Therefore, as a precaution, antibody NI-504.3E7 is expected to interfere with the co-aggregation of pTau and pCRMP2. In this context, unless otherwise explicitly stated, the term "NI-504.3E7" refers to the original antibody NI-504.3E7 and any variant thereof, and represents an equivalent CRMP2-specific binding molecule.

[0032] Extracellular neuritis plaques composed of amyloid-β (Aβ) protein and intracellular neurofibrillary tangles containing phosphorylated Tau protein are two hallmark proteins of Alzheimer's disease (AD), and the individual neurotoxicity of these proteins in AD has been extensively studied. Furthermore, the coexistence of Aβ plaques and phosphorylated Tau has been shown to be associated with a mechanism by which Aβ promotes the spread of Tau accumulation in neuritis plaques. The interaction between Aβ and Tau mediates cognitive impairment in patients with AD; see Zhang et al., Int J Biol Sci. 17 (2021), 2181-2192. It is also known that therapeutic agents developed for the treatment of AD typically 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 amyloid plaques from the brain and neurotoxic oligomeric Aβ material from presynaptic axonal terminals, thereby protecting axons from amyloid damage. This results in the retention of unphosphorylated microtubule-bound Tau within its physiological axonal localization, thus preventing the aberrant relocation of Tau from axon to somatic dendritic compartments in amyloid-affected neurons, where Tau is aberrantly phosphorylated and becomes prone to aggregation. Furthermore, extracellular material of pathological tau is removed via phagocytosis by microglia or macrophages, as observed by the reduction in CSF levels of pathological phosphate tau in patients treated with aducanumab; see, for example, International Application WO 2021 / 081101 A1 and Cummings et al. Alz.Res.Therapy 13 (2021), 98; Salloway and Cummings, Aducanumab, AmyloidLowering, and Slowing of Alzheimer Disease, in Neurology 97 (2021), 543-544.

[0033] As described above, CRMP2 can be phosphorylated or unphosphorylated, with phosphorylation regulating neurite growth. CRMP2 can be phosphorylated sequentially by GSK3β at S518, T514, and T509, but only after prior phosphorylation by Cdk5 at Ser522. Examples 2 and 4 have shown that antibody NI-504.3E7 binds to the full-length phosphorylated CRMP2, and further experiments have been performed to demonstrate that antibody NI-504.3E7 can bind to the phosphorylated CRMP2 peptide. Experiments using phosphorylated peptides to determine the binding specificity of antibody NI-504.3E7 showed that antibody NI-504.3E7 had an affinity for the CRMP2 peptide phosphorylated at position T514 that was approximately 300-fold lower than that for the unphosphorylated peptide and the peptides phosphorylated at positions T509 and S522. This indicates that the phosphorylation at T514 impairs the binding of antibody NI-504.3E7, possibly due to its close proximity to the antibody's minimal epitope 516-ASSAK-520; see Example 6 and... Figure 5 .

[0034] Antibody NI-504.3E7 was further shown to bind to the full-length recombinant CRMP2 protein in humans, mice, and rats. Internalization of antibody NI-504.3E7 was also shown in SH-SY5Y cells; see Example 12 and... Figure 11 .

[0035] As shown in Example 9, aggregated pCRMP2 and monomeric CRMP2 increase the density of neuronal dendritic spines, wherein the increase in dendritic spine density driven by CRMP2 / pCRMP2 is reversed by two different concentrations of NI-504.3E7, as determined by an ex vivo hippocampal slice culture model. This assay can be used as evidence of antibody activity, particularly as evidence of antibody interference with CRMP2 signaling, which has not been previously described.

[0036] Therefore, the present invention relates to antibodies and equivalent CRMP2 binding molecules, which may be binding fragments or derivatives of NI-504.3E7, having substantially the same binding specificity and activity as antibody NI-504.3E7. This means that the present invention relates to antibodies and equivalent CRMP2 binding molecules that (i) preferentially recognize human CRMP2 rather than CRMP1, CRMP3, CRMP4, and CRMP5, as can be tested in ELISA assays and Western blot analyses as described in Example 2, (ii) are able to reduce pCRMP2 levels in a concentration-dependent manner when CRMP2 is phosphorylated, as can be tested as described in Example 5, and / or (iii) are able to reverse the increase in neuronal dendritic spine density induced by CRMP2, as measured in an ex vivo hippocampal slice culture model, as can be tested as described in Example 9. Therefore, the present invention relates to antibodies and equivalent CRMP2 binding molecules, which may be binding fragments or derivatives of NI-504.3E7, having substantially the same binding specificity and activity as antibody NI-504.3E7 in terms of feature (i), or feature (ii), or feature (iii), or features (i) and (ii), or features (ii) and (iii), or features (ii) and (iii) or features (i), (ii) and (iii).

[0037] The characteristics can be readily determined based on the experiments and assays disclosed in the appended embodiments, wherein antibody NI-504.3E7 can be used as a reference antibody. Typically, this equivalent CRMP2 binding molecule will compete with the corresponding reference antibody for binding to CRMP2 and pCRMP2 at the same epitopes and peptides described above. Preferably, this equivalent CRMP2 binding molecule has the same epitopes as the reference antibody.

[0038] As described above, 40 variants of antibody NI-504.3E7 (NI-504.3E7_V1 to NI-504.3E7_V40) have been 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).

[0039] Therefore, in one embodiment, the antibody of the present invention can be characterized by the complementarity-determining region (CDR) or hypervariable region of the variable heavy (VH) chain and variable light (VL) chain of antibody NI-504.3E7, wherein the VH and VL comprise the amino acid sequences of SEQ ID NO: 2 (VH_3E7) and SEQ ID NO: 7 (VL_3E7) as shown in Tables II and IV.

[0040] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V1, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 2 (VH_3E7) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0041] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V2, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 2 (VH_3E7) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0042] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V3, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 12 (VH_3E7_var1) and SEQ ID NO: 7 (VL_3E7) as shown in Tables II and IV.

[0043] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V4, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 12 (VH_3E7_var1) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0044] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V5, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 12 (VH_3E7_var1) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0045] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V6, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 14 (VH_3E7_var2) and SEQ ID NO: 7 (VL_3E7) as shown in Tables II and IV.

[0046] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V7, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 14 (VH_3E7_var2) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0047] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V8, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 14 (VH_3E7_var2) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0048] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V9, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 16 (VH_3E7_var3) and SEQ ID NO: 7 (VL_3E7) as shown in Tables II and IV.

[0049] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V10, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 16 (VH_3E7_var3) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0050] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V11, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 16 (VH_3E7_var3) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0051] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V12, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 7 (VL_3E7) as shown in Tables II and IV.

[0052] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V13, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0053] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V14, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0054] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V15, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 18 (VH_3E7_var4) and SEQ ID NO: 60 (VL_3E7_var3) as shown in Tables II and IV.

[0055] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V16, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 7 (VL_3E7) as shown in Tables II and IV.

[0056] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V17, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0057] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V18, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0058] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V19, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 20 (VH_3E7_var5) and SEQ ID NO: 60 (VL_3E7_var3) as shown in Tables II and IV.

[0059] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V20, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 22 (VH_3E7_var6) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0060] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V21, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 24 (VH_3E7_var6a) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0061] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V22, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 26 (VH_3E7_var6b) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0062] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V23, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 28 (VH_3E7_var6c) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0063] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V24, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 30 (VH_3E7_var6d) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0064] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V25, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 32 (VH_3E7_var6ab) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0065] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V26, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 34 (VH_3E7_var6ac) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0066] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V27, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 36 (VH_3E7_var6ad) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0067] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V28, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 38 (VH_3E7_var6bc) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0068] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V29, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 40 (VH_3E7_var6bd) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0069] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V30, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 42 (VH_3E7_var6cd) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0070] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V31, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 44 (VH_3E7_var6bcd) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0071] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V32, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 46 (VH_3E7_var6acd) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0072] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V33, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 48 (VH_3E7_var6abd) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0073] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V34, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 50 (VH_3E7_var6abc) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0074] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V35, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 22 (VH_3E7_var6) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0075] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V36, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 22 (VH_3E7_var6) and SEQ ID NO: 60 (VL_3E7_var3) as shown in Tables II and IV.

[0076] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V37, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 52 (VH_3E7_var7) and SEQ ID NO: 56 (VL_3E7_var1) as shown in Tables II and IV.

[0077] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V38, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 52 (VH_3E7_var7) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0078] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V39, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 52 (VH_3E7_var7) and SEQ ID NO: 60 (VL_3E7_var3) as shown in Tables II and IV.

[0079] In one embodiment, the antibody of the present invention can be characterized by the CDR or hypervariable regions of the VH and VL chains of antibody NI-504.3E7_V40, wherein the VH and VL contain the amino acid sequences of SEQ ID NO: 54 (VH_3E7_var8) and SEQ ID NO: 58 (VL_3E7_var2) as shown in Tables II and IV.

[0080] While the present invention is illustrated and described by reference to human antibodies originally obtained in experiments conducted according to the invention and described in the examples, it should be understood that the invention encompasses antibodies and equivalent CRMP2-binding molecules, such as synthetic and biotechnological derivatives of antibody NI-504.3E7, meaning any engineered antibody or antibody-like CRMP2-binding molecule synthesized by chemical or recombinant techniques that retains one or more functional properties of the subject antibody, particularly its preferential recognition of human CRMP2 relative to CRMP1, CRMP3, CRMP4, and CRMP5, its ability to reduce pCRMP2 levels in a concentration-dependent manner when CRMP2 is phosphorylated, and / or its ability to reverse CRMP2-induced increases in neuronal dendritic spine density, as determined in ex vivo hippocampal slice culture models. Therefore, although the invention may be described by reference to one or more antibodies for brevity, unless otherwise stated, its synthetic and biotechnological derivatives and equivalent CRMP2-binding molecules are meant and included within the meaning of the term "antibody".

[0081] As described above and demonstrated in the embodiments, this invention establishes CRMP2 and pCRMP2 as therapeutic targets for the first time in the treatment of neurodegenerative diseases. Therefore, this invention also relates to molecules capable of targeting CRMP2 and pCRMP2 respectively and interfering with CRMP2 signaling, which are used as pharmaceuticals. Specifically, these molecules are used in the prevention of neurodegenerative diseases, cognitive disorders, delaying the progression of neurodegenerative diseases, cognitive disorders, or in the treatment of neurodegenerative diseases, cognitive disorders, and / or in methods for improving memory and learning abilities.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety. Materials, methods, and examples are illustrative only and are not intended to be limiting.

[0083] Further embodiments of the present invention will be apparent from the following description, examples and claims. Attached Figure Description

[0084] [ Figure 1-1 ] Figure 1The amino acid sequence alignment of the variable region, which is the heavy chain (VH; A) and light chain (VL; B) of the anti-CRMP2 specific human antibody NI-504.3E7 and 40 variants NI-504.3E7_V1 to V40, contains amino acid substitutions in VH, VL, and / or CDR. The complementarity-determining region (CDR) is indicated by an underlined CDR. The regions flanking the CDR are called the framework regions. The Kabat numbering scheme (see http: / / www.bioinf.org.uk / abs / ; Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983)) is used, as given in Table 1 below. Unless otherwise stated, the numbering of specific amino acid residue positions in the antibodies of the present invention or their CRMP2 binding fragments, variants, or derivatives is based on the Kabat numbering system; however, this is theoretical and may not be equally applicable to every antibody of the present invention. For example, depending on the position of the first CDR, subsequent CDRs can move in either direction. Therefore, if regarding Figure 1 In the event of any unintentional error or inconsistency in the indication of the CDR in the sequence listing, a person skilled in the art, based on the disclosure of this application (i.e., the variable heavy (VH) chain and variable light (VL) chain amino acid sequences of antibody NI-504.3E7 and its variants), is capable of determining the correct CDR sequence according to Kabat, which is used to define the claimed antibody and its CRMP2 binding fragment. The variable heavy chain VH sequence (A) of antibody NI-504.3E7 as shown in SEQ ID NO: 2 and the light chain VL sequence (B) of antibody NI-504.3E7 as shown in SEQ ID NO: 7 are described, both compared with the VH and VL sequences of 40 variants of NI.504.3E7. As further explained in the specification, conserved amino acid substitutions are preferred within the CDR and / or frame regions, taking into account the physicochemical properties of the original amino acid alone or with adjacent amino acids, as described in Mirsky et al., Mol. Biol. Evol. 32 (2014) 806-819, page 813. Figure 6 As shown, especially in AB or LG models, for example, the positions of two amino acids are interchanged.

[0085] [ Figure 1-2 ] Figure 1 (Continued)

[0086] [ Figure 1-3 ] Figure 1 (Continued)

[0087] [Figure 1-4 ] Figure 1 (Continued)

[0088] [ Figure 1-5 ] Figure 1 (Continued)

[0089] [ Figure 1-6 ] Figure 1 (Continued)

[0090] [ Figure 2 ] Figure 2 The anti-CRMP2 antibody NI-504.3E7 of this invention exhibits preferential binding to CRMP2 and weak binding to CRMP1. (A) The binding activity of NI-504.3E7 to recombinant human CRMP proteins 1-5 was assessed by ELISA. An S-shaped curve was plotted. Data are presented as mean + SEM. (B) The binding specificity of NI-504.3E7 to recombinant human CRMP proteins 1-5 was assessed by Western blotting. Numbers represent molecular weight in kilodaltons (kDa).

[0091] [ Figure 3 ] Figure 3 (A) Epitope determination of antibody NI-504.3E7 binding. Epitope mapping using an overlapping peptide array representing human CRMP2 protein shows 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) Anti-human control (secondary antibody control) shows no binding.

[0092] [ Figure 4-1 ] Figure 4 The anti-CRMP2 antibody NI-504.3E7 of this invention reduces pCRMP2 levels in a concentration-dependent manner. After phosphorylation for 1 h or 24 h in the presence of different concentrations of CRMP2-specific antibody or human IgG control (isotype control antibody), the phosphorylation levels of three selected phosphate sites (T509 (AE), T514 (FJ), S522 (KO)) were analyzed in a phosphorylation assay, which was read out by Western blot (A, F, K) (the numbers represent molecular weight in kilodaltons (kDa)). Quantification of the Western blot analysis was based on signal band intensity (BE, GJ, LO). Specifically, the signal band intensity was measured and directly displayed or normalized to the total CRMP2 protein level assessed using the commercial CRMP2 antibody C2993.

[0093] [ Figure 4-2 ] Figure 4 (Continued)

[0094] [ Figure 4-3 ] Figure 4(Continued)

[0095] [ Figure 5 ] Figure 5 The anti-CRMP2 antibody NI-504.3E7 of the present invention specifically binds to CRMP2 and the pCRMP2 peptide, as determined by ELISA. Antibody NI-504.3E7 targets the CRMP2 peptide at position T... 514 The affinity of phosphorylated CRMP2 peptides at position T is greater than that of unphosphorylated peptides and peptides at position T. 509 and S 522 The phosphorylated peptide was approximately 300-fold lower. Phosphorylation of threonine 514 showed partially impaired binding of antibody NI-504.3E7. Unphosphorylated CRMP2 protein and BSA were used as positive and negative controls, respectively. The lines depict an S-shaped curve fit. The sequence of the peptide bound by antibody NI-504.3E7 is indicated. Data are presented as mean + SEM.

[0096] [ Figure 6 ] Figure 6 The anti-CRMP2 antibody NI-504.3E7 of this invention shows that it binds to recombinant human, mouse, and rat CRMP2 proteins as orthologous proteins. The binding specificity of NI-504.3E7 was detected by Western blotting on recombinant human CRMP2 (SUMO-CRMP2), mouse CRMP2 (CRMP2-His6), and rat CRMP2 (CRMP2-His6) proteins. No binding was observed on human IgG control (isotype control antibody) and anti-human control (secondary antibody control). Numbers indicate molecular weight in kilodaltons (kDa).

[0097] [ Figure 7 ] Figure 7 In the presence of recombinant Tau and pTau proteins, the aggregation of recombinant CRMP2 and pCRMP2 increased. Thio-T positive signal (RFU) indicated the aggregation levels of different proteins or protein combinations in a 240-hour aggregation assay. Culture medium was used as a negative control. Data are presented as mean + SEM.

[0098] [ Figure 8 ] Figure 8 The increase in dendritic spine density driven by CRMP2 / phosphate-CRMP2 was reversed by two different concentrations of the anti-CRMP2 antibody NI-504.3E7 of this invention. The dendritic spine density of neurons containing pCRMP2 aggregates and CRMP2 monomers (monom.CRMP2) was assessed in a hippocampal slice culture model. Dendritic spine density was assessed by threshold image analysis. Data are presented as mean + SEM. Statistical analysis: One-way ANOVA ***p<0.01**p<0.01; compared with untreated controls, analysis was performed using the Kruskal-Wallis post-hoc test.

[0099] [ Figure 9 ] Figure 9 The present invention describes the detection of neurons in the hippocampus of Alzheimer's disease patients using the anti-CRMP2 antibody NI-504.3E7. A) Obvious neuronal cell staining was observed in Alzheimer's disease tissue using antibody NI-504.3E7, which was absent in the anti-human control. B) Larger scale of the delineated region is shown in magnified form.

[0100] [ Figure 10 ] Figure 10 The anti-CRMP2 antibody NI-504.3E7 of this invention reduces amyloid β plaques in female APPPS1 / TTBK1 transgenic mice. Threshold-based image analysis was performed on histological staining of amyloid β in the cortex and hippocampus. The number and size of amyloid β plaques were compared between NI-504.3E7 and PBS-injected APPPS1 / TTBK1 transgenic mice. Mice receiving 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). In female TTBK1 / APP mice receiving antibody NI-504.3E7, the mean plaque size in the cortex was reduced by 30% (B), and the mean plaque size in the hippocampus was reduced by 68% (D). Data are presented as mean + SEM. Group size: n=7, Statistical analysis: unpaired t-test. < 0.0001.

[0101] [ Figure 11 ] Figure 11 The anti-CRMP2 antibody NI-504.3E7 of this invention binds to differentiated SH-SY5Y cells. Positive Cy3 signaling was detected in cells stained with NI-504.3E7. SH-SY5Y cells stained with a commercial anti-CRMP2 antibody (Abcam 129082) and an anti-β-III-tubulin antibody were used as positive controls. No signal was observed with human IgG control (isotype control antibody) and anti-human control (secondary antibody control). Cell nuclei were visualized using DAPI. Images were acquired using a confocal microscope (20x objective). Scale bar: 50 μM.

[0102] [ Figure 12 ] Figure 12The anti-CRMP2 antibody NI-504.3E7 of this invention improves the long-term potentiation (LTP) in aged C57Bl / 6J mice. LTP analysis was performed in young mice and a control group (aged mice receiving PBS). Compared with young mice, LTP signaling was decreased in aged mice. After administration of the antibody to aged mice, LTP signaling increased again, thus showing 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.

[0103] [ Figure 13 ] Figure 13 The anti-CRMP2 antibody NI-504.3E7 of this invention improves cognitive function in aged C57Bl / 6J mice. The discrimination index was measured in young mice and a control group (aged mice receiving PBS), and the discrimination index was 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 to 12. Statistical analysis: **P < 0.01, compared with the young group (t-test); ###p < 0.001, compared with the PBS group (Dunnett test).

[0104] Description of the implementation plan Invention Details

[0106] This invention relates to embodiments characterized as claimed, disclosed in the specification, and further illustrated in the following examples and figures. Generally, this invention relates to recombinant, human, preferably monoclonal antibodies that bind to human brain aging response regulatory protein 2 (CRMP2), and equivalent binding molecules, such as their binding fragments and derivatives. In particular, this invention relates to anti-CRMP2 antibodies with unique binding characteristics, namely, those capable of binding both full-length unphosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2), and preferably binding to an epitope comprising the amino acid sequence 516-ASSAK-520 (SEQ ID NO: 64).

[0107] To avoid any doubt, it is important to emphasize that the use of phrases such as "in some embodiments," "in a particular embodiment," "in certain circumstances," "in some cases," "in further embodiments," and "in one embodiment" means that when reading any of the embodiments described herein, each feature of these embodiments should be considered in combination, and this disclosure must be treated in the same manner as if the combination of features of these embodiments were explained in detail in one embodiment. The same applies to any combination of embodiments and features shown in the appended claims and examples, which are also intended to be combined with features from the corresponding embodiments disclosed in the specification, where embodiments are characterized by subordination only for consistency and brevity; however, in practice, each combination of embodiments and features that can be interpreted due to (multiple) subordination must be considered as a literal disclosure and not as a choice among different options.

[0108] Unless otherwise stated, the terminology used in this article follows the definitions provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised in 2000, reprinted in 2003, ISBN 0 19 850673 2; second edition published in 2006, ISBN 0-19-852917-1 978-0-19852917-0.

[0109] The term "derivative" refers to synthetic and biotechnological derivatives of antibodies, meaning any engineered antibody and CRMP2-binding molecule synthesized through chemical or recombinant techniques that retains the functional properties of the parent antibody and the human antibody, respectively.

[0110] Furthermore, when referring to the anti-CRMP2 antibody of the present invention, equivalent binding molecules such as their binding fragments and derivatives are also covered by this definition.

[0111] The antibody is "human-derived," meaning it has a CDR region derived from a human antibody, or where the antibody's CDR includes one or more variant CDR regions derived from a human antibody, thereby maintaining the binding specificity of the human antibody. Human antibodies typically contain a human frame region, preferably a frame region derived from a human antibody, or one or more variant human frame regions derived from a human antibody. Therefore, human antibodies typically contain human VL and VH domains, or variant human VL or VH domains. Human antibodies typically contain human constant regions. The person is preferably someone who does not suffer from neurodegenerative diseases such as Alzheimer's disease or tau proteinosis. The person is preferably healthy. The person can be elderly. Elderly individuals are typically over 60, over 65, over 70, over 75, or over 80 years of age. Antibodies derived from humans can be isolated from antibody libraries, such as blood lymphocyte libraries collected from elderly individuals.

[0112] The CDR region of a variant of a human 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 a preferred embodiment, only VL-CDR3 contains one or more amino acid substitutions, preferably one, two, three, or four, more preferably one or two, and most preferably one.

[0113] The variant framework region of a human antibody typically contains one or more amino acid substitutions, preferably one, two, three, four, five or six, more preferably one or two amino acid substitutions, and most preferably one amino acid substitution.

[0114] In a preferred embodiment, variants VH and VL each contain one or more amino acid substitutions and / or deletions, preferably one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions and / or deletions. Preferably, variants of the VL chain contain one, two, three, four, or five amino acid substitutions and / or deletions, and / or variants of the VH chain contain one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions and / or deletions. Most preferably, variants VH and VL each contain one or two amino acid substitutions and / or deletions.

[0115] Preferred conserved amino acid substitutions are selected, taking into account the physicochemical properties of the original amino acid alone or with adjacent amino acids. 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.

[0116] As explained herein, variant antibodies NI-504.3E7_V1 to NI-504.3E7_V40 have been generated based on antibody NI-504.3E7. Therefore, when referring to the antibodies of this invention, the term includes not only antibody NI-504.3E7, equivalent binding molecules such as their binding fragments and derivatives, and competing antibodies, but also the corresponding variants. In particular, the results of experiments performed using the original antibody NI-504.3E7 have been confirmed using one or more variants.

[0117] Therefore, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that selectively binds to human brain aging response regulatory protein 2 (CRMP2), wherein the antibody binds to the C-terminus of CRMP2, preferably wherein the antibody is capable of binding to a CRMP2 peptide consisting of the amino acid sequences TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63) and / or wherein the antibody requires the amino acid sequence 516-ASSAK-520 (SEQ ID NO: 64) in CRMP2 for binding to the C-terminus of CRMP2. The antibody or antigen-binding fragment typically binds to CRMP2 such that when CRMP2 is phosphorylated, the level of phosphorylated CRMP2 (pCRMP2) decreases in a concentration-dependent manner. The phosphorylation assay can be performed in conjunction with Western blot analysis. The phosphorylation assay can detect the levels of T509 pCRMP2, T514 pCRMP2, and / or S522 pCRMP2. The antibody typically binds to the full-length recombinant CRMP2 protein in mice and rats, as well as the full-length recombinant CRMP2 protein in humans. Antibodies or antigen-binding fragments are typically able to reverse the CRMP2-induced increase in neuronal dendritic spine density, as measured in ex vivo hippocampal slice culture models. Antibodies are preferably human or of human origin.

[0118] The antibody selectively binds to human brain aging response regulatory protein 2 (CRMP2), wherein the antibody preferentially recognizes human CRMP2 relative to CRMP1, CRMP3, CRMP4, and CRMP5. The selective binding of the antibody can be determined using an ELISA assay and Western blot analysis as described in Example 2.

[0119] The antibody or antigen-binding molecule of the present invention may be an antibody or antigen-binding molecule that is:

[0120] - Prioritize the identification of human CRMP2 over CRMP1, CRMP3, CRMP4 and CRMP5;

[0121] - When phosphorylation of CRMP2 is measured, the level of phosphorylated CRMP2 (pCRMP2) can be reduced in a concentration-dependent manner; and / or

[0122] - It can reverse the increase in neuronal dendritic spine density induced by CRMP2, as measured in an in vitro hippocampal slice culture model.

[0123] The antibody or antigen-binding molecule inhibits the binding of the antibody to human CRMP2. This antibody has a variable region comprising a heavy chain variable (VH) region and a light chain variable (VL) region. The VH region contains complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region contains CDRs 1, 2, and 3.

[0124] (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3.

[0125] (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4.

[0126] (c) VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5.

[0127] (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8.

[0128] (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and

[0129] (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or 61 that binds to human CRMP2 and / or pCRMP2. Additionally, the binding of an antibody or antigen-binding molecule to human CRMP2 can be inhibited by an antibody comprising the VH and VL CDR amino acid sequences (a) to (f). The antibody is preferably human or of human origin.

[0130] The antibody or antigen-binding molecule of the present invention may be an antibody or antigen-binding molecule that is:

[0131] - Prioritize the identification of human CRMP2 over CRMP1, CRMP3, CRMP4 and CRMP5;

[0132] - When phosphorylation of CRMP2 is measured, the level of phosphorylated CRMP2 (pCRMP2) can be reduced in a concentration-dependent manner; and / or

[0133] - It can reverse the increase in neuronal dendritic spine density induced by CRMP2, as measured in an in vitro hippocampal slice culture model.

[0134] The binding of an antibody or antigen-binding molecule to human CRMP2 is inhibited by the antibody, which has a variable region comprising a heavy chain variable (VH) region and a light chain variable (VL) region. The VH region contains complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region contains CDRs 1, 2, and 3.

[0135] (a) VH-CDR1 contains the amino acid sequence of SEQ ID NO: 3.

[0136] (b) VH-CDR2 contains the amino acid sequence of SEQ ID NO: 4.

[0137] (c) VH-CDR3 contains the amino acid sequence of SEQ ID NO: 5.

[0138] (d) VL-CDR1 contains the amino acid sequence of SEQ ID NO: 8.

[0139] (e) VL-CDR2 contains the amino acid sequence of SEQ ID NO: 9, and

[0140] (f) VL-CDR3 contains the amino acid sequence of SEQ ID NO: 10 or 61 that binds to human CRMP2 and / or pCRMP2. Additionally, antibodies or antigen-binding molecules can inhibit the binding of antibodies containing the VH and VL CDR amino acid sequences (a) to (f) to human CRMP2.

[0141] Inhibition of antibody binding to human CRMP2 can be determined using a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to the target antigen (e.g., electrochemiluminescence assay, competitive ELISA, solid-phase radioimmunoassay (SPRIA), or blocking Western blot).

[0142] Furthermore, information regarding CRMP2 protein and anti-CRMP2 antibodies, their recombinant production, purification, modification in host cells, formulation in pharmaceutical compositions, and therapeutic uses, as well as terms and features common in the art, will be relied upon by those skilled in the art when practicing the claimed invention; see, for example, Antibodies A Laboratory Manual 2 nd This edition, published in 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, also describes antibody purification and storage; engineered antibodies, including the use of degenerate oligonucleotides, 5'-RACE, phage display and mutagenesis, immunoblotting protocols, and state-of-the-art screening and labeling techniques.

[0143] As further illustrated in the examples, the antibodies of the present invention were initially isolated from human donors and showed specific recognition of at least human full-length CRMP2, and optionally mouse and rat full-length CRMP2; see Example 7.

[0144] The CRMP2 sequence is highly conserved across mammalian species, and as a precaution, the antibody of the present invention is expected to generally bind to mammalian CRMP2, given that antibody NI-504.3E7 binds to human, mouse, and rat CRMP2. Therefore, in one embodiment, the antibody of the present invention is capable of specifically binding to mammalian CRMP2, preferably to recombinant full-length mammalian CRMP2. In a preferred embodiment, the antibody of the present invention is capable of binding to recombinant human CRMP2, particularly recombinant full-length human CRMP2. In one embodiment, the antibody of the present invention is also capable of binding to mouse and rat CRMP2, particularly recombinant full-length mouse and rat CRMP2.

[0145] Furthermore, antibody NI-504.3E7 did not bind to E. coli cell lysates, confirming that there was no cross-reactivity when recombinant CRMP2 was produced in E. coli.

[0146] As further shown in the examples, the antibodies of the present invention not only specifically bind to full-length human CRMP2, optionally mouse and rat CRMP2, but also selectively bind to CRMP2. Specifically, it has been shown (see Example 2) that the antibodies of the present invention bind to full-length human CRMP2 in Western blotting, but not to full-length human CRMP1, CRMP3, CRMP4, and CRMP5. Therefore, 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 only show weak binding to CRMP1, CRMP3, CRMP4, and / or CRMP5. In one embodiment, the antibodies of the present invention do not bind to CRMP3, CRMP4, and CRMP5, but show weak binding to CRMP1.

[0147] "Selective or specific binding to CRMP2", "selective or specific recognition of CRMP2", and "antibody with selectivity or specificity to (for) CRMP2" specifically, generally, and collectively refer to antibodies that preferentially bind to full-length (recombinant or natural) CRMP2 present, for example, in brain tissue, and optionally, also bind to denatured CRMP2, as determined by ELISA, preferably at least one order of magnitude, as shown in Example 2. Figure 2 As shown in A, and the following antibodies: antibodies that, as determined by Western blotting, are substantially non-cross-reactive with CRMP1, CRMP3, CRMP4, and CRMP5, as shown in Example 2 and Figure 2 As shown in B. As described above, the determination of antibody binding specificity can be performed, for example, by Western blotting or ELISA as described in the embodiments herein, or by equivalent assays.

[0148] As described above, CRMP2 is a phosphoprotein and can switch between non-phosphorylated (active) and phosphorylated (inactive) forms. As shown in Example 4 and Table VII, the antibody of the present invention binds both full-length non-phosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2). Therefore, in one embodiment, the antibody of the present invention is capable of binding both full-length non-phosphorylated CRMP2 and pCRMP2, as determined by ELISA assay.

[0149] Therefore, unless otherwise indicated, references to the anti-CRMP2 antibody, CRMP2 binding molecule and its fragments or derivatives, and antibodies of the present invention specific to CRMP2, mean that the antibody, CRMP2 binding molecule and its fragments or derivatives also bind phosphorylated CRMP2 (pCRMP2). Therefore, unless otherwise indicated, the terms "anti-CRMP2 antibody," "CRMP2 binding molecule and its fragments or derivatives," and "CRMP2-specific antibody" include the corresponding antibody and binding molecule and its fragments or derivatives that bind to pCRMP2.

[0150] Antibodies with high affinity for CRMP2 and / or pCRMP2, preferably for both CRMP2 and pCRMP2, are of particular interest, and to obtain a measurement of binding affinity, the EC50 of the antibodies was determined in an ELISA assay performed in Example 4. 50 The term "EC" 50 "In the context of in vitro or ex vivo assays using antibodies or their antigen-binding fragments, "refers to the concentration of the antibody or its antigen-binding fragment that induces a response of 50% of the maximum response, i.e., at the midpoint between the maximum response and the baseline."

[0151] As can be derived from Table VII, not only the binding affinity of antibody NI-504.3E7 was determined, but also the binding affinity of its 40 variants (NI-504.3E7_V1 to NI-504.3E7_V40) with amino acid substitutions in the VH and / or VL chains and / or within the CDR, as explained in further detail below. Specifically, antibody NI-504.3E7 was measured at EC50 values ​​of 0.5 nM and 4.7 nM, respectively. 50 Specific recognition of recombinant human full-length CRMP2 and pCRMP2. Values ​​for variants NI-504.3E7_V1 to NI-504.3E7_V40 are listed in Table VII.

[0152] In one embodiment, the antibody of the present invention is expressed at an EC50 of less than about 30 nM. 50The antibody identifies CRMP2, 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. In one embodiment, the antibody of the present invention is expressed at an EC50 concentration of less than about 120 nM. 50 The antibody of the present invention is preferably less than about 110 mM, preferably less than about 100 mM, preferably less than about 90 mM, 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 mM, preferably less than about 25 nM, and most preferably less than about 20 nM. Preferably, the binding affinity for CRMP2 and pCRMP2 is within the same order of magnitude, or the binding affinity for pCRMP2 is one or two orders of magnitude smaller than the binding affinity for CRMP2. Most preferably, the antibody of the present invention is formulated with the above-described EC... 50 Values ​​identify CRMP2 and pCRMP2. Therefore, antibodies that do not specifically bind to CRMP2 / pCRMP2 are, for example, those with EC50 values ​​above approximately 150 nM. 50 Antibodies binding to CRMP2 / pCRMP2, more preferably above about 400 nM, and most preferably, EC 50 It's even impossible to determine definitively. However, it also depends on the antibody form, such as whether IgG1, IgG4, or antibody fragments like Fab fragments or EC are used. 50 Values ​​may deviate from and may be higher than, for example, the values ​​described above and in the examples. Therefore, in this context, the term "about" means a value that may differ from the value determined by the reference antibody in the examples, preferably less than an order of magnitude, and most preferably within the same order of magnitude, such as EC. 50 It can be a reference value ±10 nM.

[0153] 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 phosphorylated and non-phosphorylated CRMP2, have also been confirmed by analyzing the binding affinity of the antibody to the corresponding CRMP and pCRMP peptides. Specifically, compared to (p)CRMP1((EVPA(pT)PKYA(pT)PAPSAKS(pS)PSKHQC-NH2(SEQ ID NO: 69)) and (p)CRMP4(H-DLTT(pT)PKGG(pT)PAGSARG(pS)PTRPNC-NH2(SEQ ID NO: 71)), antibody NI-504.3E7 preferentially binds to 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)) containing three phosphate sites T509, T514, and S522. 70)), as determined by ELISA. Furthermore, relative to the corresponding peptides derived from (p)CRMP1 (H-YATPAPSAKS(pS)PSKHQPPPIRC-NH2(SEQ ID NO: 66)) and (p)CRMP4 (H-GGTPAGSARG(pS)PTRPNPPVRNC-NH2(SEQ ID NO: 68)), antibody NI-504.3E7 preferentially binds to the CRMP2 peptide (TVTPASSAKTSPAKQQAPPVRC-NH2(SEQ ID NO: 67)) and the pCRMP2 peptide containing a single S522 phosphate site (H-TVTPASSAKT(pS)PAKQQAPPVRC-NH2(SEQ ID NO: 67)).

[0154] As shown in Example 6, antibody NI-504.3E7 specifically binds to the CRMP2 peptide VCEVSTPKTVTPASSAKTSPAKQQA (SEQ ID NO: 72) and different pCRMP2 peptides (VCEVSV(p)T, T, and S522) with different affinities. 509 PKTVTPASSAKTSPAKQQA(SEQ ID NO: 72),VCEVSVTPKTV(p)T 514 PASSAKTSPAKQQA(SEQ ID NO: 72) and VCESVTPKTVTPASSAKT(p)S 522PAKQQA (SEQ ID NO: 72)). Specifically, antibody NI-504.3E7 showed an affinity for the phosphorylated CRMP2 peptide at position T514 that was approximately 300 times lower than that for the unphosphorylated peptide and the peptides phosphorylated at positions T509 and S522. Therefore, in one embodiment, the antibody of the present invention binds both phosphorylated and unphosphorylated CRMP2 peptides, wherein the phosphorylated peptide is preferably phosphorylated at T509, T514, and / or S522. Preferably, the antibody of the present invention binds less of the phosphorylated CRMP2 peptide at position T514, and more preferably, those phosphorylated at positions T509 and S522, compared to the unphosphorylated CRMP2 peptide and the CRMP2 peptides phosphorylated at positions T509 and S522.

[0155] As further demonstrated in the phosphorylation assay described in Example 5, the antibody of the present invention reduces pCRMP2 levels in a concentration-dependent manner. In fact, this experiment has been exemplary performed using the variant NI-504.3E7_V20 to further support the fact that the variant antibody maintains the binding specificity and activity of the original antibody. As shown in Table VII, the binding affinity of NI-504.3E7_V20 for CRMP2 and pCRMP2 (by EC50) compared to the original antibody is shown in Table VII. 50 The value indicates a decrease. Therefore, since this variant has sufficient activity to reduce pCRMP2 levels in phosphorylation assays, other variants should also be able to achieve the same result.

[0156] This activity was not observed against the anti-CRMP2 control antibodies targeting the different epitopes described in detail above, nor against the human IgG control antibody. Therefore, this characteristic appears to be unique to the antibody of the present invention. Specifically, Western blot images show that, compared to the "antibody-free control" and the "human IgG antibody control," when phosphorylation assays of the antibody of the present invention and CRMP2 were performed under appropriate conditions for 1 h and 24 h to phosphorylate T509, T514, and S522 of the CRMP2 protein, respectively—that is, in the presence of glycogen synthase kinase-3 (GSK-3)β and cyclin-dependent kinase (Cdk)5 / p35—the amounts of CRMP2 phosphorylated by T509, T514, and S522 were reduced. Furthermore, under most conditions, the reduction depended on the concentration of the subject antibody; that is, the higher the amount of the antibody of the present invention, the stronger the reduction in CRMP2 phosphorylation.

[0157] Therefore, in one embodiment, compared to a control, when both the antibody and CRMP2 protein are phosphorylated, the antibody of the present invention is able to reduce the level of pCRMP2, preferably T509 pCRMP2, T514 pCRMP2, and / or S522 pCRMP2, in a concentration-dependent manner. Preferably, phosphorylation is performed for 1 h or 24 h under appropriate conditions in the presence of GSK-3β and CDK5 / p35, and Western blot analysis is performed to analyze the results. Therefore, in a preferred embodiment, compared to a control, and as determined by Western blot analysis, when the antibody and CRMP2 are phosphorylated for 1 h or 24 h under appropriate conditions in the presence of glycogen synthase kinase-3 (GSK-3)β and Cdk 5 / p35, the antibody of the present invention is able to reduce the level of pCRMP2, preferably selected from T509 pCRMP2, T514 pCRMP2, and S522 pCRMP2, in a concentration-dependent manner. In a preferred embodiment, a higher amount of antibody results in a stronger reduction in pCRMP2 levels, while a lower amount of antibody results in a smaller reduction in pCRMP2 levels.

[0158] There are different suitable controls, such as "antibody-free control" (where no antibody is added to the phosphorylation assay), control antibodies that do not recognize CRMP2 or pCRMP2 (e.g., human IgG antibody control), or CRMP2 antibodies that are known not to reduce pCRMP2 levels in the phosphorylation assay.

[0159] Overlapping peptide arrays revealed that antibody NI-504.3E7 binds to a linear peptide having the amino acid sequences TPKTVTPASSAKTSP (SEQ ID NO: 62) and VTPASSAKTSPAKQQ (SEQ ID NO: 63), which has a concordant sequence VTPASSAKTSP (SEQ ID NO: 65) and is part of the C-terminal domain of CRMP2; see Example 3. The C-terminal domain extends from amino acid 490 to 572. Therefore, in one embodiment, the antibody of the present invention binds to the C-terminal regions of CRMP2 and pCRMP2. In particular, in a preferred embodiment, the antibody of the present invention binds to a peptide present in the C-terminal regions of CRMP2 and pCRMP2, wherein the peptide is preferably TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63). Of course, the antibody also binds peptides containing the aforementioned sequences.

[0160] Epitope mapping shows that antibody NI-504.3E7 binds to a linear peptide containing the concordant sequence ASSAK (SEQ ID NO: 64). Therefore, in one embodiment, the antibody of the present invention binds to an epitope containing or composed of the amino acid sequence ASSAK (SEQ ID NO: 64).

[0161] As shown in Example 9, antibody NI-504.3E7 was able to reverse the increase in dendritic 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 since experiments conducted within the scope of this invention have shown that anti-CRMP2 antibodies act on dendritic spine density and preferably reduce it, this characteristic can be used as evidence of the activity of the antibodies of this invention, and it further indicates that anti-CRMP2 antibodies interfere with CRMP2 signaling in some way. Therefore, this invention also relates to molecules capable of interfering with CRMP2 signaling for use as pharmaceuticals, preferably for the prevention of neurodegenerative diseases, cognitive disorders, delaying the progression of neurodegenerative diseases, cognitive disorders, or for the treatment of neurodegenerative diseases, cognitive disorders, and / or for methods of improving memory and learning abilities.

[0162] Furthermore, in one embodiment, the antibody of the present invention is capable of reversing the increase in neuronal dendritic spine density induced by CRMP2, as determined in an ex vivo hippocampal slice culture model, preferably at a concentration of 0.4 μM to 10 μM, preferably 0.5 μM to 9 μM, preferably 0.6 μM to 8 μM, and most preferably 7.5 μM or 0.7 μM.

[0163] The increase in the density of neuronal dendritic spines is caused by the increase in pCRMP2 aggregates or CRMP2 monomers.

[0164] In one embodiment, the anti-CRMP2 antibody and the equivalent CRMP2 binding molecule exhibit the immunohistochemical characteristics and in vivo activity of any of the anti-CRMP2 antibodies further illustrated in the following examples and figures.

[0165] In particular, antibodies, their antigen-binding fragments, and derivatives have at least one of the following properties (i) to (iii):

[0166] (i) Detecting neurons in the hippocampus of AD patients;

[0167] (ii) Reduce amyloid plaque burden in female Tau tubulin kinase-1 (TTBK1) / amyloid precursor (APP) transgenic mice; and / or

[0168] (iii) Improves cognitive function in aged mice.

[0169] More specifically, IHC analysis showed that antibody NI-504.3E7 binds in hippocampal tissue and, particularly, in samples from patients with tau proteinosis, detects neuronal cells. Specifically, antibody NI-504.3E7 binds in hippocampal tissue from AD patients. Furthermore, antibody NI-504.3E7 has been shown to bind in hippocampal tissue from PSP patients, in hippocampal tissue from PiD patients, and to detect neuronal cells in hippocampal sections from PiD, FTD-PD, and FTLD-TDP patients. Further research has shown that in a transgenic mouse model of Alzheimer's disease, antibody NI-504.3E7 reduces amyloid plaque burden in the cortex and hippocampus by 63%, and reduces the size of amyloid plaques in the cortex by 30% and in the hippocampus by 68%. Additionally, in the cortex, a 50% reduction in plaque / area (area defined by the entire cortex) was observed in female TTBK1 / APP mice receiving antibody NI-504.3E7. Further demonstrations in Examples 13 and 14 have shown that the antibodies of the present invention improve 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. Indeed, these experiments have been exemplarily performed using the variant NI-504.3E7_V20 to further support the fact that the variant antibody retains the activity of the original antibody, as explained above regarding the phosphorylation assay.

[0170] Therefore, in one embodiment, the antibody of the present invention detects neurons in the hippocampus of patients with tau proteinosis, preferably those with AD, PSP, PiD, FTD-PD, and FTLD-TDP, and most preferably AD. In one embodiment, the antibody of the present invention reduces amyloid plaque burden and / or average amyloid plaque size and / or plaque / area amount in transgenic mouse models of Alzheimer's disease, preferably in female Tau tubulin kinase-1 (TTBK1) / amyloid precursor protein (APP) transgenic mice. In a preferred embodiment, the plaque burden in the cortex and / or 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 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 plaque / 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, preferably when administered at a dose of 0.1 mg / kg to 10 mg / kg, more preferably 0.2 mg / kg to 10 mg / kg, more preferably 0.3 mg / kg to 10 mg / kg, more preferably 1 mg / kg to 10 mg / kg, and most preferably at a dose of 0.3 mg / kg, 1 mg / kg, 3 mg / kg or 10 mg / kg, wherein the effect is most significant at a dose of 1 mg / kg, 3 mg / kg or 10 mg / kg.

[0171] The present invention also relates to antibodies or antigen-binding molecules that typically compete with the antibodies of the present invention for specific binding to human CRMP2 and / or pCRMP2, and which respectively retain one or more binding properties and functional features as described for the antibodies of the present invention. Preferably, the competitive antibody and antigen-binding molecule have at least one of the following features (i) to (iii), preferably at least two of the following features (i) to (iii), i.e., (i) and (ii), or (i) and (iii), or (ii) and (iii), and most preferably all three of the following features (i) to (iii):

[0172] (i) Human CRMP2 is preferentially recognized relative to CRMP1, CRMP3, CRMP4 and CRMP5, and can be tested in ELISA assays and Western blot analysis as described in Example 2;

[0173] (ii) When phosphorylation of CRMP2 is measured, the level of pCRMP2 can be reduced in a concentration-dependent manner, as can be measured as described in Example 5;

[0174] (iii) It can reverse the increase in neuronal dendritic spine density induced by CRMP2, as determined in an ex vivo hippocampal slice culture model, which can be tested as described in Example 9.

[0175] In one embodiment, the present invention relates to an antibody that recognizes the same epitope as antibody NI-504.3E7 and retains any one of the binding features (i) to (iii) described above, preferably all of them. Competitive antibodies can be identified in a simple immunoassay that demonstrates the ability of one antibody to block the binding of another antibody to a target antigen (e.g., electrochemiluminescence assay, competitive ELISA, solid-phase radioimmunoassay (SPRIA), or blocking protein blot).

[0176] This invention is illustrated using an anti-CRMP2 antibody and its antigen-binding fragment, characterized in that it contains within its variable region (i.e., the binding domain) a [missing information - likely a specific structure or feature]. Figure 1 The amino acid sequences shown represent the variable heavy (VH) and variable light (VL) chains. The corresponding nucleotide and amino acid sequences are listed in Table II below. Specifically, Figure 1 The VH and VL chains of antibody NI-504.3E7 and its 40 variants (NI-504.3E7_V1 to NI-504.3E7_V40) are shown, with amino acid substitutions made in the VH chain, VL chain, and / or CDR. CDRs are underlined in the corresponding figures and tables. The corresponding amino acid substitutions can be performed using conventional methods known in the art, such as PCR.

[0177] As usual, each chain's variable domain contains three hypervariable loops, named complementarity-determining regions (CDRs, CDR-1, -2, and -3). These CDRs are separated by structurally conserved regions called framework regions (FR-1, -2, -3, and -4), which form the "core" β-sheet structure, allowing these loops to be displayed on the surface of the variable domain. The length and composition of the CDR sequence are highly variable, especially in CDR3. CDRs are located near the complementary sites of the antibody that interact with the antigen and therefore contain antigen-binding residues. Thus, an antibody is typically defined by its six CDRs. The exemplary CDR sets in the above amino acid sequences of the VH and VL chains are depicted in... Figure 1 And in Tables II and IV. However, as discussed below, it is a fact well known to those skilled in the art that, alternatively or otherwise, the following CDRs, whose amino acid sequences are similar to those in Tables II and IV, may be used. Figure 1 The amino acid sequences shown in the table differ by one, two, three, or even more amino acids, especially in the case of CDR2 and CDR3. For example... Figure 1 As illustrated in the illustrations, those skilled in the art can readily identify CDRs based on general principles, such as those summarized in www.bioinf.org.uk / abs. In this context, although... Figure 1 The CDRs of antibodies depicted in this paper are based on those presented by Kabat et al., but those skilled in the art will know that many definitions of CDRs are commonly used, namely, (i) the Kabat definition based on sequence variability, which is the most commonly used; (ii) the Chothia definition based on the location of the structural loop region; (iii) the AbM definition, which is a compromise between the two used by the AbM antibody modeling software from Oxford Molecular; and (iv) the Contact definition, which is more recently introduced and based on the analysis of available complex crystal structures. This definition may be most useful for mutagenesis to modify the affinity of antibodies, as these residues are involved in the interaction with the antigen. For a list of CDR residues that generate contacts in each antibody, and for a summary of data for each CDR, see, for example, www.bioinf.org.uk / abs, which also references antibody modeling software such as abYmod, available at abymod.abysis.org. Table I below describes the relationship between the locations of CDRs defined by the different concepts.

[0178] [Table I]

[0179]

[0180] For the definitions stated, see also Kontermann and Dubel (eds.), Antibody Engineering Vol. 2, DOI 10.1007 / 978-3-642-01147-4_3, # Springer-Verlag Berlin Heidelberg 2010, particularly Chapter 3, Protein Sequence and Structure Analysis of Antibody Variable Domains, pp. 33-51, and Dondelinger et al., Front. Immunol. 9 (2018), 2278, specifically concerning the understanding of the meaning and implications of antibody numbering and antigen-binding surface / residue definitions; see, for example, Dondelinger et al., Figure 4 and Figure 6 This demonstrates the significant differences between the classic CDR definitions used by Kabat (ibid.), 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 (registered trademark), International Immunogenetic Information System (registered trademark), www.imgt.org). The AbM definition is a compromise between the two used by the Oxford Molecular AbM antibody modeling software.

[0181] [Chem. 1]

[0182]

[0183] The figure above illustrates an alternative definition of CDR-H1 (VH-CDR1). The Kabat and Chothia numbering schemes are displayed horizontally, and the Kabat, Chothia, AbM, and Contact definitions of the CDR are shown above and below the two numbering schemes with arrows. Therefore, based on the VH and V chain amino acid sequences provided for the two subject antibodies NI-504.3E7 as their respective variants, their epitopes, and the assays described in the examples, those skilled in the art can design and obtain equivalent antibodies and, for example, CRMP2 binding molecules whose one or more CDRs defined by Kabat differ from the corresponding CDRs of the parent subject antibodies, but substantially retain the CDRs defined by Chothia. During experiments conducted within the scope of this 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. Figure 1 As shown in Table V, certain amino acid substitutions are present in the VH and / or VL chains of the variant antibodies. Furthermore, amino acid substitutions are even present 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 variants retain their ability to bind CRMP2 and pCRMP2. Moreover, the results of experiments performed with the original antibody NI-504.3E7 as described above have been confirmed with one or more of the 40 variants.

[0184] In one embodiment, the anti-CRMP2 antibody, its antigen-binding fragment or derivative, or the CRMP2 binding molecule of the present invention comprises a variable heavy (VH) chain containing VH complementarity-determining regions (VH-CDRs) 1, 2, and 3 and / or a variable light (VL) chain containing VL-CDRs 1, 2, and 3, as defined by Kabat, wherein

[0185] NI-504.3E7

[0186] (a) VH-CDR1 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 3 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 3.

[0187] (b) VH-CDR2 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 4 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 4.

[0188] (c) VH-CDR3 contains, for example Figure 1Or the amino acid sequence of SEQ ID NO: 5 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 5.

[0189] (d)VL-CDR1 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 8 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 8.

[0190] (e)VL-CDR2 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 9 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 9, and

[0191] (f) VL-CDR3 contains, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 10 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 10; or

[0192] (NI-504.3E7_V15, NI-504.3E7_V19, NI-504.3E7_V36, and NI-504.3E7_V39)

[0193] (a) VH-CDR1 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 3 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 3.

[0194] (b) VH-CDR2 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 4 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 4.

[0195] (c) VH-CDR3 contains, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 5 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 5.

[0196] (d)VL-CDR1 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 8 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 8.

[0197] (e)VL-CDR2 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 9 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 9, and

[0198] (f) VL-CDR3 contains, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 61 (VL_3E7_var3) shown in Table III or a variant thereof, wherein the variant contains an amino acid sequence resulting from a partial change of SEQ ID NO: 61.

[0199] In a preferred embodiment, the variant CDR contains one or more amino acid substitutions, preferably one, two, three, or four, more preferably one or two amino acid substitutions, and most preferably one amino acid substitution. In a preferred embodiment, only VL-CDR3 contains one or more amino acid substitutions, preferably one, two, three, or four, more preferably one or two amino acid substitutions, and most preferably one amino acid substitution.

[0200] In another preferred embodiment, the variant of 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%, preferably 99% identical to the sequences 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. More preferably, only VL-CDR3 comprises a variant 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 NO: 61.

[0201] Alternatively or concurrently, the antibody or antigen-binding fragment of the present invention can be characterized as follows:

[0202] NI-504.3E7

[0203] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 2 (VH_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 2; and / or

[0204] (ii) VL includes, for example Figure 1Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7.

[0205] NI-504.3E7_V1

[0206] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 2 (VH_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 2; and / or

[0207] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0208] NI-504.3E7_V2

[0209] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 2 (VH_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 2; and / or

[0210] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0211] NI-504.3E7_V3

[0212] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 12 (VH_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 12; and / or

[0213] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7.

[0214] NI-504.3E7_V4

[0215] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 12 (VH_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 12; and / or

[0216] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0217] NI-504.3E7_V5

[0218] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 12 (VH_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 12; and / or

[0219] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0220] NI-504.3E7_V6

[0221] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 14 (VH_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 14; and / or

[0222] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7.

[0223] NI-504.3E7_V7

[0224] (i) The VH chain contains, for example, Figure 1Or the amino acid sequence described in SEQ ID NO: 14 (VH_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 14; and / or

[0225] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0226] NI-504.3E7_V8

[0227] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 14 (VH_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 14; and / or

[0228] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0229] NI-504.3E7_V9

[0230] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 16 (VH_3E7_var3) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 16; and / or

[0231] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7.

[0232] NI-504.3E7_V10

[0233] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 16 (VH_3E7_var3) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 16; and / or

[0234] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0235] NI-504.3E7_V11

[0236] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 16 (VH_3E7_var3) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 16; and / or

[0237] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0238] NI-504.3E7_V12

[0239] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0240] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7.

[0241] NI-504.3E7_V13

[0242] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0243] (ii) VL includes, for example Figure 1Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0244] NI-504.3E7_V14

[0245] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0246] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0247] NI-504.3E7_V15

[0248] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0249] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60.

[0250] NI-504.3E7_V16

[0251] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0252] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7.

[0253] NI-504.3E7_V17

[0254] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0255] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0256] NI-504.3E7_V18

[0257] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0258] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0259] NI-504.3E7_V19

[0260] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0261] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60.

[0262] NI-504.3E7_V20

[0263] (i) The VH chain contains, for example, Figure 1Or the amino acid sequence or a variant thereof described in SEQ ID NO: 22 (VH_3E7_var6) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 22; and / or

[0264] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0265] NI-504.3E7_V21

[0266] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 24 (VH_3E7_var6a) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 24; and / or

[0267] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0268] NI-504.3E7_V22

[0269] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 26 (VH_3E7_var6b) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 26; and / or

[0270] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0271] NI-504.3E7_V23

[0272] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 28 (VH_3E7_var6c) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 28; and / or

[0273] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0274] NI-504.3E7_V24

[0275] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 30 (VH_3E7_var6d) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 30; and / or

[0276] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0277] NI-504.3E7_V25

[0278] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 32 (VH_3E7_var6ab) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 32; and / or

[0279] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0280] NI-504.3E7_V26

[0281] (i) The VH chain contains, for example, Figure 6 Or the amino acid sequence described in SEQ ID NO: 34 (VH_3E7_var6ac) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 34; and / or

[0282] (ii) VL includes, for example Figure 6Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0283] NI-504.3E7_V27

[0284] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 36 (VH_3E7_var6ad) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 36; and / or

[0285] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0286] NI-504.3E7_V28

[0287] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 38 (VH_3E7_var6bc) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 38; and / or

[0288] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0289] NI-504.3E7_V29

[0290] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 40 (VH_3E7_var6bd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 40; and / or

[0291] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0292] NI-504.3E7_V30

[0293] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 42 (VH_3E7_var6cd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 42; and / or

[0294] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0295] NI-504.3E7_V31

[0296] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 44 (VH_3E7_var6bcd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 44; and / or

[0297] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0298] NI-504.3E7_V32

[0299] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 46 (VH_3E7_var6acd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 46; and / or

[0300] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0301] NI-504.3E7_V33

[0302] (i) The VH chain contains, for example, Figure 1Or the amino acid sequence described in SEQ ID NO: 48 (VH_3E7_var6abd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 48; and / or

[0303] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0304] NI-504.3E7_V34

[0305] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 50 (VH_3E7_var6abc) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 50; and / or

[0306] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0307] NI-504.3E7_V35

[0308] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 22 (VH_3E7_var6) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 22; and / or

[0309] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0310] NI-504.3E7_V36

[0311] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 22 (VH_3E7_var6) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 22; and / or

[0312] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60.

[0313] NI-504.3E7_V37

[0314] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 52 (VH_3E7_var7) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 52; and / or

[0315] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56.

[0316] NI-504.3E7_V38

[0317] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 52 (VH_3E7_var7) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 52; and / or

[0318] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0319] NI-504.3E7_V39

[0320] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 52 (VH_3E7_var7) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 52; and / or

[0321] (ii) VL includes, for example Figure 1Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60.

[0322] NI-504.3E7_V40

[0323] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 54 (VH_3E7_var8) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 54; and / or

[0324] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0325] In a preferred embodiment, variants VH and VL each comprise one or more amino acid substitutions and / or deletions, preferably one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions and / or deletions. Preferably, variants of the VL chain comprise one, two, three, four, or five amino acid substitutions and / or deletions, and / or variants of the VH chain comprise one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions and / or deletions, preferably two, three, four, five, six, seven, eight, or ten amino acid substitutions and / or deletions. Most preferably, variants VH and VL each comprise one or two amino acid substitutions and / or deletions. For example, variant VL comprises an amino acid deletion, wherein the N-terminal amino acid is deleted via post-translational modification, and in one embodiment, the first serine residue is deleted during the signal peptide deletion.

[0326] In another preferred embodiment, the variants of the VH and VL chains respectively comprise the following: SEQ ID NO:2, SEQ ID NO:7, 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, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO: The sequence shown in 60 has at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% identical amino acid sequences.

[0327] In these embodiments, preferably, one or more of the CDRs defined according to Kabat remain substantially unchanged. However, under the simplified assumption that complementary sites correspond to CDRs, the Chothia definition of CDRs can be used alternatively or as an alternative, as they are well correlated with the structural loops present in the variable region. Therefore, in order to provide an anti-CRMP2 antibody equivalent to the subject antibody NI-504.3E7 or any of the variants V1 to V40, preferably one or more of the above, preferably no more than at least one or both of the two amino acid substitutions, and most preferably one amino acid substitution, if prepared in a CDR defined according to Kabat, is prepared outside of a CDR defined by Chothia and / or IMGT, and most preferably outside the overlapping portion of the CDRs defined according to Kabat and Chothia.

[0328] For example, regarding amino acid substitutions within the CDR, variable heavy and light chains, and framework amino acid sequences, conserved amino acid substitutions are preferably made, for example, according to the analysis and description of the most frequently exchanged amino acids, as presented by Mirsky et al., Mol. Biol. Evol. 32 (2014), 806-819; see Mirsky et al., page 813. Figure 1Specifically, in VH-CDR1, S can be replaced by T; in VH-CDR3, V can be replaced by E, T can be replaced by S, and / or M can be replaced by V; in VL-CDR1, R can be replaced by K, R can be replaced by E, and / or T can be replaced; in VL-CDR2, S can be replaced by A and / or A can be replaced by G; and in VL-CDR3, P can be replaced by S. As described above, preferably, the above-mentioned Mirsky et al. (2014) are selected. Figure 1 The model shown is one of LG and AB, or preferably both of the same class of amino acid substitutions, wherein the LG model is preferred because it tends to preserve the characteristics of the amino acids, and wherein the amino acid substitutions are preferably selected such that the physicochemical properties of the original amino acids are substantially preserved, i.e., hydrophobicity, polarity or charge, or, for example, in the case of two or more amino acid substitutions, they compensate for each other, thereby jointly providing the physicochemical properties of the surface.

[0329] Of course, besides theoretical considerations, there are experimental methods for identifying CDR variants within reasonable timeframes and inappropriate loads. For example, Tiller et al., in Front Immunol. 8 (2017), 986, describe a simplified affinity maturation of antibody variable domains induced by natural diversity mutagenesis. In fact, several years earlier, Rajpal et al. reported in PNAS 102 (2005), 8466-8471 a general method for significantly enhancing antibody affinity by using combinatorial libraries, illustrating their method with the anti-TNF-α antibody D2E7 (HUMIRA (copyright)), which identified 38 substitutions at 21 CDR sites that resulted in higher affinity binding to TNF-α. Recently, Cannon et al., in PLOS Computational Biology, https: / / doi.org / 10.1371 / journal.pcbi.1006980 May 1, 2019, described experimentally guided computational antibody affinity maturation. This method utilizes de novo docking, modeling, and rational design of computer-aided affinity maturation, combined with alanine scanning, to allow fine-tuning of the protein-protein docking model. This subsequently enabled the identification of two single-point mutations that enhanced the affinity of the hybridoma-derived antibody AB1 for its antigen (mouse CCL20).

[0330] Therefore, although each antibody is unique and can have different characteristics, once a lead candidate is provided, those skilled in the art can obtain an equivalent anti-CRMP2 antibody that retains the desired characteristics of the antibody, such as those described for the anti-CRMP2 antibodies shown in the examples and specifically defined in the claims, taking into account the teachings of the invention as disclosed in this application and the computational design and experimental methods developed to date. In this context, it is well known that variant antibodies substantially retain the binding specificity of the parent antibody, such as binding to peptides / epitopes as defined above; binding to CRMP2 and pCRMP2; selectively binding CRMP2 without specifically binding CRMP1, CRMP3, CRMP4, and CRMP5; reducing pCRMP2 levels in a concentration-dependent manner in phosphorylation assays; reversing the increase in neuronal dendritic spine density induced by CRMP2, as measured in an ex vivo hippocampal slice culture model; and / or competing with the parent antibody (i.e., with antibody NI-504.3E7 and any one of variants NI-504.3E7_V1 to NI-504.3E7_V40) for binding to the corresponding epitope.

[0331] As is known in the art, the CDR3 (VH-CDR3) of the variable heavy chain appears to primarily determine antigen specificity; see, for example, Xu and Davis, Immunity 13 (2000), 37-45. In this context, it is noted that the diversity of heavy chain CDR3 drives specificity, while VH-CDR1 and VH-CDR2 residues exhibit broad cross-reactivity and can be improved by somatic hypermutation; see Davis, Semin. Immunol. 16 (2004), 239-243. Therefore, in one embodiment, the antibody of the present invention, having the immunological characteristics of a reference antibody and capable of competitively participating in the binding of the antibody to CRMP2 at the corresponding epitope, comprises at least VH-CDR3 or the following VH-CDR3 of the corresponding reference antibody in its variable region: its amino acid sequence being at least 90% identical, preferably 95%, and more preferably 96%, 97%, 98%, 99%, or 100% identical to the reference VH-CDR3. For example, a variant of the reference antibody may retain the VH-CDR3 of the reference (parental) antibody, while VH-CDR1 and / or VH-CDR2 may contain one or more amino acid substitutions; see above.

[0332] However, in addition to the theoretical and practical considerations presented in the literature, experiments conducted within the scope of this invention have shown that the resulting variant 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 variants still bind phosphorylated and non-phosphorylated CRMP2. Furthermore, the results of experiments conducted within the scope of this invention have been confirmed with one or more variants.

[0333] Therefore, in a further preferred embodiment, the amino acid substitution is present in VL-CDR3. Preferably, VL-CDR3 comprises one or more amino acid substitutions, preferably one, two, three, or four, more 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 VL-CDR3, preferably at position 91 of the VL chain, and most preferably D91E.

[0334] In another further preferred embodiment, the variant CDR comprises at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% of the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 61, respectively.

[0335] Therefore, in one embodiment, regarding NI-504.3E7, the characteristic is...

[0336] (a) VH-CDR1 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 3 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 3.

[0337] (b) VH-CDR2 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 4 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 4.

[0338] (c) VH-CDR3 contains, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 5 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 5.

[0339] (d)VL-CDR1 includes, for example Figure 1Or the amino acid sequence of SEQ ID NO: 8 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 8.

[0340] (e)VL-CDR2 includes, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 9 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 9, and

[0341] (f) VL-CDR3 contains, for example Figure 1 Or the amino acid sequence of SEQ ID NO: 10 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 10.

[0342] The variant CDR contains one or more amino acid substitutions, preferably one, two, three, or four, more preferably one or two, and most preferably one. In a preferred embodiment, the variant CDR is VL-CDR3 and contains one or more amino acid substitutions, preferably one, two, three, or four, more 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 most preferably D91E.

[0343] In one embodiment, regarding NI-504.3E7, the variant CDR comprises at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% of the amino acid 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. In a preferred embodiment, the variant CDR comprises at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% of the amino acid sequence shown in SEQ ID NO: 10.

[0344] In another preferred embodiment, the variants of the VH and VL chains each comprise one or more amino acid substitutions, preferably one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Preferably, the variants of the VL chain comprise one, two, three, four, or five amino acid substitutions, and / or the variants of the VH chain comprise one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, preferably two, three, four, five, six, seven, eight, or ten amino acid substitutions. In one embodiment, the variants of the VH and VL chains each comprise one or two amino acid substitutions.

[0345] In one embodiment, the listed VH chain variants comprise amino acid substitutions of T7S, Q19R, P28T, Y29F, V40S, V40A, I71T, M80T, R82Y, H84Q, and / or Y86S. In particular, in a preferred embodiment, the VH chain variants comprise amino acid substitutions:

[0346] (i) M80T and Y86S,

[0347] (ii) V40S, M80T and Y86S,

[0348] (iii) I71T, M80T and Y86S,

[0349] (iv) V40S, I71T, M80T, and Y86S,

[0350] (v) V40A, I71T, M80T, and Y86S,

[0351] (vi) T7S, Q19R, V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0352] (vii) Q19R, V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0353] (viii) T7S, V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0354] (ix) T7S, Q19R, V40A, I71T, M80T, H84Q, and Y86S,

[0355] (x) T7S, Q19R, V40A, I71T, M80T, R82Y, and Y86S,

[0356] (xi) V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0357] (xii) Q19R, V40A, I71T, M80T, H84Q and Y86S,

[0358] (xiii) Q19R, V40A, I71T, M80T, R82Y and Y86S,

[0359] (xiv) T7S, V40A, I71T, M80T, H84Q, and Y86S,

[0360] (xv) T7S, V40A, I71T, M80T, R82Y and Y86S,

[0361] (xvi) T7S, Q19R, V40A, I71T, M80T, and Y86S,

[0362] (xvii) T7S, V40A, I71T, M80T, and Y86S,

[0363] (xviii) Q19R, V40A, I71T, M80T, and Y86S,

[0364] (ixx) V40A, I71T, M80T, R82Y and Y86S,

[0365] (xx) V40A, I71T, M80T, H84Q and Y86S,

[0366] (xxi) T7S, Q19R, V40S, I71T, M80T, R82Y, H84Q, and Y86S, or

[0367] (xxii) T7S, Q19R, P28T, Y29F, V40A, I71T, M80T, R82Y, H84Q and Y86S.

[0368] In one embodiment, the listed VL chain variants comprise amino acid substitutions of L37Q, L69T, N75S, L78Q, and / or D91E. Particularly, in a preferred embodiment, the VL variants comprise amino acid substitutions:

[0369] (i) L69T and L78Q,

[0370] (ii) L37Q, L69T, N75S, and L78Q, or

[0371] (iii) L37Q, L69T, N75S, L78Q and D91E.

[0372] As shown in Tables II and V, the amino acid substitutions are present in the 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_3 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, VH_3E7_var8) and variants of the VL chain of antibody NI-504.3E7 (VL_3E7_var1, VL_3E7_var2, VL_3E7_var3). Preferred allocations of the variant VH and VL chains are described in Table IV.

[0373] In one embodiment, the variants of the VH and VL chains each contain one or two amino acid substitutions, preferably any of those listed above. In another preferred embodiment, the variants of the VH and VL chains respectively comprise the chains specified in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 12 and 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, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: The sequence shown in 60 has at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% identical amino acid sequences.

[0374] Regarding antibody NI-504.3E7, the variants of the VH and VL chains respectively contain at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% of the same amino acid sequence as the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 7 respectively.

[0375] However, preferably, the antibody of the present invention contains, in one or both of its immunoglobulin chains, the following... Figure 1 and one, two, or all three CDRs of the variable regions shown in Tables II and III, or with such Figure 1 One, two, or all three CDRs that are identical to the CDRs of the variable regions shown in Table III at 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Alternatively, one or more FRs from the frame region (FR) and the corresponding FR (which is located in...) Figure 1 The CDRs shown in Table II are 80% the same, preferably the same as the FRs (which are...). Figure 1 The sequences (85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the underlined CDRs in Table II) are also present. In some implementations, there are 1, 2, 3, or all four FRs (each identical to...). Figure 1 (and the FR shown in Table II is at least 90%, 90-95%, and / or 95-99% the same).

[0376] [Table II]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384] [Table III]

[0385]

[0386] [Table IV]

[0387]

[0388] [Table V]

[0389]

[0390] In further, additional, or alternative embodiments of the invention, anti-CRMP2 antibodies, their antigen-binding fragments, synthetic or biotechnological variants may be optimized to have appropriate binding affinity and stability properties to the target. Thus, at least one amino acid in the CDR or variable region (which is readily modified, such modification being selected from glycosylation, oxidation, deamination, peptide bond cleavage, isoflavone formation, and / or unpaired cysteine) is replaced by a mutant amino acid lacking such a change, or at least one carbohydrate moiety is missing or chemically or enzymatically 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; Haberger et al., MAbs. 6 (2014), 327-339.

[0391] Immunoglobulins or their encoding cDNA may be further modified. Therefore, in a further embodiment, the method of the present invention includes any one of the steps of generating chimeric antibodies, murine antibodies, single-chain antibodies, Fab fragments, bispecific antibodies, fusion antibodies, labeled antibodies, or analogs of any of these. 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), first edition; second edition, Edward A. Greenfield, Dana-Farber Cancer Institute (Copyright) 2014, ISBN 978-1-936113-81-1. For example, using enzymes such as papain (to generate the Fab fragment) or pepsin (to generate the F(ab')2 fragment), the Fab and F(ab')2 fragments can be recombined or generated by proteolytic cleavage of immunoglobulin molecules. The F(ab')2 fragment contains a variable region, a light chain constant region (CL), and a CH1 domain of the heavy chain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving the coupling of the immunoglobulin's immunospecific portion to a detection reagent such as a radioisotope.

[0392] In one embodiment, the antibody of the present invention may therefore be provided in the form of a single-chain Fv fragment (scFv), F(ab') fragment, F(ab) fragment and F(ab')2 fragment, Fd, Fv, single-chain antibody, disulfide bond-stabilized Fv (dsFv) and nanobody and / or a chimeric mouse-human or murine antibody.

[0393] As mentioned above, CRMP2 has been shown to co-localize with hyperphosphorylated Tau and is therefore present in the corresponding intracellular neurofibrillary tangles. Therefore, in one embodiment, the use of recombinant Fab (rFab) and scFv containing anti-Tau antibodies may be advantageous, as they may more readily penetrate the cell membrane.

[0394] Immunotherapy using different antibody forms, such as scFv, single-domain antibody fragments (VHH or sdAb), bispecific antibodies, intracellular antibodies, and nanobodies, has shown therapeutic efficacy in several animal models of Alzheimer's disease (AD), Parkinson's disease (PD), Lewy body dementia (DLB), frontotemporal dementia (FTD), Huntington's disease (HD), infectious spongiform encephalopathy (TSE), and multiple sclerosis (MS). Recombinant antibody fragments have been shown to neutralize extracellular and intracellular toxic misfolded proteins involved in the pathogenesis of neurodegenerative diseases, thus representing a promising tool for developing antibody-based immunotherapies for these diseases. See the review by Manoutcharian et al., Curr Neuropharmacol 15 (2017), 779-788.

[0395] The perceptual advantages of using engineered antibody forms of small Fab and scFv lacking effector function include more efficient crossing of the blood-brain barrier and minimizing the risk of triggering inflammatory side effects. Furthermore, in addition to retaining the binding specificity of full-length antibodies, scFv and single-domain antibodies can also be expressed intracellularly as single genes in mammalian cells as intracellular antibodies, with the potential to alter the folding, interaction, modification, or subcellular localization of their targets; see reviews, e.g., Miller and Messer, Molecular Therapy 12 (2005), 394-401. Additionally, alternative CRMP2 binding molecules are provided, such as engineered ankylosing spondylamine repeats (DARPins); see Hanenberg et al., J Biol Chem. 289 (2014), 27080-27089, which describe amyloid β-peptide-specific DARPins as novel potential therapies for Alzheimer's disease. However, as shown in the examples, IgG antibodies are preferred according to the invention.

[0396] The five main 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 called the γ chain; IgM has a μ chain; IgA has an α chain; IgE has an ε chain; and IgD has a δ chain; see reviews, e.g., Schroeder et al., Structure and function of immunoglobulins. J. Allergy Clin. Immunol. 125 (2010), S41-S52. Furthermore, there are different subclasses, with IgA further divided into IgA1 and IgA2, and IgG further divided into IgG1, IgG2, IgG3, and IgG4. In addition, there are two types of light chains, kappa (κ) and lambda (λ).

[0397] In principle, the antibodies of the present invention can be of any class and subclass, and can contain any type of light chain, as long as the binding specificity against CRMP2 (e.g., as shown in Tables VI or VII, and as illustrated in the appended examples for the corresponding reference antibodies) remains unaffected in class. However, it is preferred to use complete IgG antibodies, wherein the antibody contains constant domains. Thus, in one embodiment, the constant domains of the immunoglobulin heavy chain and / or light chain present in the antibody of the present invention belong to the IgG, IgM, IgA, IgD, or IgE type, preferably the IgG type. In one embodiment, the constant domains of the immunoglobulin heavy chain and / or light chain present in the antibody of the present invention are subclasses of IgA1, IgG1, IgG1, IgG2, IgG3, or IgG4, preferably subclasses of IgG1, IgG2, IgG3, or IgG4, and most preferably subclass IgG2. In one embodiment, the light chain is a kappa(κ) or lambda(λ) light chain.

[0398] The constant domain can be natural, i.e., originally cloned together with the variable domain, or heterologous, such as the mouse constant domain in the case of envisioned animal studies. In the present case, the initial isolated antibody is an IgG2 antibody, wherein the recombinant human antibody of the present invention is preferably of the IgG4 or IgG1 type, preferably IgG1. Thus, preferably, the constant domain is human and has a different IgG subtype, such as IgG2 versus IgG4, or IgG2 versus IgG1, or different allotypes and alleles, compared to the antibody constant domain naturally present in humans. The definition of “allotype” requires that the antibody reagent can be used to determine the allotype by serological methods. If only the sequence level is measured, then the polymorphism must be described as “allele.” This does not preclude establishing an allotype correspondence if the allele-allotype correspondence has been experimentally demonstrated, or if a single sequence is identical to a demonstrated sequence.

[0399] In a preferred embodiment of the invention, the constant domain is heterologous to at least one of the CDR and 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. Alternatively or additionally, the heterologous portion of the antibody may be a mammalian secreted signal peptide. In other words, in one embodiment, the anti-CRMP2 antibody and CRMP2 binding fragment or derivative thereof of the invention are (i) fusion proteins comprising polypeptide sequences heterologous to the VH region and / or VL region or at least one CDR; and / or (ii) non-natural variants of immunoglobulin-derived polypeptides comprising a heavy chain constant region containing one or more amino acid deletions, substitutions, and / or additions relative to the wild-type polypeptide. For example, the human constant domain of the recombinant human antibody of the invention may be an IgG allotype or a different allotype from the constant domain of the parental antibody naturally produced by memory B cells, for example, to avoid or reduce immunogenicity that may occur due to alloimmunization; see, for example, Jefferis and Lefranc, MAbs 1 (2009), 332-338. Therefore, in one embodiment, if the parental antibody is IgG1 subclass, the human constant domain of the recombinant human antibody of the present invention is IgG2, IgG3, or IgG4 subclass. In one embodiment, if the parental antibody is IgG2 subclass, the human constant domain of the recombinant human antibody of the present invention is IgG1, IgG3, or IgG4 subclass. In one embodiment, if the parental antibody is IgG3 subclass, the human constant domain of the recombinant human antibody of the present invention is IgG1, IgG2, or IgG4 subclass. In one embodiment, if the parental antibody is IgG3 subclass, the human constant domain of the recombinant human antibody of the present invention is IgG1, IgG2, or IgG4 subclass. In one embodiment, if the parental antibody is IgG4 subclass, the human constant domain of the recombinant human antibody of the present invention is IgG1, IgG2, or IgG3 subclass. In a preferred embodiment, the parental antibody is IgG2 subclass; therefore, the human constant domain of the recombinant human antibody of the present invention is IgG1, IgG3, or IgG4 subclass, preferably IgG1 or IgG4 subclass, most preferably IgG1 subclass or isotype.

[0400] In alternative or other embodiments, if the parent antibody contains a kappa(κ) light chain, the light chain of the recombinant human antibody of the present invention may 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 may be changed to a kappa(κ) light chain.

[0401] In addition to the four IgG subclasses mentioned above, human heavy and light chain genes also exhibit extensive structural polymorphisms and are tightly linked, inherited as haplotypes. Allotype variants can be immunogenic and elicit antibody responses as a result of alloimmunization. Therefore, allotype conversion can be of particular significance in providing non-immunogenic antibody therapy. To date, extensive allotypes (polymorphisms) are known, but the focus is on serologically defined allotypes. Allotypes of IgG proteins are defined by the expression of unique epitopes recognized by unique serological reagents. Allotypes expressed on constant regions of the IgG heavy chain are named Gm (genetic markers), along with subclasses such as G1m, and allotype numbers (or letters), such as 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 Irani et al., Molecular Immunology 67 (2015), 171–182. Figure 1 In section A, the contents are incorporated herein by reference. Therefore, in one embodiment, the antibody of the present invention is any, but not limited to, the following allotypes: 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 any of the above allotypes, then the recombinant human antibody of the present invention belongs to any of the above allotypes, but is one of the parent antibodies.

[0402] The four highly conserved subclasses IgG1, IgG2, IgG3, and IgG4 differ in their constant regions, particularly in their hinge and upper CH2 domains. These regions are involved in binding to both the IgG-Fc receptor (FcgR) and C1q. Therefore, the different subclasses have different effector functions in triggering FcgR-expressing cells, leading to phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc region also contains the binding epitope of the neonatal Fc receptor (FcRn), responsible for prolonging half-life, placental transport, and bidirectional transport of IgG across mucosal surfaces. However, FcRn is also expressed in bone marrow cells, where it participates in both phagocytosis and antigen presentation, along with the classic FcgR and complement. These characteristics, IgG polymorphisms, and how post-translational modifications of antibodies in glycosylated forms affect 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 immunoglobulin heavy and / or light chain constant domains present in the antibodies of the present invention belong to the IgG type.

[0403] Therefore, in certain embodiments of the invention, it is preferred that a specific IgG type, such as an IgG1 isotype and / or a constant region of the antibody, or its antigen-binding fragment, variant, or derivative, has been altered to provide desired biochemical properties. In particular, in one embodiment, the Fc portion of the antibody may be mutated to alter, i.e., to reduce or increase immune effector function or increase its half-life using techniques known in the art. Thus, in one embodiment, the Fc portion of the antibody is mutated to reduce immune effector function, and in another embodiment, the Fc portion of the antibody is mutated to increase immune effector function. In yet another embodiment, the antibody is mutated to increase its half-life.

[0404] For example, constant region modifications consistent with this invention may moderate complement binding, thereby reducing the serum half-life and nonspecific association of conjugated cytotoxins. Other modifications to the constant region can be used to modify disulfide bonds or oligosaccharide moieties, which allow for enhanced tissue-antigen interactions due to increased antigen specificity or antibody flexibility. Furthermore, mutations can be made in the Fc region to result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent phagocytosis (ADCP) via increased FcγRIIIa binding and / or decreased FcγRIIIb binding, and via increased FcγRIIa binding and / or FcγRIIIa binding, respectively. For example, the GASDALIE Fc mutant (G236A / S239D / A330L / I332E) exhibits a higher affinity for FcγRIIIa. Another possibility is to enhance complement-dependent cytotoxicity (CDC) via increased C1q binding and / or hexamerization.

[0405] In other embodiments, certain antibodies used in the diagnostic and therapeutic methods described herein have constant regions, such as the IgG heavy chain constant region, which are altered to eliminate glycosylation, referred to elsewhere herein as glycosylated or “agly” antibodies. Such “agly” antibodies can be prepared by enzymatic methods as well as by engineering common glycosylation sites in the constant region. “Agly” antibodies are believed to have reduced effector function, and therefore improved safety and stability characteristics in vivo. Methods for producing glycosylated antibodies with desired effector function can be found, for example, in International Application WO 2005 / 018572A2, which is incorporated herein by reference in its entirety. A further method for reducing the effector function of an antibody is to reduce the binding of FcγR and C1q through mutations in the Fc region.

[0406] Summary, for example, is given in the review by Wang et al., Protein Cell 9 (2018), 63-73, where Table 1 provides examples of modifications that regulate antibody effector function and antibody half-life, and the mutations described therein are incorporated herein by reference. The resulting physiological characteristics, bioavailability, and other biochemical effects of the modifications, such as recognition and binding to CRMP2, biodistribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without excessive experimentation.

[0407] As mentioned above, in some cases, inflammatory responses should be avoided, and therefore the effector function of the antibody's constant domain can be attenuated or completely eliminated. For example, IgG4 antibodies are potential candidates for immunotherapy when reduced effector function is desired. IgG4 Abs are dynamic molecules capable of undergoing a process called Fab arm exchange (FAE). This results in functional monovalent bispecific antibodies (bsAbs) with unknown specificity, thus potentially reducing therapeutic efficacy. However, stable S228P mutations can prevent IgG4 FAEs, thereby restoring the therapeutic efficacy of IgG4 antibodies; see Silva et al., J Biol Chem 290 (2015), 5462-5469. Furthermore, recombinant human IgG antibodies (hIgG) that completely do not bind to the Fcγ receptor (FcγR) and complement protein C1q, and therefore possess eliminated immune effector function, can be used for various therapeutic applications. The combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) or the SPLE mutation was found to eliminate FcγRIIa binding and showed elimination of detectable binding to FcγRI, IIIa, and IIIa for both IgG1 and IgG4. Furthermore, the LALA-PG mutation was superior to the LALA mutation alone because it disabled Fc function in both mouse and human IgG. For the 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.

[0408] Another early approach to reducing effector function was to alter the glycosylation site at N297 through mutations such as N297A, N297Q, and N297G. Antibody half-life can be increased by introducing the following mutations: M252Y / S254T / T256E or M428L / N434S; see Wang et al. 2018.

[0409] As shown in Examples 13 and 14, administration of antibody NI-504-3E7 resulted in 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 determinant of the observed antibody biological effects. This is consistent with the finding that antibody NI-504.3E7 is capable of detecting CRMP2 intracellularly, as demonstrated in Example 12 with fully differentiated SH-SY5Y cells, where effector function is not required. 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 or IgG1 class having any of the above-described 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 IgG1 subclass having the LALA mutation, and most preferably the LALA-PG mutation.

[0410] This invention also relates to antibody or antigen-binding fragments comprising heterologous polypeptides. For example, the antibody polypeptides of this invention may comprise, consist substantially of, or consist of fusion proteins. Fusion proteins are chimeric molecules comprising, for example, an immunoglobulin CRMP2 binding domain having at least one target binding site and at least one heterologous portion, i.e., a portion not naturally associated with it in nature. The amino acid sequences can typically be present in individual proteins aggregated together in the fusion polypeptide, or they can typically be present in the same protein but arranged in a novel manner in the fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis, or by generating and translating polynucleotides in which the peptide regions encode in a desired relationship.

[0411] When applied to polynucleotides or peptides, the term "heterogeneous" means that the polynucleotide or peptide is derived from an entity that is different from the rest of the entity being compared with it. For example, as used herein, a "heterogeneous peptide" fused with an antibody or its antigen-binding fragment, variant, or analogue is derived from a non-immunoglobulin peptide of the same species, or from an immunoglobulin or non-immunoglobulin peptide of a different species.

[0412] As described above, chimeric molecules constitute fusion proteins. The antibodies of the present invention can be chimeric rodent-human antibodies, preferably chimeric mouse-human antibodies, which are particularly useful for animal diagnostic methods and research. In one embodiment, the antibody of the present invention is a chimeric mouse-human antibody, preferably wherein the antibody comprises a mouse constant region, most preferably the mouse IgG2a constant region. The production of chimeric antibodies is described, for example, in International Application WO 89 / 09622.

[0413] The present invention also relates to one or more polynucleotides encoding the antibody, antigen-binding fragment or derivative thereof or immunoglobulins VH and VL of the present invention, preferably wherein the polynucleotide is cDNA.

[0414] In a preferred embodiment of the invention, the polynucleotide comprises, substantially constitutes, or is constituted a nucleic acid having a polynucleotide sequence encoding a VH or VL chain of an anti-CRMP2 antibody as shown in Table II. In this regard, those skilled in the art will readily understand that the polynucleotide encoding the light chain and / or heavy chain may be encoded by one or more polynucleotides. Therefore, in one embodiment, the polynucleotide comprises, substantially constitutes, or is constituted a nucleic acid having a polynucleotide sequence having VH and VL chains of an anti-CRMP2 antibody as depicted in Table II.

[0415] In one embodiment of the invention, the polynucleotide is linked to a heterologous nucleic acid, such as an expression control sequence like a promoter, a transcription and / or translation enhancer sequence, an internal ribosome binding site, or a nucleic acid encoding a peptide leader sequence for recombinant expression in a host.

[0416] Therefore, the present invention relates to a polynucleotide encoding an anti-CRMP2 antibody, in a preferred embodiment, a human recombinant anti-CRMP2 antibody or a CRMP2 binding fragment or a derivative thereof, wherein the polynucleotide encodes a VH chain comprising CDR1, 2, and 3 as defined by Kabat and / or a VL chain comprising CDR1, 2, and 3, wherein

[0417] (a) VH-CDR1 includes, for example Figure 1 The amino acid sequence of SEQ ID NO: 3 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 3,

[0418] (b) VH-CDR2 includes, for example Figure 1 The amino acid sequence of SEQ ID NO: 4 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 4,

[0419] (c) VH-CDR3 contains, for example Figure 1 The amino acid sequence of SEQ ID NO: 5 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 5,

[0420] (d)VL-CDR1 includes, for example Figure 1 The amino acid sequence of SEQ ID NO: 8 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 8.

[0421] (e)VL-CDR2 includes, for example Figure 1 The amino acid sequence of SEQ ID NO: 9 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 9, and

[0422] (f) VL-CDR3 contains, for example Figure 1 The amino acid sequence of SEQ ID NO: 10 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 10.

[0423] In a preferred embodiment, the variant 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 the VL-CDR contains 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 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 variant CDR contains at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% of the sequences 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 variant CDR contains at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% of the amino acid sequence shown in SEQ ID NO: 10.

[0424] The present invention further relates to a polynucleotide encoding an anti-CRMP2 antibody, wherein, in a preferred embodiment, a human recombinant anti-CRMP2 antibody or a CRMP2 binding fragment or a derivative thereof, the polynucleotide encoding

[0425] (i) VH chains containing CDRs 1, 2, and 3 as defined by Kabat, and / or VL chains containing VL CDRs 1, 2, and 3, wherein

[0426] (a) VH-CDR1 includes, for example Figure 1The amino acid sequence of SEQ ID NO: 3 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 3,

[0427] (b) VH-CDR2 includes, for example Figure 1 The amino acid sequence of SEQ ID NO: 4 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 4,

[0428] (c) VH-CDR3 contains, for example Figure 1 The amino acid sequence of SEQ ID NO: 5 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 5,

[0429] (d)VL-CDR1 includes, for example Figure 1 The amino acid sequence of SEQ ID NO: 8 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 8.

[0430] (e)VL-CDR2 includes, for example Figure 1 The amino acid sequence of SEQ ID NO: 9 shown in Table III, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change to SEQ ID NO: 9, and

[0431] (f) VL-CDR3 contains, for example Figure 1 The amino acid sequence of SEQ ID NO: 61 shown in Table III or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 61,

[0432] In a 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, and most preferably one amino acid substitution. In another preferred embodiment, the variant comprises at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, and preferably 99% identical amino acid sequences to those 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.

[0433] In another or alternative embodiment, the present invention relates to a polynucleotide encoding an anti-CRMP2 antibody, and in a preferred embodiment, a human recombinant anti-CRMP2 antibody or a CRMP2 binding fragment or a derivative thereof, wherein the polynucleotide encodes an anti-CRMP2 antibody, a CRMP2 binding fragment or a derivative thereof.

[0434] VH chain and / or VL chain, where

[0435] (a) The VH chain contains, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 2 as shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 2; and / or

[0436] (b) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 as shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7.

[0437] In another preferred embodiment, variants VH and VL each contain one or more amino acid substitutions, preferably one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Preferably, variants of the VL chain contain one, two, three, four, or five amino acid substitutions, and / or variants of the VH chain contain one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, preferably two, three, four, five, six, seven, eight, or ten amino acid substitutions. Most preferably, variants VH and VL each contain one or two amino acid substitutions.

[0438] In one embodiment, the listed VH chain variants comprise amino acid substitutions of T7S, Q19R, P28T, Y29F, V40S, V40A, I71T, M80T, R82Y, H84Q, and / or Y86S. In particular, in a preferred embodiment, the VH chain variants comprise amino acid substitutions:

[0439] (i) M80T and Y86S,

[0440] (ii) V40S, M80T and Y86S,

[0441] (iii) I71T, M80T and Y86S,

[0442] (iv) V40S, I71T, M80T, and Y86S,

[0443] (v) V40A, I71T, M80T, and Y86S,

[0444] (vi) T7S, Q19R, V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0445] (vii) Q19R, V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0446] (viii) T7S, V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0447] (ix) T7S, Q19R, V40A, I71T, M80T, H84Q, and Y86S,

[0448] (x) T7S, Q19R, V40A, I71T, M80T, R82Y, and Y86S,

[0449] (xi) V40A, I71T, M80T, R82Y, H84Q and Y86S,

[0450] (xii) Q19R, V40A, I71T, M80T, H84Q and Y86S,

[0451] (xiii) Q19R, V40A, I71T, M80T, R82Y and Y86S,

[0452] (xiv) T7S, V40A, I71T, M80T, H84Q, and Y86S,

[0453] (xv) T7S, V40A, I71T, M80T, R82Y and Y86S,

[0454] (xvi) T7S, Q19R, V40A, I71T, M80T, and Y86S,

[0455] (xvii) T7S, V40A, I71T, M80T, and Y86S,

[0456] (xviii) Q19R, V40A, I71T, M80T, and Y86S,

[0457] (ixx) V40A, I71T, M80T, R82Y and Y86S,

[0458] (xx) V40A, I71T, M80T, H84Q and Y86S,

[0459] (xxi) T7S, Q19R, V40S, I71T, M80T, R82Y, H84Q, and Y86S, or

[0460] (xxii) T7S, Q19R, P28T, Y29F, V40A, I71T, M80T, R82Y, H84Q and Y86S.

[0461] In one embodiment, the listed VL chain variants comprise amino acid substitutions of L37Q, L69T, N75S, L78Q, and / or D91E. Particularly, in a preferred embodiment, the VL variants comprise amino acid substitutions:

[0462] (i) L69T and L78Q,

[0463] (ii) L37Q, L69T, N75S, and L78Q, or

[0464] (iii) L37Q, L69T, N75S, L78Q and D91E.

[0465] As shown in Tables II and V, the amino acid substitutions are present in the 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_3 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, VH_3E7_var8) and variants of the VL chain of antibody NI-504.3E7 (VL_3E7_var1, VL_3E7_var2, VL_3E7_var3). Preferred allocations of the variant VH and VL chains are described in Table IV.

[0466] In another preferred embodiment, the variants of the VH and VL chains each contain at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% of the same amino acid sequence as the sequences shown in SEQ ID NO:2 and SEQ ID NO:7, respectively.

[0467] In one embodiment, the present invention relates to a polynucleotide encoding an anti-CRMP2 antibody, and in a preferred embodiment, to a human recombinant anti-CRMP2 antibody or a CRMP2 binding fragment or a derivative thereof, wherein the polynucleotide encodes a VH chain and / or a VL chain, wherein

[0468] NI-504.3E7_V1

[0469] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 2 (VH_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 2; and / or

[0470] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0471] NI-504.3E7_V2

[0472] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 2 (VH_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 2; and / or

[0473] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0474] NI-504.3E7_V3

[0475] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 12 (VH_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 12; and / or

[0476] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7;

[0477] NI-504.3E7_V4

[0478] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 12 (VH_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 12; and / or

[0479] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0480] NI-504.3E7_V5

[0481] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 12 (VH_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 12; and / or

[0482] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0483] NI-504.3E7_V6

[0484] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 14 (VH_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 14; and / or

[0485] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7;

[0486] NI-504.3E7_V7

[0487] (i) The VH chain contains, for example, Figure 1Or the amino acid sequence described in SEQ ID NO: 14 (VH_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 14; and / or

[0488] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0489] NI-504.3E7_V8

[0490] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 14 (VH_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 14; and / or

[0491] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0492] NI-504.3E7_V9

[0493] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 16 (VH_3E7_var3) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 16; and / or

[0494] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7;

[0495] NI-504.3E7_V10

[0496] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 16 (VH_3E7_var3) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 16; and / or

[0497] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0498] NI-504.3E7_V11

[0499] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 16 (VH_3E7_var3) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 16; and / or

[0500] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0501] NI-504.3E7_V12

[0502] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0503] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7;

[0504] NI-504.3E7_V13

[0505] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0506] (ii) VL includes, for example Figure 1Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0507] NI-504.3E7_V14

[0508] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0509] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0510] NI-504.3E7_V15

[0511] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 18 (VH_3E7_var4) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 18; and / or

[0512] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60;

[0513] NI-504.3E7_V16

[0514] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0515] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 7 (VL_3E7) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 7;

[0516] NI-504.3E7_V17

[0517] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0518] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0519] NI-504.3E7_V18

[0520] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0521] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0522] NI-504.3E7_V19

[0523] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 20 (VH_3E7_var5) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 20; and / or

[0524] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60;

[0525] NI-504.3E7_V20

[0526] (i) The VH chain contains, for example, Figure 1Or the amino acid sequence or a variant thereof described in SEQ ID NO: 22 (VH_3E7_var6) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 22; and / or

[0527] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0528] NI-504.3E7_V21

[0529] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 24 (VH_3E7_var6a) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 24; and / or

[0530] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0531] NI-504.3E7_V22

[0532] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 26 (VH_3E7_var6b) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 26; and / or

[0533] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0534] NI-504.3E7_V23

[0535] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 28 (VH_3E7_var6c) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 28; and / or

[0536] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0537] NI-504.3E7_V24

[0538] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 30 (VH_3E7_var6d) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 30; and / or

[0539] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0540] NI-504.3E7_V25

[0541] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 32 (VH_3E7_var6ab) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 32; and / or

[0542] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0543] NI-504.3E7_V26

[0544] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 34 (VH_3E7_var6ac) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 34; and / or

[0545] (ii) VL includes, for example Figure 1Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0546] NI-504.3E7_V27

[0547] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 36 (VH_3E7_var6ad) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 36; and / or

[0548] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0549] NI-504.3E7_V28

[0550] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 38 (VH_3E7_var6bc) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 38; and / or

[0551] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0552] NI-504.3E7_V29

[0553] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 40 (VH_3E7_var6bd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 40; and / or

[0554] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0555] NI-504.3E7_V30

[0556] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 42 (VH_3E7_var6cd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 42; and / or

[0557] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0558] NI-504.3E7_V31

[0559] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 44 (VH_3E7_var6bcd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 44; and / or

[0560] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0561] NI-504.3E7_V32

[0562] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 46 (VH_3E7_var6acd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 46; and / or

[0563] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0564] NI-504.3E7_V33

[0565] (i) The VH chain contains, for example, Figure 1Or the amino acid sequence described in SEQ ID NO: 48 (VH_3E7_var6abd) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 48; and / or

[0566] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0567] NI-504.3E7_V34

[0568] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 50 (VH_3E7_var6abc) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 50; and / or

[0569] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0570] NI-504.3E7_V35

[0571] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 22 (VH_3E7_var6) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 22; and / or

[0572] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0573] NI-504.3E7_V36

[0574] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 22 (VH_3E7_var6) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 22; and / or

[0575] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60;

[0576] NI-504.3E7_V37

[0577] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 52 (VH_3E7_var7) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 52; and / or

[0578] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 56 (VL_3E7_var1) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 56;

[0579] NI-504.3E7_V38

[0580] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 52 (VH_3E7_var7) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 52; and / or

[0581] (ii) VL includes, for example Figure 1 Or the amino acid sequence described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58;

[0582] NI-504.3E7_V39

[0583] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 52 (VH_3E7_var7) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 52; and / or

[0584] (ii) VL includes, for example Figure 1Or the amino acid sequence or a variant thereof described in SEQ ID NO: 60 (VL_3E7_var3) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 60;

[0585] NI-504.3E7_V40

[0586] (i) The VH chain contains, for example, Figure 1 Or the amino acid sequence described in SEQ ID NO: 54 (VH_3E7_var8) shown in Table II, or a variant thereof, wherein the variant comprises an amino acid sequence resulting from a partial change in SEQ ID NO: 54; and / or

[0587] (ii) VL includes, for example Figure 1 Or the amino acid sequence or a variant thereof described in SEQ ID NO: 58 (VL_3E7_var2) shown in Table II, wherein the variant comprises an amino acid sequence resulting from a partial change of SEQ ID NO: 58.

[0588] In another preferred embodiment, variants VH and VL each contain one or more amino acid substitutions, preferably one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Preferably, variants of the VL chain contain one, two, three, four, or five amino acid substitutions, and / or variants of the VH chain contain one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, preferably two, three, four, five, six, seven, eight, or ten amino acid substitutions. Most preferably, variants VH and VL each contain one or two amino acid substitutions.

[0589] In another preferred embodiment, the variants of the VH and VL chains respectively comprise the sequences specified in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12 and 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, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO: The sequence shown in 60 has at least 90%, preferably 91%, preferably 92%, preferably 93%, preferably 94%, preferably 95%, preferably 96%, preferably 97%, preferably 98%, preferably 99% identical amino acid sequences.

[0590] Furthermore, this invention relates to polynucleotides linked to heterologous nucleic acids, wherein the polynucleotides are selected from the group consisting of:

[0591] (a) A polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or the fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively, and wherein CRMP2 is bound when the VH pairs with a light chain variable region (VL) comprising the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 58 or SEQ ID NO: 60;

[0592] (b) A polynucleotide encoding an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or fragment thereof comprising a VL, the VL comprising CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, or SEQ ID NO: 8, 9, and 61, respectively, and wherein CRMP2 is bound when the VL pairs with a VH, the VH comprising 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. The amino acid sequence shown in SEQ ID NO: 50, SEQ ID NO: 52 or SEQ ID NO: 54;

[0593] (c) Polynucleotides, which encode

[0594] (i) An immunoglobulin heavy chain or a fragment thereof, comprising a VH containing CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; and

[0595] (ii) An immunoglobulin light chain or a fragment thereof, comprising a VL containing CDRs 1, 2 and 3 having the amino acid sequences shown in SEQ ID NO: 8, 9 and 10 or SEQ ID NO: 8, 9 and 61, respectively;

[0596] (d) A polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof containing VH, wherein VH comprises 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 CRMP2 is bound when VH pairs with VL, and VL comprises SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 54. The amino acid sequence shown in SEQ ID NO: 60;

[0597] (e) A polynucleotide encoding an immunoglobulin light chain or fragment thereof comprising VL, wherein VL comprises the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 58 or SEQ ID NO: 60, wherein VL binds CRMP2 when paired with VH, wherein VH comprises 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: 60, SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 6 ... The amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 54;

[0598] (f) A polynucleotide encoding an immunoglobulin heavy chain or fragment thereof containing VH and an immunoglobulin light chain or fragment thereof containing VL, wherein VH comprises 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 VL comprises the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54, and VL comprises the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54. The amino acid sequence shown in SEQ ID NO: 60;

[0599] (g) A polynucleotide as in any of (a)-(f), wherein the CDR comprises one or more, preferably no more than two amino acid substitutions, and / or the variable region sequence is 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% are at least 90% the same.

[0600] Tables III and II provide the assignments of SEQ ID NO to the corresponding CDR and the VH and VL chains, with preferred combinations of the VH and VL chains shown in Table IV.

[0601] In a preferred embodiment, when paired as VH and VL, immunoglobulin binds to CRMP2, as measured by direct or capture ELISA or an assay equivalent to the assay described in the examples.

[0602] Furthermore, the present invention relates to vectors, and the vectors comprise one or more of these polynucleotides, preferably wherein the vector is an expression vector, and one or more polynucleotides are operatively linked to an expression control sequence.

[0603] Polynucleotides can be produced and, if desired, manipulated using methods well-known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Molecular Cloning: A Laboratory Manual (4th Edition): Three-volumeset; Green and Sambrook (2012) ISBN 10: 1936113422 / ISBN 13: 9781936113422 Cold Spring Harbor Laboratory Press; Updated (2014) ISBN 978-1-936113-42-2 and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998) and the Updated, which are incorporated herein by reference in their entirety), to generate antibodies with different amino acid sequences, for example, to produce amino acid substitutions, deletions, and / or insertions.

[0604] Once a polynucleotide encoding the heavy or light chain of the antibody molecule of the present invention, or a portion thereof (preferably containing a variable domain of the heavy or light chain), is obtained, a vector for generating the antibody molecule can be produced using recombinant DNA technology with techniques well known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody-encoding sequences and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo gene recombination. Therefore, the present invention provides reproducible vectors comprising nucleotide sequences operatively linked to a promoter encoding the antibody molecule of the present invention, or its heavy or light chain, or a variable domain of the heavy or light chain. Such vectors may include nucleotide sequences encoding constant regions of the antibody molecule (see, for example, International Applications WO 86 / 05807 A1 and WO 89 / 01036 A1; and U.S. Patent No. 5,122,464), and the variable domain of the antibody may be cloned into such vectors for expression of the entire heavy or light chain.

[0605] The terms “vector” or “expression vector” are used herein to refer to a vector used according to the invention, such as a medium for introducing into a host cell and expressing a desired gene within the host cell. Typically, vectors compatible with the invention may contain selection markers, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells. Markers may provide protrophic status for auxotrophic hosts, resistance to bactericides (e.g., antibiotics), or resistance to heavy metals (such as copper). The selection marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Optimal synthesis of mRNA may also require additional elements. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and termination signals. For the expression of double-stranded antibodies, a single vector or multiple vectors encoding the heavy and light chains may be co-expressed in a host cell for the expression of the entire immunoglobulin molecule, as detailed below.

[0606] Host cells can 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. Both vectors may contain the same selection marker, which allows for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both the heavy and light chain polypeptides may be used. In this case, the light chain is advantageously placed before the heavy chain to avoid excessive amounts of the non-toxic heavy chain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. The expression vectors can be transferred to host cells using conventional techniques, and the transfected cells can then be cultured using conventional techniques to produce antibodies used in the methods described herein. Therefore, the present invention also relates to host cells comprising one or more polynucleotides of the present invention or one or more vectors.

[0607] As used herein, “host cell” refers to a cell containing a vector constructed using recombinant DNA technology that encodes at least one heterologous gene. In the description of methods 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 stated. In other words, recovering peptides from “cells” can mean recovering peptides from a whole cell that is rotating at a reduced speed, or from a cell culture containing both culture medium and suspended cells.

[0608] Antibodies used for 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 glycosylated engineered yeast, insect cell lines, and transgenic plants promise antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments, including bispecific antibodies without any glycosylation, have been successfully produced in bacteria and are already in clinical testing. The first therapeutic antibody product from a non-mammalian source is expected to be available in the coming years. Frenzel et al., Front Immunol. 4 (2013), 217, published July 29, 2013, doi: 10.3389 / fimmu.2013.00217, provides a review of current antibody production systems applicable to the preparation of the human recombinant anti-CRMP2 antibody or CRMP2 binding fragment or derivatives thereof of the present invention, including their availability for various applications, and the transient expression of human antibodies in mammalian cells is described by 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.

[0609] Once the antibody molecules of the present invention have been recombinantly expressed, the whole antibodies of the present invention, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures in the art, including, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for specific antigens after protein A, and fractional column chromatography), centrifugation, differential solubility such as ammonium sulfate precipitation, or by 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 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Therefore, the present invention also relates to a method for preparing anti-CRMP2 antibodies and / or fragments thereof or immunoglobulin chains thereof, said method comprising:

[0610] (a) Culture host cells as defined above, containing the polynucleotides or vectors as defined above, under conditions that allow expression of anti-CRMP2 antibodies, CRMP2 binding fragments, or their immunoglobulin chains; and

[0611] (b) Isolate anti-CRMP2 antibody, CRMP2 binding fragment or its immunoglobulin chain from the culture.

[0612] Furthermore, the present invention also relates to anti-CRMP2, CRMP2 binding fragments, derivatives thereof, and immunoglobulin chains encoded by the polynucleotides of the present invention described above and obtainable by the recombinant production method thereof; see above.

[0613] The antibodies of the present invention can also be used in diagnostic methods. Since the antibodies of the present invention bind to both pCRMP2 and CRMP2, total CRMP2 can be detected. Therefore, 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 a CRMP2-binding fragment thereof, wherein the increased level of total CRMP2 indicates the corresponding disease in the subject compared to a control (e.g., a sample from a healthy subject or a sample from a subject suffering from a diagnostic disease associated with an increased level of total CRMP2).

[0614] The anti-CRMP2 antibody of the present invention is administered to a subject diagnosed with this disease. Alternatively, the subject is diagnosed according to the method of the present invention, the information is directly or indirectly transmitted to the subject or a physician or medical institution, and if the subject has been diagnosed with a disease related to CRMP2 pathology, the subject is treated with an agent capable of improving, treating, or reducing the progression of at least one symptom of the disease in the subject. Preferably, the agent is an anti-CRMP2 antibody, and most preferably, the antibody of the present invention.

[0615] Subjects to be diagnosed may be asymptomatic or preclinical in relation to the disease. In one implementation, subjects to be diagnosed and control subjects are age-matched. The sample to be analyzed may be any bodily fluid suspected of containing CRMP2 / pCRMP2, such as blood, CSF, or tissue.

[0616] The level of CRMP2 / pCRMP2 can be assessed by any suitable method known in the art, including, for example, analysis of 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), rapid protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC), followed by tandem mass spectrometry (MS / MS), laser density measurement, and IHC.

[0617] Furthermore, anti-CRMP2 antibodies or CRMP2 binding fragments or derivatives thereof can be used for in vivo imaging of CRMP2 / pCRMP2. Therefore, 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).

[0618] In some embodiments, the antibody peptide comprises an amino acid sequence or one or more portions that do not normally associate with an antibody. Therefore, the invention further covers antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, conjugated to diagnostic or therapeutic agents. Antibodies can be used for diagnosis, for example, to demonstrate the presence of disease associated with abnormal levels / locations of pCRMP2 / CRMP2 as described above, to indicate the risk of having disease associated with abnormal levels / locations of pCRMP2 / CRMP2, to monitor the development or progression of disease associated with abnormal levels / locations of pCRMP2 / CRMP2, for example, as part of a clinical testing procedure, such as to determine the efficacy of a given treatment and / or prevention regimen. Detection can be facilitated by conjugating 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 antibodies of the present invention or their CRMP2 binding fragments (such as single-chain Fv antibody fragments) may contain flexible linker sequences or may be modified to add functional portions or detectable labels (e.g., PEG, drugs, toxins, or labels such as fluorescence, (chemiluminescence / bioluminescence), radioactivity, enzymes, NMR, heavy metals, tags, flags, etc.); for general techniques, see, for example, Antibodies A Laboratory Manual 2nd edition, 2014 by 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 mimics, see Falck and Muller, Antibodies 7 (2018), 4; doi:10.3390 / antib7010004.

[0619] Furthermore, the anti-CRMP2 antibody or CRMP2 binding fragment or its derivative of the present invention may contain organ-specific targeting entities, particularly brain-targeting entities, and / or be contained in or conjugated in media such as exosomes or nanoparticles for delivery to the brain.

[0620] As is well known in the art, the blood-brain barrier (BBB) ​​limits the efficacy of drugs for central nervous system (CNS) diseases. For example, monoclonal antibodies cannot effectively cross the BBB, reaching a maximum of 0.11% at 1 h post-injection (Banks et al. (2002), Peptides 23, 2223-2226). The BBB is a specialized structural, physiological, and biochemical barrier and acts as the first interface between the variable environments of blood and extracellular fluid in the CNS. The BBB regulates nervous system homeostasis by tightly controlling the movement of small or large molecules from the blood to the brain. It allows only selective transport of molecules essential for brain function. Specifically, over 98% of small molecule drugs and almost 100% of large molecule drugs cannot be delivered to the brain (Redzic (2011) Fluids Barriers CNS 8, 3; Pardridge (2005) NeuroRx, 2, 3-14). Therefore, peptides and antibodies, their antigen-binding fragments, variants, or derivatives can be modified to enable penetration of the BBB.

[0621] For example, the antibody and binding fragment can be fused with a cell-penetrating peptide (CPP), which is eligible as a brain-targeting entity and is typically a short cationic and / or amphiphilic peptide with the ability to transport related molecular cargoes (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 instructions on how to fuse antibodies with CPPs are provided, for example, in Gastonet et al. (2019) Sci. Rep. 9, 18688 doi:10.1038 / s41598-019-55091-0. Furthermore, polyamine modification has been shown to significantly increase the penetration 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 insulin receptor (IR). Therefore, RMT receptors are also brain-targeting entities. For example, bispecific antibodies have emerged as promising scaffolds, delivering therapeutic antibodies to the brain by engineering them to incorporate one arm specific to the BBB RMT receptor (which drives antibody delivery across the BBB) and another arm specific to CNS therapeutics. Essentially, bispecific antibodies can be generated by fusing antibody fragments such as Fab, scFv, or single-domain antibodies to the N or C terminus of a conventional IgG molecule, or through heterodimerization strategies such as the “knobs-into-holes” technology developed by Genentech; see Neves et al. (2016) TrendsBiotech. 34, 36-48 for details.

[0622] Therefore, the anti-CRMP2 antibody of the present invention can be a bispecific antibody that binds to CRMP2 and the BBB RMT receptor.

[0623] In another approach, lipid nanoparticles / nanoexosomes can be used to deliver the antibodies or binding fragments of the present invention across the blood-brain barrier or toward the brain or kidneys. For example, nanoliposomes dual-modified with anti-CRMP2 monoclonal antibodies and anti-RMT antibodies, such as anti-TfR monoclonal antibodies conjugated with biotin-streptavidin, can be used to improve delivery across the blood-brain barrier. This 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.

[0624] Furthermore, as summarized in Tosi et al. (2013) (Curr.Med.Chem.20, 2212-25), biodegradable nanoparticles formulated from poly(D,L-lactide-co-glycolic acid) (PLGA) have been extensively studied for sustained and targeted delivery of various pharmaceutical agents. Therefore, the antibody and binding fragment of this invention are conjugated to nanoparticles and nanoexosomes, respectively.

[0625] Then, according to standard techniques known in the art, anti-CRMP2 antibodies or CRMP2-binding fragments, synthetic or biotechnological derivatives thereof, particularly human recombinant antibodies, are provided for a variety of applications, optionally as fusion proteins and / or labeled as described above; see, for example, Antibodies A Laboratory Manual, 2nd edition, 2014 by ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Current advances in the design, preparation and formulation of therapeutic antibodies are described in Sifniotis et al., Antibodies 2019, 8(2), 36; https: / / doi.org / 10.3390 / antib8020036, which also discusses the development of computational methods for the strategic design of antibodies with regulatory functions.

[0626] This invention relates to compositions comprising the CRMP2 binding molecule described above, such as antibodies or CRMP2 binding fragments, variants or biotechnological derivatives thereof as defined above, or polynucleotides, carriers or cells. In one embodiment, the composition of this invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

[0627] The present invention also provides pharmaceutical and diagnostic compositions, respectively, in the form of packages or kits, the packages or kits comprising one or more containers filled with one or more of the above-mentioned components (e.g., the anti-CRMP2 antibody of the present invention, CRMP2 binding fragments, biotechnological derivatives or variants thereof, polynucleotides, vectors, or cells). Associated with such containers may be a notification in the form prescribed by a government agency regulating the preparation, use, or sale of pharmaceutical or biological products, reflecting that agency's approval for the preparation, use, or sale for human administration. Additionally or alternatively, the kits include reagents and / or instructions for use in appropriate immunologically based diagnostic assays. The compositions, such as the kits of the present invention, are of course particularly suitable for the risk assessment, diagnosis, prevention, and treatment of diseases or conditions associated with CRMP2, and particularly suitable for the treatment of conditions commonly associated with CRMP2, as discussed above.

[0628] 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) by the University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable drug carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to subjects at appropriate doses. The administration of suitable compositions can be achieved in various ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, external, or intradermal administration, or by spinal or brain delivery. Aerosol formulations such as nasal spray formulations comprise purified aqueous or other solutions of active agents, preservatives, and isotonic agents. Preferably, such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

[0629] The dosing regimen will be 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 body size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health condition, and other medications administered concurrently; see, for example, Bai et al., Clinical Pharmacokinetics 51 (2012), 119-135.

[0630] As explained in detail above, the antibodies of the present invention have been shown to improve cognitive function in aged mice and reduce amyloid β-plaque burden in the mouse cortex and hippocampus. Therefore, the present invention provides for the first time a therapeutically useful anti-CRMP2 antibody. Accordingly, in one embodiment, the present invention relates to antibodies used as pharmaceuticals, or antigen-binding fragments, derivatives, or variants thereof.

[0631] Experiments demonstrating improved cognitive function and reduced amyloid-β load in aged mice have been performed in a mouse model of Alzheimer's disease (AD). Furthermore, pCRMP2 is known to co-localize with pTau in neurofibrillary tangles, and experiments conducted within the scope of this invention have confirmed the co-aggregation of pCRMP2 and pTau. Additionally, staining of hippocampal tissue from patients with various tau protein disorders using the antibodies of this invention has been observed. Therefore, as a precaution, it is anticipated that the antibodies of this invention and their corresponding CRMP2-binding fragments may be used not only for the treatment of AD but also for the treatment of other neurodegenerative diseases, particularly those related to Tau and / or Aβ pathology (more specifically to cognitive impairment), diseases associated with memory and learning impairment, and neurodegenerative diseases in general.

[0632] The antibody of the present invention can alleviate or eliminate at least one symptom of neurodegenerative disease in a subject. Symptoms may include hyperphosphorylation of CRMP2, accumulation of pCRM2 (particularly in neurofibrillary tangles), depletion of CRMP2, co-aggregation of pCRMP2 and pTau, and / or accumulation of amyloid β plaques in the subject's brain.

[0633] Neurodegenerative diseases are chronic conditions characterized by the progressive loss of neurons in the brain and spinal cord, with dementia-related tau proteinopathy being the most prominent and prevalent group. Neurodegenerative tau proteinopathy shares a common pathological lesion consisting of intracellular aggregates of aberrant filaments primarily composed of pathologically hyperphosphorylated Tau in neurons and / or glial cells. The clinical features of tau proteinopathy are heterogeneous and are characterized by dementia and / or motor syndromes. The progressive accumulation of filamentous Tau inclusions may combine with other deposits (such as β-amyloid in AD) leading to neuronal and glial degeneration. Due to the heterogeneity of their clinical manifestations, a potential, incomplete list of tau protein diseases also associated with memory loss can be provided, including Alzheimer's disease (AD), amyotrophic lateral sclerosis and Parkinson's syndrome-dementia complex (ALS-PDC), aerophilic granulomatosis (AGD), British amyloid angiopathy, cerebral amyloid angiopathy, Creutzfeldt-Jakob disease (CJD), boxing dementia, diffuse neurofibrillary tangles with calcification, fibrillary dysplasia (FTD), fibrillary dysplasia-17 (FTDP-17), frontotemporal degeneration (FLD), Hallervorden-Spatz disease, PiD, prion cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, tangles dementia, and multiple infarct dementia; see reviews, e.g., Lee et al. (2001) Annu. Rev. Neurosci. 24, 1121-1159, where Table 1 lists unique members of tau protein diseases, or Sergeant et al. (2005), Bioch. Biophy. Acta 1739, 179-97, of which Figure 1 The list in the document.

[0634] In one embodiment, the present invention relates to antibodies or corresponding CRMP2 binding fragments for the treatment of neurological disorders, preferably those related to Tau and / or Aβ pathology (particularly those related to cognitive impairment), those related to memory and learning impairment, and neurodegenerative disorders in general. In a preferred embodiment, the present invention relates to antibodies or corresponding CRMP2 binding fragments for the treatment of neurological disorders, including but not limited to AD, ALS-PDC, AGD, British amyloid angiopathy, cerebral amyloid angiopathy, CJD, boxing dementia, diffuse neurofibrillary tangles with calcification, FTD, FTDP-17, FLD, Hallervorden-Spatz disease, PiD, prion cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, tangled dementia, multiple infarct dementia, and Lewy body disease. Mild cognitive impairment, cognitive impairment, Lewy body dementia (DLB), Parkinson's disease, ALS, spinal cord injury, traumatic brain injury, Huntington's disease, multiple sclerosis, corticobasal degeneration (CBD), stroke, cerebrovascular disease, neurological diseases, CNS diseases, neuropathic pain, chronic pain, low back pain, schizophrenia, depression, anxiety disorder, bipolar disorder, autism spectrum disorder, attention deficit / hyperactivity disorder, learning disability, motor disorder, obsessive-compulsive disorder, personality disorder, sleep disorder, delirium, developmental disorder, intellectual disability, and post-traumatic stress disorder. In a further preferred embodiment, the present invention relates to antibodies or corresponding CRMP2 binding fragments for the treatment of Tau protein diseases, including the aforementioned diseases, preferably AD, ALS-PDC, AGD, British amyloid angiopathy, cerebral amyloid angiopathy, CJD, boxing dementia, diffuse neurofibrillary tangles with calcification, FTD, FTDP-17, FLD, Hallervorden-Spatz disease, PiD, prion cerebral amyloid angiopathy, progressive subcortical gliosis, PSP, tangles dementia, and multiple infarct dementia, most preferably AD, PSP, PiD, FTD, and FLD, and most preferably AD.

[0635] Therefore, the present invention also relates to methods for treating diseases associated with Tau and / or Aβ pathology (particularly cognitive impairment), diseases associated with memory and learning impairment, and neurodegenerative diseases in general, comprising administering to a subject in need a therapeutically effective amount of any of the aforementioned CRMP2-binding molecules, particularly the human antibodies of the present invention. The present invention provides a method for treating a subject with a neurodegenerative disease by administering a therapeutically effective amount of any 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 subject's neurodegenerative disease. Preferably, the method is used for the treatment of neurodegenerative diseases, preferably tau protein diseases, including the aforementioned diseases, preferably AD, PSP, PiD, FTD, and FLD, and most preferably AD.

[0636] Additionally, in one embodiment, the present invention relates to a peptide having an epitope of (p)CRMP2 that is specifically recognized by any antibody of the present invention, preferably specifically recognized by antibody NI-504.3F4 in which one or more amino acids are substituted, deleted, and / or added, 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 and comprises the amino acid sequence ASSAK (SEQ ID NO: 64), optionally wherein the peptide further comprises a heterologous amino acid sequence or functional portion as defined above. Most preferably, the peptide is one shown in SEQ ID NO: 62 or 63.

[0637] In one embodiment of the invention, this peptide can be used for antibody screening, for example, screening for CRMP2-binding molecules equivalent to antibody NI-504.3E7 and / or antibodies competing with the antibodies of the present invention, or this peptide can be used as an immunogen to generate antibodies for diagnostic purposes, for example in mice or rabbits. As described above, the peptides of the present invention can be formulated in arrays, kits, and compositions, respectively. In this context, the invention also relates to kits as described above, but further includes the peptides of the present invention.

[0638] Numerous references are cited throughout the text of this specification. The contents of all cited references (including references cited throughout this application, published patents, published patent applications, including background sections and manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference; however, no prior art relating to the invention is acknowledged in any cited reference.

[0639] A more complete understanding can be obtained by referring to the following specific embodiments, which are provided herein for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0640] The following section presents the results of experiments performed using the original antibody NI-504.3E7. However, these results have been confirmed with one or more variants, namely, one or more of any one of NI-504.3E7_V1 to NI-504.3E7_V40.

[0641] Example 1: Isolation, identification, cloning, and recombinant expression of anti-CRMP2 antibody

[0642] To identify and isolate potential CRMP and CRMP2-specific antibodies that may have a protective effect in older adults, cDNA sequences encoding human antibodies that bind recombinant human CRMP2 and pCRMP2 were derived from a delabeled blood lymphocyte library collected from healthy older adults, using proprietary reverse translational medicine. TM (RTM) technology platform, Neurimmune AG, 8952 Schlieren / Zurich, Switzerland. Positive matches were subjected to reverse screening to exclude clones that cross-reacted with unrelated proteins. Selected CRMP2-reactive B cell clones were cloned into cDNA sequences of the variable regions of the IgG heavy and light chains (see Table II for selected antibodies) and subcloned into expression vectors encoding the constant domain sequence of human IgG1. Expression constructs encoding the IgG1 heavy and light chains were transiently expressed in the ExpiCHO cell line and purified by protein A affinity chromatography. Table VI summarizes the results of the first screening.

[0643] [Table VI]

[0644]

[0645] Example 2: NI-504.3E7 selectively binds to human CRMP2

[0646] The binding specificity of antibody NI-504.3E7 to full-length CRMP1, CRMP2, CRMP3, CRMP4 and CRMP5 was tested by direct ELISA and Western blotting.

[0647] Figure 1

[0648] For direct ELISA, 96-well microplates (Costar) were coated with 5 μg / mL recombinant human CRMP1-5 protein (CRMP1 (Origene), CRMP2 (Origene), CRMP3 (Origene), CRMP4 (Origene), and CRMP5 (Origene)) in PBS (Gibco) and incubated overnight at 4°C.

[0649] The plates were blocked in blocking buffer (PBS-T (phosphate-buffered saline, Fisher Bioreagents, Cat. BP 399; containing 2% BSA in 0.1% Tween 20 (Sigma-Aldrich)) at room temperature for 1 h. After three washes with PBS-T using a Bio Tek Microplate Staker 3, the plates were incubated with a 30 μL / well serial dilution of antibody NI-504.3E7 at room temperature for 1 h, followed by three more washes in PBS-T. The binding of antibody NI-504.3E7 was detected using HRP-conjugated anti-human Fc secondary antibody (Jackson ImmunoResearch, catalog number 709-036-098) at room temperature for 1 h, followed by three washes with PBS-T. The chromogenic substrate TMB (Clinical Science) was then converted for use in a plate reader (Thermo Fisher Scientific) at 450 °C. Absorbance measurement at nm. To estimate the half-maximum concentration of EC. 50 The logarithmic transform absorbance values ​​were fitted using a logarithmic (agonist) versus response-variable slope (four-parameter) curve, applying the formula Y=Bottom + (Top-Bottom) / (1+10^((LogEC50-X)*HillSlope)), in GraphPad Prism version 9.3. Data is displayed as mean + SEM.

[0650] Figure 1

[0651] For Western blot analysis, 0.2 μg of recombinant human CRMP1 (Origene), CRMP2 (Origene), CRMP3 (Origene), CRMP4 (Origene), and CRMP5 (Origene) were denatured at 95°C for 5 min in LDS sample buffer (NuPAGE) + 4% β-mercaptoethanol (Sigma-Aldrich). The samples were loaded onto 4–12% Bis-Tris protein gels (NuPAGE) and run in 1x MOPS SDS run buffer (NuPAGE) at 200 V for 45 min. Proteins were then transferred to a PVDF membrane (NuPAGE) using iBlot and held for 7 min at 20 V. The membrane was blocked with PBS-T (phosphate-buffered saline, Fisher Bioreagents, Cat. BP399; containing 2% BSA in 0.1% Tween 20 (Sigma-Aldrich)) at room temperature for 1 h, and the antibody NI-504.3E7 was incubated with 2% BSA in PBS-T at 5 μg / mL at room temperature for 1 h. After three washes with PBS-T, the membrane was incubated with HRP-conjugated anti-human-Fc secondary antibody (Jackson Immuno Research, catalog number 709-036-098) at room temperature for 1 h, washed three more times with PBS-T, and incubated with ECL (Amersham) for 2 min. The chemiluminescence signal was detected using an Image Quant LAS 4000 (Amersham).

[0652] Figure 1

[0653] In ELISA assays, antibody NI-504.3E7 was observed to preferentially bind to CRMP2 relative to CRMP1, CRMP3, CRMP4, and CRMP5. Specifically, antibody NI-504.3E7 has been shown to bind with high affinity (i.e., at 4 nM EC50) to CRMP2. 50 ) binds to CRMP2, where only EC32 at 69 nM was observed. 50 Weak binding with CRMP1; see also Figure 1 A. Furthermore, even weaker bindings were observed with CRMP3, CRMP4, and CRMP5. The same results were obtained by Western blot analysis. Specifically, antibody NI-504.3E7 showed strong binding with CRMP2, very weak binding with CRMP1, and no binding with CRMP3, CRMP4, and CRMP5; see [link to relevant documentation]. Figure 1 B. Therefore, antibody NI-504.3E7 selectively binds to CRMP2.

[0654] Example 3: Linear epitope at the C-terminus of NI-504.3E7 combined with CRMP2

[0655] To evaluate the binding epitopes of antibody NI-504.3E7, epitope mapping was performed using a cellulose membrane array with overlapping peptides of 15 amino acids in length and 11 amino acids in overlap, covering the full-length CRMP2 protein. This membrane was manufactured by JPTMembrane Technologies (Germany). The membrane was blocked with 1x RotiBlock (Roth) at room temperature for 1 h, and antibody NI-504.3E7 was incubated overnight at 4°C in 1x RotiBlock at 0.04 μg / mL. After three washes with 1x TBS-T (TBS 20x, Biocave Medical, catalog number TWB 945M; containing 0.1% Tween20, Sigma-Aldrich), the membrane was incubated with HRP-conjugated anti-human H+L secondary antibody (Jackson ImmunoResearch, catalog number 709-036-149) at room temperature for 1 h and washed three times with TBS-T. The antibody NI-504.3E7 bound to the membrane was detected using an ECL (Amersham) for 2 minutes, and the chemiluminescence signal was detected using an Image Quant LAS 4000 (Amersham).

[0656] like Figure 1 As shown, by using an overlapping peptide array, antibody NI-504.3E7 has been found to bind to two linear peptides 128 and 129 (SEQ ID NO: 62 and 63) that share the common sequence VTPASSAKTSP (SEQ ID NO: 65). Furthermore, the minimal epitope of antibody NI-504.3E7 contains the amino acid sequence 516-ASSAK-520 (SEQ ID NO: 64).

[0657] Example 4: NI-504.3E7 combines non-phosphorylated CRMP2 and phosphorylated CRMP2

[0658] ELISA assays were performed to determine the binding specificity of antibody NI-504.3E7 and its 40 variants for CRMP2 and pCRMP2. The variants contain one or more amino acid substitutions within the VH chain, VL chain, and / or CDR. ELISA assays were performed with CRMP2 and pCRMP2 as described in Example 2. EC50 assays were performed on antibody NI-504.3E7 and antibody variants NI-504.3E7_V1 to V40. 50 The values ​​are listed in Table VII.

[0659] [Table VII]

[0660]

[0661] As shown in Table VII, antibody NI-504.3E7 was administered at EC50 concentrations of 0.5 nM and 4.7 nM. 50 Specific recognition of recombinant human full-length CRMP2 and pCRMP2. As can be seen, all variants still bind to CRMP2 and pCRMP2, thus confirming that one or more amino acid substitutions do not substantially alter the antibody's binding affinity.

[0662] Example 5: NI-504.3E7 reduces pCRMP2 levels in a concentration-dependent manner.

[0663] To clarify whether CRMP2 phosphorylation changes in the presence of the antibody or isotype control of the present invention at increasing concentrations, a combination of phosphorylation assay and Western blot analysis was performed. This assay was also performed exemplary using the variant NI-504.3E7_V20 to further support the fact that the variant antibody retains the binding specificity and activity of the original antibody. Specifically, this experimental setup was performed to observe T509 pCRMP2 when phosphorylation assays were performed on CRMP2 and antibody NI-504.3E7_V20 or anti-CRMP2 control antibody (NI-504.B) or isotype control antibody. Figure 1 AE), T514 pCRMP2 ( Figure 1 FJ) and S522pCRMP2 Figure 1 KO) level.

[0664] CRMP2 protein (Alexotech) was incubated for 1 h with CRMP2 antibodies (NI-504.3E7 and NI-504.B) at different concentrations (3x less, equimolar, or 3x more than CRMP2) and a human IgG control (isotype control antibody). CRMP2 was phosphorylated at 30°C for 1 h or 24 h using a phosphorylation mixture containing phosphorylation buffer (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)). CRMP2 phosphorylation levels were analyzed by Western blotting. 200 ng of phosphorylated CRMP2 protein was denatured at 95°C for 10 min in LDS sample buffer + 4% β-mercaptoethanol (NuPAGE). The sample was loaded onto a 4-12% Bis-Tris protein gel (NuPAGE) and the gel was run at 200V for 45 min in 1x MES SDS run buffer (NuPAGE). The protein was transferred to a nitrocellulose membrane (NuPAGE) using iBlot and held for 7 min at 20V. The membrane was blocked overnight at 4°C with PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP 399; containing 2% BSA (Sigma-Aldrich) in 0.1% Tween 20 (Sigma-Aldrich)). The next day, the membrane was incubated with anti-CRMP2 antibody C2993 (Sigma-Aldrich), pCRMP2 (Thr-514) antibody (Cell Signaling, catalog number 9397S), pCRMP2 (Thr-509) antibody (Thermo Fisher Scientific, catalog number PA 5-37551), or pCRMP2 (Ser-522) antibody (ECMBioscience, catalog number CP 2191) at room temperature for 1 h, and washed three times with PBS-T. The membrane was then incubated with HRP-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, catalog number 111-035-045) at room temperature for 1 h. After washing three times with PBS-T, the membrane was incubated with ECL (Amersham) for 1 min, and the chemiluminescence signal was detected using an Image Quant LAS 4000 (Amersham). Signal band density was analyzed using the Java-based image processing program ImageJ.

[0665] Antibody C2993 was used to analyze the total CRMP2 level in the samples to normalize the pCRMP2 level, showing that the total CRMP2 level was comparable in all samples as well as in the untreated loading control (200 ng / well) from Alexotech. Figure 1 The graphs shown in C, E, H, J, M, and O have been normalized to those total CRMP2 levels.

[0666] As shown in the figures, antibody NI-504.3E7_V20 reduced T509 pCRMP2 levels in a concentration-dependent manner after 1 h or 24 h of phosphorylation. Furthermore, compared to antibody NI-504.B and the isotype control, antibody NI-504.3E7_V20 reduced T514 pCRMP2 levels after 1 h or 24 h of phosphorylation. Compared to antibody NI-504.B and the isotype control, antibody NI-504.3E7_V20 reduced S522 pCRMP2 levels after 1 h of phosphorylation. These results indicate that antibody NI-504.3E7_V20 reduces pCRMP2 levels in a concentration-dependent manner. Further replications of this study have confirmed these results and also demonstrated a concentration-dependent reduction in S522 levels after 24 h.

[0667] To ensure that antibody binding affinity was not reduced or modified by the phosphorylation procedure, which lasted up to 24 h, separate phosphorylation assays were performed in the absence of CRMP2 protein. Antibodies NI-504.3E7_V20 and NI-504.B, along with an isotype control antibody, were incubated for 24 h under standard phosphorylation conditions. Binding affinity was assessed using a direct CRMP2 ELISA. In summary, all antibodies showed comparable binding before and after phosphorylation, and their CRMP2 binding was unaffected by the phosphorylation procedure.

[0668] Example 6: NI-504.3E7 specifically binds to CRMP2 and pCRMP2 peptides

[0669] To assess whether antibody NI-504.3E7 binds to CRMP2 in its phosphorylated stage, four different peptides (Bacheme) covering the antibody epitopes (either lacking phosphorylation sites or phosphorylated at specific sites (T509, T514, S522)) were tested by ELISA. The peptide sequences are as follows:

[0670] Peptide 4152960: VCESVTPKTVTPASSAKTSPAKQQA (SEQ ID NO: 72)

[0671] Peptide 4152961: VCEVSV(p)T509 PKTVTPASSAKTSPAKQQA (SEQ ID NO: 72)

[0672] Peptide 4152962: VCEVSTPKTV(p)T 514 PASSAKTSPAKQQA (SEQ ID NO: 72)

[0673] Peptide 4152963: VCEVSTPKTVTPASSAKT(p)S 522 PAKQQA (SEQ ID NO: 72)

[0674] For direct ELISA, 96-well microplates (Costar) were coated with 30 μL / well of each peptide in PBS at 5 μg / mL and incubated overnight at 4°C. The ELISA plates were blocked in PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP399; containing 2% BSA in 0.1% Tween 20 (Sigma-Aldrich)) at room temperature for 1 h, incubated with serially diluted 30 μL / well of antibody NI-504.3E7 at room temperature for 1 h, and then washed three times with PBS-T. The binding antibody was detected using HRP-conjugated anti-human Fc secondary antibody (Jackson ImmunoResearch, Cat. 709-036-098), and the chromogenic substrate TMB was switched (Clinical Science). The absorbance was measured at 450 nm using a plate reader (Thermo Fisher Scientific). To estimate the half-maximal concentration (MCC) of EC... 50 The logarithmic transform absorbance values ​​were fitted using a logarithmic (agonist) versus response-variable slope (four-parameter) curve, applying the formula Y=Bottom + (Top-Bottom) / (1+10^((LogEC50-X)*HillSlope)), in GraphPad Prism version 9.3. Data is displayed as mean + SEM.

[0675] like Figure 1 As shown, antibody NI-504.3E7 exhibits approximately 300-fold lower affinity for the phosphorylated CRMP2 peptide (peptide 4152962) at position T514 than for the unphosphorylated peptide and the peptides phosphorylated at positions T509 (peptide 4152961) and S522 (peptide 4152963). The phosphorylation at T514 appears to partially impair the binding of antibody NI-504.3E7 due to the antibody epitope.

[0676] Example 7: NI-504.3E7 can bind to the full-length recombinant CRMP2 protein in humans, mice, and rats.

[0677] To determine the binding of antibody NI-504.3E7 to CRMP2 from different species, 0.5 μg of recombinant human SUMO-CRMP2 (Boston Biochem), recombinant mouse CRMP2-His6 (MyBioSource, catalog number MBS1185591), and recombinant rat CRMP2-His6 (MyBioSource, catalog number MBS961395) were denatured at 70°C for 10 min in LDS sample buffer + 4% β-mercaptoethanol (NuPAGE). The samples were loaded onto 4–12% Bis-Tris protein gels (NuPAGE), and the gels were run in 1xMES SDS running buffer (NuPAGE) at 200 V for 60 min. Proteins were then transferred to nitrocellulose membranes (NuPAGE) using iBlot and held for 7 min at 20 V. The membrane was blocked with 5% milk in 1x TBS-T (TBS 20x, Biocave Medical, catalog number TWB945M; containing 0.1% Tween20, Sigma-Aldrich) at room temperature for 1 h, and the antibody NI-504.3E7 was incubated overnight at 4°C with 5 μg / mL in 1% milk in TBS-T. After washing three times with TBS-T, the membrane was incubated with HRP-conjugated anti-human H+L secondary antibody (Jackson ImmunoResearch, catalog number 709-036-149) at room temperature for 1 h, and washed three times with TBS-T. The membrane was incubated with ECL (Amersham) for 2 min, and the chemiluminescence signal was detected using an Image Quant LAS 4000 (Amersham).

[0678] like Figure 1 As shown, compared with isotype IgG1 and human IgG1 controls, antibody NI-504.3E7 specifically binds to all analyzed CRMP2 species, namely human, mouse, and rat CRMP2. Therefore, antibody NI-504.3E7 can bind to the full-length recombinant CRMP2 protein in humans, mice, and rats.

[0679] Example 8: Coagulation of recombinant (p)Tau and (p)CRMP2 in ThioT assay

[0680] Based on Takata, et al., Am J. Pathol. 175 (2009), 17-24 (of which...) Figure 1ThioT (Sigma-Aldrich) assays were performed to detect whether recombinant (p)Tau and (p)CRMP2 proteins not only co-localized but also co-aggregated, showing co-localization of hyperphosphorylated Tau and phosphorylated CRMP2. ThioT is known to bind to a variety of filaments, despite their different amino acid sequences, strongly suggesting that ThioT recognizes common structural features within the filaments. The ThioT assay measured changes in fluorescence intensity upon ThioT binding to the filaments. It does not bind to the corresponding monomeric structures. In the present case, ThioT assays were performed on CRMP2, pCRMP2, Tau+pCRMP2, and pTau+pCRMP2, with fluorescence signals measured over 240 h (5 min exposure). The Tau protein was obtained from rPeptide, Watkinsville, USA, and the human CRMP2 was a custom protein obtained from Boston Biochem, Inc. (Cambridge USA), including an N-terminal SUMO tag, expressed in *E. coli*. Specifically, amyloid fibrillation was measured by increasing ThioT fluorescence in samples containing CRMP2 (CRMP2, 6.4 μM, Boston Biochem), phosphorylated CRMP2 (pCRMP2, 6.4 μM), Tau (Tau, 4.35 μM, full-length Tau-441 protein (rPeptide, catalog number T-1001)), and combinations of phosphorylated CRMP2 (CRMP2, 6.4 μM) or phosphorylated Tau (pTau, 4.35 μM) and phosphorylated CRMP2 (pCRMP2, 6.4 μM). For CRMP2 and pCRMP2 samples, 50 μM ThioT was used, while for combined samples (Tau+pCRMP2, pTau+pCRMP2), 50 μM ThioT and 50 μM heparin were used. Aggregation assays were performed in plate form at room temperature with shaking at 300 rpm. Before each measurement, the plate was oscillated at 600 rpm for 5 seconds, and then excited at 456 nm followed by fluorescence measurement at an emission wavelength of 490 nm. The data are presented as the average of repeated values ​​measured at specified time points + SEM.

[0681] like Figure 1 As shown, ThioT binds only weakly to CRMP2 (as determined by ThioT fluorescence), indicating that CRMP2 does not aggregate. Slightly higher ThioT binding was observed during pCRMP2 incubation, and the ThioT fluorescence signal increased during incubation of pCRMP2 with Tau, indicating more aggregate formation. The highest signal was obtained when pCRMP2 was incubated with pTau, indicating co-aggregation of pCRMP2 and pTau.

[0682] Example 9: Antibody NI-504.3E7 reverses CRMP2-induced increase in dendritic spine density in a hippocampal slice culture model.

[0683] The effect of antibody NI-504.3E7 on CRMP2 / pCRMP2 (i.e., pCRMP2 aggregates and CRMP2 monomers)-driven neuronal dendritic spine density was analyzed in an ex vivo hippocampal section culture (HSC) model. Organoid 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 hemispheres were separated, the midbrain was removed, and the bilateral hippocampi were removed. The hippocampi were sliced ​​into 400 μm thick sections using a tissue mincer. Experimental procedures were performed according to publication Suendermann et al., Methods Mol Biol (2012), 277-293. Each condition was tested in duplicate. A total of 2 x 5 sections for each condition were transferred to Multidish 6-well plates (Nunclon). TM The plate contains culture medium and Millicell cell culture insert (Millipore, catalog number PICM 0RG 50). Hippocampal sections are cultured, treated with combinations of the target protein and antibody NI-504.3E7, infected, and fixed. Hippocampal sections are prepared on day 0, cultured for 10 days with the medium changed every 2-3 days, and then treated with pCRMP2 aggregates or CRMP2 monomers with or without a specified concentration of antibody NI-504.3E7. After medium change and infection, antibody NI-504.3E7 is diluted in NB medium containing N1 supplement for 4 days on day 10, and then diluted again for 2 days on day 14. Monomeric yeast CRMP2 was diluted in NB medium supplemented with N1. Recombinant CRMP2 was phosphorylated at 37°C for 1 day in a phosphorylation buffer containing Tris-HCl (Sigma-Aldrich), MgCl2 (Sigma-Aldrich), DTT (Sigma-Aldrich), GSK3β (SignalChem), CDK5 / p35 (SignalChem), and ATP (SignalChem) to obtain pCRMP2 aggregates. The aggregation of phosphorylated CRMP2 was carried out at 1 mg / mL in 5 mM Tris-HCl (pH 7.4) containing 5 mM heparin for 10 days at room temperature.

[0684] On day 14, the culture sections were treated with GFP Sinbis virus (Sinbis Expression System Manual, Invitrogen catalog number K 750-01) using the droplet method, with 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 h with fixative (phosphate-buffered saline (Gibco), 25% paraformaldehyde (Sigma-Aldrich, catalog number 158127), 25% sucrose (Sigma-Aldrich)), then mounted on glass slides, embedded in a confocal matrix, and imaged using a Zeiss LSM 510 confocal laser scanning microscope. Semi-automatic threshold analysis of dendritic spine density was performed by determining the number of spines per μm of dendritic length. A total of 29–58 images were analyzed for each treatment condition, with 11–52 spines detected on each dendritic segment. Data are presented as mean + SEM. Univariate ANOVA analysis: *** p<0.01 ** p<0.01; Kruskal-Wallis post-hoc test compared to untreated.

[0685] like Figure 1 As shown, pCRMP2 aggregates or CRMP2 monomers increased the density of neuronal dendritic spines compared to the untreated / aggregated buffer pool. The increase in dendritic spine density driven by CRMP2 / pCRMP2 was reversed by antibody NI-504.3E7 at concentrations of 7.5 μM and 0.7 μM. Therefore, antibody NI-504.3E7 was able to reverse the increase in dendritic spine density induced by both aggregated and monomeric CRMP2 in a hippocampal slice culture model.

[0686] Example 10: Detection of neuronal cells in AD hippocampal slices using NI-504.3E7

[0687] The binding of antibody NI-504.3E7 to tissue sections was analyzed by immunohistochemistry (IHC). To analyze the binding of antibody NI-504.3E7 to human Alzheimer's disease (AD) tissue, deparaffinized and rehydrated FFPE brain sections from human AD donors were boiled in citrate buffer (20 mM trisodium citrate dihydrate, Sigma-Aldrich, catalog number C7129 and 80 mM trisodium citrate dihydrate, Sigma-Aldrich, catalog number 71405) for 15 minutes, washed with water, and then incubated with methanol containing 3% hydrogen peroxide (Sigma-Aldrich) to block endogenous peroxidase, followed by washing again with water. The sections were then blocked in blocking buffer (5% goat serum (Vector Laboratories); 5% horse serum (Vector Laboratories); 4% bovine serum albumin (Sigma-Aldrich) in PBS (Gibco) at room temperature for 1 h. Antibody NI-504.3E7 was diluted to 6.67 nM in 2% BSA in PBS-T (phosphate-buffered saline, Fisher Bioreagents, catalog BP 399; containing 0.1% Tween 20 (Sigma-Aldrich)) and incubated overnight at 4°C on slides, followed by three washes with water. Detection was performed for 1 h at room temperature using Biotin-SP AffiniPure goat anti-human IgG (H+L) (Jackson ImmunoResearch, catalog 109-065-088), followed by Vectastain ABC kit (Vector Laboratories) at room temperature for 20 min, and then stained with diaminobenzidine (DAB, Thermo Fisher Scientific, catalog 1856090) and hematoxylin (Roth, catalog T865) for cell nuclei.

[0688] like Figure 1 As shown, antibody NI-504.3E7 was used to detect neurons in the hippocampus of AD patients. Similar observations were made in hippocampal tissues from patients with PSP, PiD, FTD-PiD, and FTLD-TDP, and the results are summarized in Table VIII below.

[0689] [Table VIII]

[0690]

[0691] Example 11: NI-504.3E7 reduces β-amyloid plaques in a transgenic mouse model of Alzheimer's disease.

[0692] The in vivo effects of antibody NI-504.3E7 were analyzed in a Tau tubulin kinase 1 (TTBK1) / amyloid precursor (APP) double transgenic Alzheimer's disease mouse model. Specifically, the in vivo efficacy of antibody NI-504.3E7 was evaluated by weekly intraperitoneal injection of 30 mg / kg into female APPPS1 / TTBK1 transgenic mice (APPPS1 #034829-JAX, Jackson Laboratories; TTBK1 #B6 / 129-TTBK1-Tg Line 141, Tsuneya Ikezu, Nebraska University) for a period of 9 months (3 to 12 months of age). To allow for repeated injections into the mouse model, the antibody was formulated in PBS and used for injection. The antibody and PBS volumes were adjusted by animal body weight. At the end of their lifespan, the mice were perfused with cold PBS, their brains were fixed in paraffin, and sagittal sections were prepared at 3 μm.

[0693] For amyloid plaque analysis, deparaffinized and rehydrated FFPE mouse brain sections covering the cortex and hippocampus were boiled in citrate buffer for 15 minutes and washed with water. Endogenous peroxidase was blocked by incubating the sections with 3% hydrogen peroxide solution (Sigma-Aldrich) followed by washing with water again. The sections were then blocked in PBS containing 5% goat serum (Vector Laboratories), 5% horse serum (Vector Laboratories), and 4% bovine serum albumin (Sigma-Aldrich). The sections were then incubated with 6.67 nM anti-β-amyloid antibody 6E10 (Biolegend), washed with water, and detected by a combination of 2.3 μg / ml secondary antibody Biotin-SP AffiniPure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, catalog 115-065-003) with Vectastain ABC kit (Vector Laboratories), diaminobenzidine (Thermo Fisher Scientific, catalog 1856090), and hematoxylin nuclear staining (Roth, catalog T 865). For image analysis, three 3 μm thick sections spaced 60 μm apart were used for detection. The number and size of plaques in the depicted brain regions were assessed using the Java-based image processing program ImageJ, with thresholding determined. Amyloid-β plaque load represents the percentage of area covered by plaques stained with the corresponding 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.

[0694] like Figure 1 As shown in A and C, mice receiving antibody NI-504.3E7 exhibited a 63% reduction in amyloid plaque burden in the cortex and hippocampus. Furthermore, not only was the amyloid plaque burden reduced, but the size of the plaques in the cortex and hippocampus also decreased. Figure 1 B and C). Additionally, in the cortex, a 50% reduction in the amount of plaque / area (area defined by the entire cortex) was observed in female TTBK1 / APP mice receiving antibody NI-504.3E7. In female TTBK1 / APP mice receiving antibody NI-504.3E7, the mean plaque size in the cortex was reduced by 30% ( Figure 1 B), the average plaque size in the hippocampus even decreased by 68% ( Figure 1 D).

[0695] Example 12: NI-504.3E7 can bind CRMP2 in fully differentiated SH-SY5Y cells.

[0696] To further analyze the mechanism of action of antibody NI-504.3E7, its binding in differentiating cells has been observed. For this analysis, SH-SY5Y cells were maintained in MEM medium (Thermo Fisher Scientific, catalog number 41090028) supplemented with 1% MEM NEAA (Gibco), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 1% penicillin-streptomycin (Gibco), and 10% heat-inactivated FBS (Gibco). SH-SY5Y cell differentiation was performed in two steps, with a 7-day pre-differentiation period during which 10 μM all-trans retinoic acid (Sigma-Aldrich, catalog number R2625) was added to the medium. 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 15 μm slides (Ibidi, catalog number 80825) and cultured until fully differentiated into MEM medium (Thermo Fisher Scientific, catalog number 41090028) supplemented with 1% MEMNEAA (Gibco), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 1% penicillin-streptomycin (Gibco), 50 ng / ml BDNF (Sigma-Aldrich, catalog number SRP3014), 10 ng / ml NGF (RnD Systems), and 24 nM cholecalciferol (Sigma-Aldrich, catalog number C9756) for 10 days. 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, cells were permeabilized and blocked for 1 h in PBS containing 10% FBS and 0.2% Triton-X-100 (Sigma-Aldrich). Primary antibodies were incubated overnight at 4°C with 50 nM human IgG control (isotype control antibody), 50 nM NI-504.3E7, 1 μg / ml anti-β-III-tubulin (Abcam, catalog number AB 78078), or 1% anti-CRMP2 (Abcam, catalog number AB 129082) diluted in blocking buffer (PBS containing 10% FBS).After two washes with PBS, cells were stained at room temperature for 1 h with secondary antibodies: anti-mouse Cy3 (Jackson Immuno Research, catalog number 715-165-150), anti-rabbit Cy3 (Jackson Immuno Research, catalog number 711-165-152), or anti-human Cy3 (Jackson Immuno Research, catalog number 709-165-149), at a concentration of 2.7 μg / ml in blocking buffer for each antibody. Cell nuclei were stained with DAPI (Thermo Fisher Scientific) in PBS for 5 min and imaged using an inverted confocal microscope (20x / dry objective, Leica DMI6000 AFC, Model SP8).

[0697] like Figure 1 As shown, antibody NI-504.3E7 binds to differentiated SH-SY5Y cells, where no signal was observed in the human IgG control and anti-human control. These results demonstrate the binding of the target, CRMP2, in cells.

[0698] Example 13: NI-504.3E7 improves long-term potentiation (LTP) in aged mice.

[0699] In addition to its ability to reduce amyloid plaque burden in a transgenic mouse model of Alzheimer's disease, as shown in Example 11, the ability of the NI-504.3E7 antibody family to improve long-term potentiation (LTP) has been demonstrated through one of its variants. Specifically, similar to the phosphorylation assay described in Example 5, this assay was also performed exemplarily with antibody NI-504.3E7_V20 to further support the fact that the variant antibody retains the original antibody activity.

[0700] The effect of antibody NI-504.3E7_V20 has been analyzed in aged C57Bl / 6J male mice (90 weeks old). The antibody, in chimeric mouse IgG2a form, was formulated in PBS and injected. Antibody and PBS volumes were adjusted by animal body weight and delivered weekly via intraperitoneal injection for 5 weeks, with different doses of antibody delivered: 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg. At the end of their lifespan, the mice were anesthetized with 5% isoflurane and then decapitated. The brain was dissected from the skull and immediately immersed in cold, freshly prepared artificial cerebrospinal fluid (aCSF) 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, under continuous oxygenation (95% O2, 5% CO2) (pH 7.4) for a total duration of 3–4 minutes. Tip sections (350 μm thick) were prepared using a vibratory microtome (VT1000S; Leica Microsystems, Bannockburn, IL). Before recording, 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, and 10 mM glucose) at room temperature for at least 1 h. For electrophysiological recording, a single slice is placed in the recording chamber (room temperature) and, for the remainder of the experiment, immersed at a constant rate (2 mL / min). -1Continuous perfusion with aerated aCSF (95% O2, 5% CO2; pH 7.4). Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 radiating layer using a glass micropipette filled with aCSF. fEPSPs were induced by electrical stimulation of the Schaffer lateral communication pathway at 0.1 Hz (i.e., single pulse every 10 s) using glass stimulation electrodes placed in the radiating layer (borosilicate capillary glass with filament, standard wall; outer diameter: 1.5 mm; inner diameter: 0.86 mm; length: 75 mm; reference: Harvard Apparatus W3 30-0060). A stable baseline fEPSP was then recorded over 10 minutes by stimulation at 30% of the maximum field amplitude (single pulse every 10 s, i.e., 0.1 Hz). After stabilization for 10 minutes, LTP was assessed by applying a conditioning stimulus consisting of a set of 100 Hz stimuli. Following conditioned stimulation, a 1-hour testing period was recorded, during which a response was re-elicited every 10 seconds (0, .Hz) with a single stimulus at the same stimulus intensity. Signals were amplified using an Axopatch 200B amplifier (Molecular Devices, Union City, CA), digitized via a Digidata 1322A interface (Axon Instruments, Molecular Devices, US), and sampled at 10kHz. Recordings were acquired using Clampex (Molecular Devices) and analyzed using Clampfit (Molecular Devices). LTP was calculated as the percentage of the baseline fEPSP slope recorded within the 10 minutes prior to LTP induction. The mean slope response during the 40–60 minutes following LTP induction was used for analysis. 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.

[0701] like Figure 1 As shown, in aged mice, LTP was decreased compared to young mice; see PBS control. However, after administration of the antibody to aged mice, their LTP increased compared to the PBS control and almost reached the LTP score of young mice, especially after administration of the antibody at 3 mg / kg, 10 mg / kg, and 30 mg / kg. Therefore, the antibody NI-504.3E7_V20 has been shown to improve LTP in aged mice at a dose of 3 mg / kg.

[0702] Example 14: NI-504.3E7 improves cognitive function in aged mice

[0703] In addition to the ability of the NI-504.3E7 antibody family to reduce amyloid plaque load in a transgenic mouse model of Alzheimer's disease as shown in Example 11, and its ability to improve LTP in aged mice as shown in Example 13, the ability of the NI-504.3E7 antibody family to improve cognitive function in aged mice has been demonstrated through one of its variants. Specifically, similar to the phosphorylation assay described in Example 5 and the LTP assay described in Example 13, this assay was also performed exemplarily with antibody NI-504.3E7_V20 to further support the fact that the variant antibody retains the original antibody activity.

[0704] The effects of antibody NI-504.3E7_V20 on cognitive function were analyzed in aged C57Bl / 6J male mice. Experiments were conducted using 7-week-old (young) and 73-week-old (aged) mice. The antibody and a mediator (PBS) were administered intraperitoneally weekly for 5 weeks. Cognitive function was assessed using the Novel Object Recognition (NOR) test. During the first day of acclimatization, each mouse was placed in the test chamber and allowed free movement for 10 minutes, after which it was returned to its living cage. Acclimatization was repeated twice at intervals of at least 4 hours. In the second day of the acquisition test, two objects of the same color and shape were placed in the test chamber, and similarly, each mouse was allowed free movement for 10 minutes. In the third day of the assessment test, one of the objects used in the acquisition test (the familiar object) was replaced with a new object of different color and shape, and similarly, each mouse was allowed free movement for 10 minutes, and the process was recorded by a camera. The assessment test began 24 hours after the acquisition test. Based on the video images recorded during the assessment test, the time spent exploring each object was measured using a stopwatch. The discrimination index was calculated using the following formula: (Time spent exploring new objects - Time spent exploring familiar objects) / (Time spent exploring new objects + Time spent exploring familiar objects). Data is presented as mean + SEM. Group size: n=12. Statistical analysis: **P<0.01, compared with the younger group (t-test); ###p<0.001, compared with the PBS group (Dunnett test).

[0705] like Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 Figure As shown, the discrimination index was decreased in aged mice compared to younger mice; see PBS control. However, the discrimination index significantly increased after administration of the antibody at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg. Therefore, it has been shown that the antibody NI-504.3E7_V20 has improved the discrimination index in aged mice at 1 mg / kg, and these results demonstrate that the antibody NI-504.3E7 improves cognitive function in aged mice.

Claims

1. A human monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment selectively binds human brain aging response regulatory protein 2 (CRMP2), wherein the antibody binds an epitope comprising the amino acid sequence ASSAK (SEQ ID NO: 64), preferably wherein the antibody is capable of binding a CRMP2 peptide composed of the amino acid sequences TPKTVTPASSAKTSP (SEQ ID NO: 62) or VTPASSAKTSPAKQQ (SEQ ID NO: 63).

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody is capable of binding full-length unphosphorylated CRMP2 and phosphorylated CRMP2 (pCRMP2), preferably wherein when phosphorylation of the antibody and CRMP2 is measured, the antibody is capable of reducing the level of pCRMP2 in a concentration-dependent manner, and / or wherein the antibody is capable of binding full-length recombinant CRMP2 protein from humans, mice, and rats, preferably wherein the antibody reduces the level of pCRMP2 in a concentration-dependent manner. < 10.0 nM EC50 combined with full-length human CRMP2, and / or with approximately < 100.0 nM EC50 binds to pCRMP2, as determined by ELISA.

3. A human monoclonal antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment selectively binds to human brain aging response regulatory protein 2 (CRMP2), and wherein the antibody or antigen-binding fragment includes a heavy chain variable (VH) region and a light chain variable (VL) region in its variable region, wherein the VH region includes complementarity-determining regions (CDRs) 1, 2, and 3, and the VL region includes CDRs 1, 2, and 3, wherein... (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 61; or One or more of the CDRs may contain one or more amino acid substitutions, and preferably the antibody or antigen-binding fragment contains an amino acid sequence selected from the VH and VL regions in its variable region: (0) SEQ ID NO: 2 and SEQ ID NO: 7, (1) SEQ ID NO: 2 and SEQ ID NO: 56, (2) SEQ ID NO: 2 and SEQ ID NO: 58, (3) SEQ ID NO: 12 and SEQ ID NO: 7, (4) SEQ ID NO: 12 and SEQ ID NO: 56, (5) SEQ ID NO: 12 and SEQ ID NO: 58, (6) SEQ ID NO: 14 and SEQ ID NO: 7, (7) SEQ ID NO: 14 and SEQ ID NO: 56, (8) SEQ ID NO: 14 and SEQ ID NO: 58, (9) SEQ ID NO: 16 and SEQ ID NO: 7, (10) SEQ ID NO: 16 and SEQ ID NO: 56, (11) SEQ ID NO: 16 and SEQ ID NO: 58, (12) SEQ ID NO: 18 and SEQ ID NO: 7, (13) SEQ ID NO: 18 and SEQ ID NO: 56, (14) SEQ ID NO: 18 and SEQ ID NO: 58, (15) SEQ ID NO: 18 and SEQ ID NO: 60, (16) SEQ ID NO: 20 and SEQ ID NO: 7, (17) SEQ ID NO: 20 and SEQ ID NO: 56, (18) SEQ ID NO: 20 and SEQ ID NO: 58, (19) SEQ ID NO: 20 and SEQ ID NO: 60, (20) SEQ ID NO: 22 and SEQ ID NO: 58, (21) SEQ ID NO: 24 and SEQ ID NO: 58, (22) SEQ ID NO: 26 and SEQ ID NO: 58, (23) SEQ ID NO: 28 and SEQ ID NO: 58, (24) SEQ ID NO: 30 and SEQ ID NO: 58, (25) SEQ ID NO: 32 and SEQ ID NO: 58, (26) SEQ ID NO: 34 and SEQ ID NO: 58, (27) SEQ ID NO: 36 and SEQ ID NO: 58, (28) SEQ ID NO: 38 and SEQ ID NO: 58, (29) SEQ ID NO: 40 and SEQ ID NO: 58, (30) SEQ ID NO: 42 and SEQ ID NO: 58, (31) SEQ ID NO: 44 and SEQ ID NO: 58, (32) SEQ ID NO: 46 and SEQ ID NO: 58, (33) SEQ ID NO: 48 and SEQ ID NO: 58, (34) SEQ ID NO: 50 and SEQ ID NO: 58, (35) SEQ ID NO: 22 and SEQ ID NO: 56, (36) SEQ ID NO: 22 and SEQ ID NO: 60, (37) SEQ ID NO: 52 and SEQ ID NO: 56, (38) SEQ ID NO: 52 and SEQ ID NO: 58, (39) SEQ ID NO: 52 and SEQ ID NO: 60, and (40) SEQ ID NO: 54 and SEQ ID NO: 58, or A pair of VH and VL amino acid sequences, wherein the VH and / or the VL may contain one or more amino acid substitutions and / or deletions, preferably wherein the VH and / or the VL are at least 90% identical to the amino acid sequence of any one of (0) to (40).

4. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment is capable of reversing the increase in neuronal dendritic spine density induced by CRMP2, as measured in an ex vivo hippocampal slice culture model, preferably wherein the increase in neuronal dendritic spine density is caused by pCRMP2 aggregates or CRMP2 monomers.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment (a) Contains a constant region, preferably said constant region belongs to the IgG type, preferably to the IgG1 class or isotype; (b) Containing a heterologous polypeptide, optionally wherein said antibody or antigen-binding fragment is a chimeric mouse-human antibody, preferably wherein said antibody contains a mouse IgG2a constant region; and / or (c) Select 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.

6. An antibody or antigen-binding molecule, said antibody or antigen-binding molecule competitively binding specifically to human CRMP2 and / or pCRMP2 with any one of claims 1 to 5, wherein said antibody - Prioritize the identification of human CRMP2 over CRMP1, CRMP3, CRMP4 and CRMP5; - When phosphorylation of CRMP2 is measured, the level of phosphorylated CRMP2 (pCRMP2) can be reduced in a concentration-dependent manner; and / or - It can reverse the increase in neuronal dendritic spine density induced by CRMP2, as measured in an ex vivo hippocampal slice culture model.

7. One or more polynucleotides encoding an antibody or antigen-binding fragment or immunoglobulin VH or VL of any one of claims 1 to 6, preferably wherein said polynucleotide is cDNA and / or operably linked to a heterologous nucleic acid, preferably expressing a control sequence.

8. One or more vectors comprising the polynucleotide of claim 7.

9. A host cell comprising the polynucleotide of claim 7 or the vector of claim 8.

10. A method for preparing an anti-CRMP2 antibody or its antigen-binding fragment or immunoglobulin chain, the method comprising: (a) Culturing the cells as described in claim 9; and (b) Isolate the antibody or its antigen-binding fragment or immunoglobulin chain from the culture.

11. An antibody, antigen-binding fragment, or immunoglobulin chain, wherein the antibody, antigen-binding fragment, or immunoglobulin chain is encoded by the polynucleotide of claim 9, or can be obtained by the method of claim 10.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 6 or 11, wherein the antibody or antigen-binding fragment (i) Detectably labeled using a label selected from the group consisting of: enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, tags, flags, and heavy metals; (ii) Attached to a drug; (iii) Contains polyethylene glycol; (iv) Contains organ-specific targeting entities; and / or (v) It is contained in or conjugated with a carrier such as an exosome or nanoparticle for delivery to a specific organ.

13. A peptide having an epitope of (p)CRMP2 specifically recognized by an antibody according to any one of claims 1 to 6 or 11, wherein the peptide consists of 10 to 25 consecutive amino acids of CRMP2 and comprises the amino acid sequence ASSAK (SEQ ID NO: 64), optionally wherein the peptide further comprises a heterologous amino acid sequence or functional moiety.

14. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 6, 11 or 12, a polynucleotide according to claim 7, a vector according to claim 8 or a host cell according to claim 9, preferably wherein the composition is: (i) a pharmaceutical composition, and further comprising a pharmaceutically acceptable carrier; or (ii) A diagnostic composition, designed as a kit, optionally further comprising reagents conventionally used for immunologically-based diagnostic methods.

15. The antibody or antigen-binding fragment of any one of claims 1 to 6, 11 or 12, or the composition of claim 14, for the treatment of neurodegenerative diseases such as Alzheimer's disease and tau proteinosis.

16. A molecule capable of interfering with the signaling of the brain aging response regulatory protein 2 (CRMP2), used as a drug, preferably used in the prevention of neurodegenerative diseases, cognitive disorders, delaying the progression of neurodegenerative diseases, cognitive disorders, or in the treatment of neurodegenerative diseases, cognitive disorders, and / or in methods for improving memory and learning abilities.

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