Novel Anti-nogo-a antibodies

Abstract

To provide novel human-derived monoclonal neutralizing Nogo-A specific antibodies as well as fragments, derivatives and variants thereof as well as methods related thereto; and to provide polynucleotides, vectors, host cells and kits related to the Nogo-A specific antibodies.SOLUTION: Provided is a human-derived recombinant monoclonal anti-Nogo-A antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof binding to the Nogo-A-Δ20 domain and being capable of inducing dose-dependent neurite outgrowth and / or angiogenesis in stroke penumbra.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to novel human-derived antibodies, as well as fragments, derivatives and bioengineered variants thereof, that specifically bind and neutralize Nogo-A and are useful in the treatment of central nervous system diseases and trauma, such as retinopathies. [Background technology]

[0002] Central nervous system (CNS) tissues, such as the retina, have a limited ability to regenerate damaged tissue. CNS regeneration is inhibited by various cell-intrinsic inhibitors of growth signaling as well as extracellular mechanisms. These include growth inhibitors that are potent in the glial scar and myelin. Nogo-A is considered one of the myelin-associated factors that limit the amount of recovery and plasticity in injured CNS tissue, both in the vasculature and neurons (Waelchli et al., PNAS, 2013). It is a member of the reticulon protein family and contains at least two biologically active and pharmacologically distinct domains, Nogo-66 and Nogo-AΔ20, both of which have been shown to have potent inhibitory activity against neurite outgrowth (GrandPre et al., Nature 417 (2002), 547-51; Oertle et al., J. Neurosci. 23 (2003), 5393-406). Therefore, blocking the inhibitory activity of Nogo-A has been shown to be an important pharmaceutical target for treating disorders or conditions involving damage or degeneration of vascular and neuronal elements of CNS tissues by improving and promoting vascular and neuronal repair and growth (reviewed in Pernet, BBA - Molecular Basis of Disease, 2017).

[0003] In this context, it is a potent inhibitor of neurite outgrowth (later known as nogo in rats). It has been reported that mouse monoclonal antibody IN-1, raised against rat myelin protein NI-220 / 250 (which was found to be encoded by the A gene), promotes axonal regeneration and functional recovery after CNS injury (Schnell and Schwab, Nature 343 (1990), 269-272; Bregman et al. al, Nature 378 (1995), 498-501; Thallmair et al, Nature Neuroscience 1 (1998), 124-131; and Chen et al, Nature 403 (2000), 434-439). Further attempts have been made to develop therapeutically effective monoclonal antibodies targeting Nogo-A. For example, WO 2004 / 052932 describes the murine antibody 11C7, which has been shown to efficiently block Nogo-A-induced inhibition in vitro and in vivo (Oertle et al, (2003) supra; Liebscher et al, Annals of Neurology, 58 (2005), 706-719). For example, in live animals, administration of 11C7 has been shown to stimulate axonal growth and locomotor recovery after spinal cord lesions in rats and promotes vascular regeneration after ischemic injury in the CNS (Liebscher et al. (2005) cited above; Joly et al., Glia 66 (2018), 2079-2093; Rust (Wahl et al., PNAS 116 (2019), 14270-14279). Furthermore, a study by Lindau et al., Brain (2013) observed that intrathecal application of antibody 11C7 to the sensorimotor cortex after corticospinal tract transection or unilateral subtotal photothrombotic stroke (Wahl et al., Science 344 (2014), 1250-5) resulted in a significant functional recovery of forelimb fine motor function in adult rats. Significant functional recovery of arm function with intrathecal application of anti-Nogo-A antibodies was also observed in macaque monkeys with cervical spinal cord or motor cortex injuries (Freund et al., Nat Med. 12 (2006), 790-2; Hamadjida et al., Exp Brain Res. 223 (2012), 321-40). Furthermore, inactivation of Nogo-A can be shown to improve visual plasticity and recovery after retinal injury (see, e.g., Mdzomba et al., Cell Death and Disease (2018) 9:727).

[0004] Further monoclonal anti-Nogo-A antibodies are disclosed in WO 2005 / 061544 (murine antibody 2A10 and its humanized version H1 L11), WO 2007 / 068750 and WO 2009 / 056509 (ATI355, derived from monoclonal antibody 6A3 produced in HuMabmouse™, a genetically reconstituted mouse produced by Medarex Inc., in which human immunoglobulin genes have replaced the corresponding mouse genes). Some of these antibodies are the subject of clinical trials for the treatment of spinal cord injury (SCI), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS); see Schmandke et al., (2014) supra, and Kucher et al., Neurorehabil. Neural Repair. (2018), 578-589. ATI335, also known as NG-101, is currently being investigated in a multicenter, international, placebo-controlled Phase II trial for safety and preliminary efficacy in patients with acute cervical spinal cord injury (SCI), in particular, if the antibody therapy can improve motor function and quality of life in tetraplegic patients, in which the antibody is administered by intrathecal bolus injection of 45 mg (see, e.g., ClinicalTrials.gov Identifier: NCT03935321).

[0005] In summary, the development of monoclonal anti-Nogo-A antibodies to date shows great promise for the prophylactic or therapeutic treatment of disorders or conditions involving damage or injury or degeneration of the central nervous system (CNS), including the retina, such as spinal cord injury (SCI), stroke, or retinopathy, e.g., membrane vascular disease.

[0006] However, in the case of monoclonal antibodies, the origin of the product is an important factor that can affect immunogenicity. Although murine antibodies have been shown to induce stronger immune responses in humans compared to chimeric, humanized, and human monoclonal antibodies, it should be noted that chimeric, humanized, and human monoclonal antibodies can also induce a high rate of immunogenicity depending on the dosing regimen and patient population. Indeed, some human antibodies developed using phage display, and even fully "human" antibodies derived from transgenic mice, can have significant anti-drug antibody (ADA) responses (see, e.g., Harding et al., MAbs. 2010 May-Jun;2(3):256-265, and "Immunogenicity Assessment for Therapeutic Protein Products," USDepartment of (See Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) August 2014 Clinical / Medical.) Thus, in some cases, treatment was discontinued due to persistent ADA positivity in a significant number of patients (e.g., Kuriakose et al., J. Immunology Research (2016), Article ID 1298473, http: / / dx.doi.org / 10.1155 / 2016 / 1298473, and Davda et al. J. ImmunoTherapy of Cancer (2019) 7:105, https: / / doi.org / 10.1186 / s40425-019-0586-0).

[0007] In this regard, the immunogenicity of monoclonal antibodies can also result from impurities and heterogeneity of the antibody preparation, for example, from chemical degradation products of the antibody and the resulting lack of stability of the antibody molecule (see, for example, Doevendans and Schellekens, Antibodies 8 (2019), 21; https: / / doi.org / 10.3390 / antib8010021.). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2004 / 052932 [Patent Document 2] International Publication No. 2005 / 061544 [Patent Document 3] International Publication No. 2007 / 068750 [Patent Document 4] International Publication No. 2009 / 056509 [Non-patent literature]

[0009] [Non-Patent Document 1] Waelchli et al,PNAS,2013 [Non-patent document 2] GrandPre et al, Nature 417(2002), 547-51 [Non-patent document 3] Oertle et al, J. Neurosci. 23 (2003), 5393-406 [Non-patent document 4] Pernet,BBA-Molecular Basis of Disease,2017 [Non-patent document 5] Schnell and Schwab,Nature 343(1990),269-272 [Non-patent document 6] Bregman et al,Nature 378(1995),498-501

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[0010] The present invention relates to embodiments as characterized in the claims, disclosed herein and illustrated in the following examples and figures, namely, to Nogo-A specific human-derived monoclonal antibodies and Nogo-A binding fragments thereof, and equivalent synthetic variants and biotechnological derivatives of the antibodies exemplified herein, which are particularly useful in the prophylactic or therapeutic treatment of a variety of disorders or conditions involving injury, damage or degeneration of the central nervous system (CNS), including retinal and peripheral nervous system (PNS) tissue.

[0011] As illustrated in the Examples, in the complex process of antibody discovery, it has been fortunate to discover antibodies that can enhance, for example, the growth of neurites in adult rats after extensive unilateral motor cortex strokes by enhancing neurite outgrowth in the presence of growth-inhibitory CNS myelin. We have cloned and identified a high-affinity human monoclonal anti-Nogo-A antibody capable of neutralizing the biological activity of Nogo-A by enhancing limb functional recovery or by increasing angiogenesis in the penumbra after stroke injury in a mouse model of stroke. This antibody is at least as effective as the previously established mouse anti-Nogo-A antibody "11C7," considered the "gold standard," in e.g., resulting in functional recovery of advanced forelimb use after stroke in stroke studies. Specifically, experiments conducted within the scope of the present invention demonstrate that angiogenesis is induced by the anti-Nogo-A antibody of the present invention within the penumbra of adult mice after a permanent stroke of the motor cortex (see, e.g., Example 9). Thus, the anti-Nogo-A antibody of the present invention can generally be characterized by its pro-angiogenic effect and its ability to promote vascular repair / growth in the ischemic penumbra for up to three weeks after injury. Additionally or alternatively, the anti-Nogo antibodies of the present invention may be characterized as being capable of forming vascular endothelial cells and having a significant effect of increasing the number of newly formed vascular endothelial cells compared to controls (see, e.g., Example 9).

[0012] In summary, experiments performed in accordance with the present invention have successfully identified anti-Nogo-A antibodies that are effective in neurite outgrowth and regeneration, as well as functional and vascular repair after, for example, stroke.

[0013] Furthermore, as can be shown in further experiments performed in accordance with the present invention, the anti-Nogo-A antibodies of the present invention are highly soluble (up to at least 20 mg / ml in PBS) and particularly stable in common buffers such as phosphate-buffered saline (PBS), e.g., repeated freeze-thaw cycles (PBS solution, pH 7.4, 7 mg / ml) did not result in detectable levels of aggregation and degradation products (see, e.g., Example 10 and Figure 9).

[0014] Surprisingly, antibodies of the invention bind to the Nogo-A delta 20 (d20) domain (stretching over 160 amino acids) and are at least as effective as the anti-Nogo-A antibody 11C7, but recognize an epitope distinct from that of 11C7 and the other known anti-Nogo-A antibodies ozanezumab and ATI355, and do not compete with antibody 11C7 for binding to Nogo-A. In particular, as shown in Example 3, antibody NG004 of the invention binds to several additional amino acids C- and N-terminal to the extended d20plus region of the inhibitory region (human amino acid positions 543-866). Epitope mapping identified a sequence within the d20 region of human Nogo-A comprising amino acids 141-INAALQE-147 (SEQ ID NO: 21), corresponding to amino acids 683-689 of the native Nogo-A protein, as the minimal epitope recognized by the antibody NG004 of the invention (see Example 4 and Figure 3). Thus, in one embodiment, the antibody binds to a Nogo-A epitope comprising the amino acid sequence INAALQE (SEQ ID NO: 21) (see Example 4). The present invention therefore relates to an antibody or binding fragment thereof with the same binding specificity as antibody NG004, i.e., the ability to enhance neurite outgrowth in the presence of growth-inhibitory CNS myelin and / or increase penumbra angiogenesis after stroke injury in a mouse model of stroke, preferably binding to the Nogo-A Δ20 (d20) domain, in particular the amino acid sequence 141-INAALQE-147 (SEQ ID NO: 21). The mentioned characteristics can be readily identified according to the experiments and assays disclosed in the accompanying Examples, in which the antibody NG004 can be used as a reference antibody. Typically, such an antibody competes with the corresponding reference antibody for binding to Nogo-A at the same epitope and peptide, respectively.

[0015] Thus, in one embodiment, an antibody of the invention may be derived from antibody NG004 and characterized by complementarity determining regions (CDRs) or hypervariable regions of the variable heavy (VH) and variable light (VL) chains comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:7 or SEQ ID NO:12, as shown in Figure 1A and described below in the legend to Figure 1. In another embodiment, an antibody of the invention may be derived from antibody NG034 and characterized by CDRs or hypervariable regions of the VH and VL chains comprising the amino acid sequences of SEQ ID NO:12 and SEQ ID NO:17, as shown in Figure 1B and described below in the legend to Figure 1.

[0016] Further experiments conducted within the scope of the present invention demonstrated that the antibodies of the present invention are capable of immunostaining cells and tissues expressing Nogo-A. Specifically, immunofluorescence staining showed that NG004 positively stained the human oligodendrocyte cell line MO3.13 and the rat neuronal cell line Neuroscreen-1 (NS-1), which express Nogo-A intracellularly and on the cell surface, as well as oligodendrocytes and motor neurons in rat CNS tissue, to a similar extent as the positive control antibodies 11C7 and ozanzumab. During experiments conducted in accordance with the present invention, it was further demonstrated that the antibody NG004 of the present invention is at least as efficient as the previously used gold standard antibody 11C7, with an IC50 of less than 5 nM, or even less than 12 nM, for inducing neurite outgrowth in the presence of growth-inhibitory CNS myelin-containing Nogo-A (see Example 8).

[0017] Thus, based on the results obtained in the experiments carried out within the scope of the present invention, a novel class of anti-Nogo antibodies is provided that is therapeutically useful in the treatment of disorders associated with unwanted Nogo-A activity.

[0018] Although the invention will be illustrated and described with reference to human-derived antibodies obtained for the first time in the experiments performed in accordance with the invention and described in the Examples, it should be understood that an antibody or antibody fragment of the invention includes synthetic and biotechnological derivatives of antibodies, meaning any engineered antibody or antibody-like Nogo-A binding molecule synthesized by chemical or recombinant techniques that retains one or more functional properties of the subject antibody, in particular its neutralizing activity against Nogo-A. Thus, although the invention will for brevity be described with reference to antibodies, unless otherwise stated, the term "antibody" refers to and is encompassed within its meaning, including synthetic and biotechnological derivatives thereof, and equivalent Nogo-A binding molecules.

[0019] Further embodiments of the present invention will become apparent from the following description and examples. [Brief explanation of the drawings]

[0020] [Figure 1]Amino acid sequences of the variable regions, i.e., heavy and kappa light chains (VH, VL), of the anti-Nogo-A specific human antibodies NG004 (A) and NG034 (B) of the present invention. The framework regions (FR) and complementarity determining regions (CDR) are indicated, with the CDRs underlined. The Kabat numbering scheme was used (see: http: / / www.bioinf.org.uk / abs / ; Kabat et al., US Department of Health and Human Services, "Sequence of Proteins of Immunological "Numbering of CDRs of Interest" (1983). The Kabat numbering scheme is given in Table 1 on page 28 of WO 2015 / 092077, mentioned in the aforementioned web references and incorporated herein by reference. Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody of the invention or a Nogo-A-binding fragment, variant or derivative thereof are according to the Kabat numbering system, although this is theoretical and does not apply equally to all antibodies of the invention. For example, depending on the position of the first CDR, the next CDR can be shifted in either direction. Thus In the event of unintentional errors or discrepancies with respect to the representation of the CDRs in Figure 1 and / or the sequence listing, a person skilled in the art is well placed to determine the correct CDR sequences according to Kabat based on the disclosure of the present application, i.e., the amino acid sequences of the variable heavy (VH) and variable light (VL) chains of antibodies NG004 and NG034, which Kabat shall be used to define the claimed antibodies and Nogo-A binding fragments thereof. The sequences of the variable heavy chain VH and light chain VL of antibody NG004 as set forth in SEQ ID NO:2 and SEQ ID NO:7 (A) and antibody NG034 as set forth in SEQ ID NO:12 and SEQ ID NO:17 (B) are shown.As further explained herein, within the CDR and / or framework regions, conservative amino acid substitutions are preferred that take into account either the physicochemical properties of the original amino acid alone or the physicochemical properties of the original amino acid and the adjacent amino acid, as shown in Mirsky et al., Mol. Biol. Evol. 32 (2014) 806-819, page 813, Figure 6, particularly the AB model or LG model, where, for example, the positions of two amino acids are exchanged. [Figure 2] Binding specificity of antibodies NG004 and NG034 to recombinantly expressed human and rat Nogo-A d20+ region and rat corpus callosum oligodendrocytes. (A) NG004 binds to the d20+ region of human Nogo-A with high affinity / avidity. The EC50 value of NG004 is 0.26 nM. (B) NG004 binds weakly to the d20+ region of rat Nogo-A. (C) NG034 binds to the d20+ region of human Nogo-A with high affinity / avidity (NG034 EC50 value is 0.298 nM). (D) NG034 binds to the d20+ region of rat Nogo-A with high affinity / avidity (NG034 EC50 value is 0.229 nM). (E) NG004 and NG034 positively stain rat corpus callosum oligodendrocytes, as shown by immunofluorescence staining on fixed rat brain tissue sections, resulting in a staining pattern similar to that of the control antibody, ozanzumab. No staining is observed with the secondary donkey anti-human Cy3-labeled (DoxHu Cy3) antibody alone. [Figure 3] Nogo-A binding epitope of antibody NG004 assessed by pepscan analysis. Pepscan image of NG004. NG004 binding occurred with peptides 34, 35, and 36 (white boxes), which cover amino acids 141 to 147 of the d20plus region of Nogo-A (peptide 34: 133-EEIKEPENINAALQE-147 SEQ ID NO: 22; peptide 35: 137-EPENINAALQETEAP-151 SEQ ID NO: 23; peptide 36: 141-INAALQETEAPYISI-155 SEQ ID NO: 24; consensus binding sequence: 141-INAALQE-147 SEQ ID NO: 21). [Figure 4] Cross-competition assay of antibodies NG004 and NG034 for competitive binding to Nogo-A with antibodies ozanzumab and 11C7. NG004 shows no competitive binding with antibodies ozanzumab (A) and 11C7 (B). NG034 shows no competitive binding with NG004 and 11C7 in the human d20plus region (C). [Figure 5] In vivo target engagement of NG004 was analyzed by intrathecal treatment of rats with NG004 for 1 week, followed by analysis of Nogo-A and Nogo-B protein levels in the CNS by immunofluorescence staining. (A) NG004 downregulates endogenous Nogo-A levels in the CNS. (B) NG004 upregulates endogenous Nogo-B levels in the CNS. (C) NG004 upregulates endogenous NgR1 levels in the CNS. [Figure 6] The effect of NG004 on long-term potentiation (LTP) was analyzed in an ex vivo assay in mouse hippocampus. (A) Antibody 11C7, used as a positive control, increases LTP by blocking Nogo-A. FG12 is an inactive control antibody. (B) Antibody NG004 exerts similar ex vivo activity as 11C7, i.e., increases LTP. (C) A higher dose of NG004 (25 μg / ml) increases the magnitude and onset of action. [Figure 7] In vitro neurite outgrowth assay of N1E mouse neuroblastoma cells in the presence or absence of growth-inhibitory CNS myelin extract and anti-Nogo-A antibody. (A,B) NG004 stimulates neurite outgrowth in the presence of rat spinal cord extract (SCE) in a dose-dependent manner, very similar to antibody 11C7. (C,D) NG004 and NG034 stimulate neurite outgrowth in the presence of non-human primate CNS extract (CNSE) very similar to antibody ATI355. The respective inactive control antibody 3.1 IgG1 has no effect. [Figure 8]In vivo stroke model in adult mice; extent of vascular repair in the ischemic penumbra surrounding the focal stroke core 3 weeks after stroke. (A) NG004 increases vascular area within the ischemic penumbra compared to control antibody FG12 / B5. (B) NG004 increases the number of vascular branches compared to control antibody FG12 / B5. (C) NG004 increases vessel length within the ischemic penumbra compared to control antibody FG12 / B5. (D) NG004 increases the proliferation rate of CD31+ endothelial cells. Effect sizes for all parameters are similar for NG004 and 11C7. [Figure 9] Size exclusion chromatography analysis of NG004 at different pH values ​​after repeated freeze-thaw cycles shows that the antibody is very stable. [Figure 10] Functional recovery after ischemic stroke and 2 weeks of anti-Nogo-A treatment. Rats received a unilateral photothrombotic stroke and were treated continuously (2 ml) intrathecally via osmotic minipump with either two different anti-Nogo-A antibodies (11C7 [4 mg / ml]; NG004 [4 mg / ml or 8 mg / ml]) or a control antibody (BrdU antibody, 4 mg / ml) for 2 weeks. Horizontal ladder success score assessment (forelimb impairment: number of correct steps / total steps) for the different treatment groups. (A) Timeline of weekly horizontal ladder performance after injury. (B) Performance on day 63 post-injury. Animals treated with 8 mg of NG004 showed significant improvement compared to anti-BrdU-treated animals. Animals treated with 4 mg of NG004 showed a clear trend toward improvement. [Figure 11] C1q binding was compared in an ELISA-based CDC assay using NG004 isotypes (IgG1 and IgG4), rituximab (IgG1, Mabtera), and natalizumab (IgG4, Tysibra). NG004 IgG4 S228P exhibited reduced reactivity to C1q, behaving similarly to other IgG4s (natalizumab). DETAILED DESCRIPTION OF THE INVENTION

[0021] In general, the present invention relates to human-derived monoclonal antibodies capable of binding to and neutralizing Nogo-A, as well as fragments, derivatives and variants thereof. More specifically, the present invention relates to the embodiments characterized in the claims, disclosed in the specification and further illustrated in the examples and figures below. Due to their human origin, i.e., maturation of the original antibodies in the human body, and their neutralizing capacity against Nogo-A, the antibodies have high therapeutic value and are preferably substantially non-immunogenic in humans.

[0022] Unless otherwise stated, terms used herein include those defined in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2, 2nd Edition, Published 2006, ISBN 0-19-852917-1 978-0-19852917-0 is given.

[0023] Furthermore, unless otherwise stated, the terms and expressions used herein to characterize the present invention are given the definitions provided in WO2015 / 092077, particularly subsection "I. Definitions" on pages 16-42, including Table 1 of CDR definitions on page 28, the disclosure of which is expressly incorporated herein by reference. The same applies to the general embodiments disclosed in WO2015 / 092077 for antibodies, polynucleotides, etc. In addition, without acknowledging that the scientific publications and patent applications cited in the "Background Art" represent prior art with respect to the claimed invention, their disclosures regarding Nogo-A and anti-Nogo-A antibodies, their recombinant production in host cells, purification, modification, formulation in pharmaceutical compositions and therapeutic uses, as well as terms and characteristics common in the art, may be relied upon by a person skilled in the art when carrying out the claimed invention; see, for example, Antibodies A Laboratory Manual 2nd edition, 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, which also describes antibody purification and storage, antibody manipulation such as the use of degenerate oligonucleotides, 5'-RACE, phage display and mutagenesis, immunoblotting protocols, and state-of-the-art screening and labeling techniques.

[0024] The terms "neutralizing" and "neutralizing antibody" are used in their usual manner in the art to refer to an antibody that reduces or eliminates at least some biological activity of an antigen or live microorganism, respectively. For example, an anti-Nogo-A antibody of the invention is a neutralizing antibody if it eliminates or reduces the activity of Nogo-A in sufficient amounts, e.g., in an assay as described in the Examples. Neutralization is generally defined by the 50% inhibitory concentration (IC50) and can be statistically assessed based on the area under the neutralization titration curve (AUC). IC50 values ​​for exemplary anti-Nogo-A antibodies of the invention are illustrated and described herein, e.g., in Figures 5-8. Specifically, the neutralizing ability of the antibodies of the invention is, and can be, analyzed, for example, as shown in Examples 6-9, in that the antibodies downregulate endogenous Nogo-A in vivo in the CNS as measured by immunohistochemistry, increase long-term synaptic plasticity (long-term potentiation, LTP) as measured in an LTP assay in mouse hippocampus, stimulate neurite outgrowth in an in vitro neurite outgrowth assay, and induce angiogenesis in the penumbra of an in vivo mouse stroke model.

[0025] Thus, the present invention generally relates to human-derived recombinant monoclonal anti-Nogo-A antibodies and antigen-binding fragments thereof that neutralize the biological activity of Nogo-A and can induce neurite outgrowth in a dose-dependent manner in the presence of a growth-inhibitory CNS extract, as demonstrated, for example, in Example 8, and / or induce angiogenesis of the stroke penumbra, as demonstrated, for example, in Example 9.

[0026] Glial-derived axonal growth inhibitory proteins limit functional repair after injury to the adult CNS. In particular, Nogo-A and, for example, MAG and OMgp, interact with Nogo receptors, such as neuronal (co-)receptors, e.g., Nogo receptor-1 (NgR1). NG004 is an inhibitor that interacts with the NogoA receptor, the sphingolipid receptor S1PR2 or leucine-rich repeat, and the immunoglobulin-like domain-containing protein also known as LINGO-1, resulting in the inhibition of axon growth. For example, upon interaction with an inhibitory protein (e.g., NogoA), the NgR1 complex transmits signals that result in growth cone collapse and inhibition of neurite outgrowth. As described above, in vivo studies of intrathecal administration of NG004 in rats demonstrated a significant reduction in NogoA in CNS tissue compared with control antibody treatment. Thus, in contrast to the known mechanism of inhibiting receptor binding of the ligand NogoA, the antibodies of the present invention also exert their potent biological effects by depleting the ligand from the system, i.e., downregulating NogoA levels in the CNS.

[0027] In vivo experiments using intrathecal administration of NG004 in rats further demonstrated that the Nogo-A receptor NgR1 was upregulated (see Figure 5C), suggesting that NG004 binds to Nogo-A in vivo, downregulating CNS Nogo-A levels and upregulating its receptor NgR1 as a compensatory mechanism. Therefore, therapeutic approaches using anti-Nogo-A antibodies, preferably antibodies NG004 or NG034, may be further improved when combined with molecules that also inhibit Nogo-A receptor binding to Nogo-A, such as NgR1, S1PR2, or LINGO-1. In particular, the anti-Nogo-A antibodies of the present invention, i.e., NG004 and NG034, remove Nogo-A from the system, leaving Nogo-A receptors such as NgR1, S1PR2, or LINGO-1 unstimulated. This mechanism is similar to that of AXER-204, a recently developed soluble human fusion protein that acts as a decoy or trap for myelin-associated growth inhibitors such as MAG, OMgp, and Nogo-A, inhibiting their signaling and promoting neuronal growth (see Bradbury and Oliveira, Brain 143 (2020), 1618-1622). Therefore, it is prudent to consider that the anti-Nogo antibodies of the present invention may be used to treat diseases that can be treated with AXER-204.

[0028] Thus, in a further aspect, the present invention relates to a combination therapy comprising an anti-Nogo-A antibody, preferably NG004 or NG034, in combination with a molecule that inhibits the binding of Nogo-A to its receptor complex or a blocker of a post-receptor signaling pathway, for use in treating a disease or injury of the peripheral (PNS) and / or central (CNS) nervous system as defined herein. Molecules that inhibit Nogo-A receptor binding are known in the art. For example, an isolated polypeptide fragment that inhibits NgR1-mediated neurite outgrowth inhibition is described in WO 2007 / 089601, or the lateral olfactory tract usher substance (LOTUS), which binds to NgR1 and blocks Nogo-A binding to NgR1, resulting in the suppression of Nogo-A-induced axonal growth inhibition, as described in Kurihara and Takei, Neural Regen Res. 10 (2015), 46-48. Furthermore, the anti-LINGO-1 antibody Li81 (opicinumab) blocks LINGO-1 function and exhibits robust remyelination activity in animal models. This antibody is currently being investigated in a phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (see Hanf et al., mAbs 12(1)(2020), 1713648).

[0029] Similar to Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) have axonal inhibitory roles, and therefore treatment with anti-Nogo-A antibodies of the invention can be combined with anti-MAG antibodies and / or anti-OMgp antibodies (see, e.g., Yu et al., Transl. Stroke Res. 4 (2013), 477-483, and Irving et al., J Cereb Blood Flow Metab. 25 (2005), 98-107).

[0030] Furthermore, as demonstrated for example for antibody NG004 in Example 8, the antibodies of the invention exhibit low inhibitory concentrations (IC 50 ) have particularly high neutralizing activity. Specifically, the IC of the antibody of the present invention for stimulating neurite outgrowth 50 The IC value was shown to be 11.16 nM, which is the IC measured for the reference antibody 11C7, which has been used previously as the gold standard. 50 Thus, in one embodiment, an anti-Nogo-A antibody or antigen-binding fragment thereof exhibits an IC50 value for inducing neurite outgrowth in a neurite outgrowth inhibition assay. 50 A value of less than 15 nM, preferably less than 12 nM, is indicated.

[0031] As further illustrated in the Examples and Figures, e.g., Figure 2, the antibodies of the invention were originally isolated from human donors and are shown to bind to human Nogo-A. Thus, in one embodiment, the anti-Nogo-A antibodies and Nogo-A binding fragments of the invention are derived from the antibody NG004 and preferentially recognize the human Nogo-A d20plus peptide over corresponding antigens from other species, such as rat or mouse. The binding properties, such as specificity and affinity, of the antibodies of the invention have been tested in several experimental assays described and shown herein, e.g., in Examples 3 to 5 and Figures 2 to 4. In this regard, the EC of the antibodies of the invention was measured in an ELISA performed in Example 3 to provide a measure of binding affinity. 50 It has been demonstrated that the antibodies of the present invention inhibit EC 50 Specifically, antibody NG004 exhibits a particularly high apparent binding affinity for binding to the human Nogo-A d20plus peptide, as confirmed by the EC 50 The EC value of NG034 is 0.26 nM, whereas rat Nogo-A binds only weakly (see Example 3). In another embodiment, the antibody of the invention is derived from the antibody NG034, which recognizes human and rat Nogo-A with high affinity. Specifically, the EC value of NG034 is 50is 0.298 nM for human binding and 0.229 nM for rat binding to the d20plus region (see Example 3). Binding of NG004 to human and rat Nogo-A expressed on HEK cells was further confirmed by immunoprecipitation assays followed by Western blot detection.

[0032] Therefore, the antibodies of the present invention preferably have IC50 activity for inducing neurite outgrowth in a neurite outgrowth inhibition assay. 50 and / or an EC value for binding to the human or rat d20plus region of less than 15 nM, preferably less than 12 nM, more preferably about 11 nM. 50 The IC value may be characterized as being less than 0.5 nM, preferably less than 0.4 nM, more preferably about 0.3 nM. However, depending on the antibody format, e.g., whether an IgG1, IgG4, or antibody fragment such as a Fab fragment is used, the IC 50 and EC 50 Values ​​may deviate, e.g., may be higher or lower than those stated above and in the Examples. Thus, in this context, the term "about" means a value that may differ from the value determined for the reference antibody in the Examples, preferably by less than an order of magnitude, and most preferably by within the same order of magnitude, e.g., IC 50 can be the reference value ± 10 nM, EC 50 can be the reference value ±0.3 nM.

[0033] As demonstrated in the competition assays of Example 5 and Figure 4, the subject antibodies do not exhibit competitive binding to Nogo-A with at least the antibody 11C7, and preferably also with ozanzumab, as shown for NG004. Thus, in one embodiment, an antibody or antigen-binding fragment thereof of the invention additionally or alternatively does not compete with the anti-Nogo-A antibody 11C7 for binding to Nogo-A, and preferably does not compete with ozanzumab. Competition between antibodies is determined by an assay in which the immunoglobulin under test is inhibited by the specific binding of a reference antibody to a common antigen, such as Nogo-A. Many types of competitive binding assays are known (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988), (2014) supra). Preferably, the competitive binding assay is performed under the conditions described in Example 5.

[0034] The neurite outgrowth inhibitor Nogo-A contains three inhibitory domains: two shared with the splice variant Nogo-B (Nogo-66 located between the two transmembrane domains and the N-terminal tip of the NIR domain) and one shared with the splice variant Nogo-C (Nogo-66). The unique domain highly inhibitory to neurite outgrowth of Nogo-A is located in exon 3 of Nogo-A and is called the delta20 domain (d20; human amino acid positions 566-748) (Oertle et al., J. Neurosci. 23 (2003), 5393-5406). As demonstrated in Example 3, the antibodies of the present invention bind to a fragment containing the d20 domain (Nogo-A-Δ20 domain) plus several additional amino acids at the C- and N-termini of the inhibitory domain (human amino acid positions 543-866), the so-called d20plus domain. Thus, in one embodiment of the invention, the antibody binds to Nogo-A within the region between amino acid positions 543 and 866 of human Nogo-A, and preferably binds to an epitope and / or peptide comprising or consisting of the amino acid sequence 141-INAALQE-147 (SEQ ID NO: 21), which corresponds to amino acids 683 to 689 of human Nogo-A (see Example 4).

[0035] The present invention relates to a variable heavy chain (VH) having, in its variable region, i.e., binding domain, the amino acid sequence shown in Figures 1A and 1B, respectively. H ) and variable light chain (V L ) The corresponding nucleotide and amino acid sequences are shown in Table II below.

[0036] As usual, the variable domain of each chain contains three hypervariable loops called complementarity-determining regions (CDRs, CDR-1, CDR-2, and CDR-3). The CDRs are separated by structurally conserved regions called framework regions (FR-1, FR-2, FR-3, and FR-4), which form a "core" β-sheet structure that displays these loops on the surface of the variable domain. The length and composition of CDR sequences are highly variable, especially in CDR3. CDRs approximate the paratope of the antibody that interacts with the antigen and therefore contain antigen-binding residues. Therefore, it is common to define antibodies by their six CDRs. V H Chain and V L An exemplary set of CDRs in the above amino acid sequences of the chains are shown in Figures 1A and 1B. However, as discussed below, those skilled in the art are well aware of the fact that, additionally or alternatively, for CDR2 and CDR3, CDRs whose amino acid sequences differ by one, two, three or more amino acids from those shown in either one of Figures 1A and 1B may be used. As noted in the figure legend of Figure 1, those skilled in the art can readily identify CDRs according to general principles, such as those summarized at www.bioinf.org.uk / abs. In this regard, although the CDRs of the antibody shown in Figure 1 are shown by Kabat et al., those skilled in the art are aware of several definitions of CDRs that are commonly used, namely, the following: (i) the most commonly used Kabat definition based on sequence diversity; (ii) the Chothia definition based on the location of structural loop regions; (iii) the definition of AbM used in Oxford Molecular's AbM antibody modeling software, which is a compromise between the two above; and (iv) The recently introduced Contact definition, based on analysis of available complex crystal structures. This definition is likely to be most useful for performing mutagenesis to alter antibody affinity, since these are the residues involved in interactions with the antigen. For a list of CDR contact residues for each antibody, along with summary data for each CDR, see, e.g., www.bioinf.org.uk / abs (which also mentions antibody modeling software such as abYmod, available at abymod.abysis.org).

[0037] Table I below shows the relationships between the CDR positions defined by the different concepts.

[0038] [Table 1]

[0039] For the above definitions, please also refer to Kontermann and Duebel (eds.), Antibody Engineering Vol. 2, DOI 10.1007 / 978-3-642-01147-4_3, #Springer-Verlag Berlin Heidelberg 2010, especially Chapter 3, Protein Sequence and Structure Analysis of Antibody. See pages 33-51 of Variable Domains, and Dondelinger et al., Front. Immunol. 9 (2018), 2278, which deals in particular with understanding the importance and significance of antibody numbering and antigen binding surface / residue definition; see, e.g., Kabat, Chothia (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917), Contact (Mac See Dondelinger et al., Figures 4 and 6, which illustrate the differences between the classical CDR definitions by Callum et al., J. Mol. Biol. 262 (1996), 732-745) and IMGT (IMGT®, the international ImMunoGeneTics information system®, www.imgt.org). The AbM definition is a compromise between the two, used by Oxford Molecular's AbM antibody modeling software.

number

[0040] The top diagram shows an alternative definition of CDR-H1 (VH-CDR1): the Kabat and Chothia numbering schemes are shown horizontally, and the Kabat, Chothia, AbM, and Contact definitions of the CDRs are indicated by arrows above and below the two numbering schemes.

[0041] In one embodiment, the invention relates to a human-derived monoclonal anti-Nogo-A antibody, or a Nogo-A binding fragment, synthetic or biotechnological derivative thereof, wherein the fragment or derivative comprises a variable heavy chain (VH) comprising VH complementarity-Determining Regions (CDRs) 1, 2 and 3, and a variable light chain (VL) comprising VL CDRs 1, 2 and 3 as defined by Kabat, (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; or (g) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (h) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (i) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (j) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (k) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (l) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof, wherein the variant comprises one or two amino acid substitutions.

[0042] Additionally or alternatively, the antibody or antigen-binding fragment thereof of the present invention comprises: (a) the VH chain comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) VL comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; or (c) VH comprises the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (d) VL comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; Preferably, the amino acid sequences of the VH and VL chains are characterized by being at least 90% identical to SEQ ID NO: 2 and SEQ ID NO: 7, respectively. In this embodiment, preferably, one or more CDRs according to the Kabat definition are maintained substantially unchanged. However, under the simplifying assumption that the paratopes correspond to the CDRs, the Chothia definition of CDRs may additionally or alternatively be used, since they correlate very well with the structural loops present in the variable regions. Thus, in the case of CDRs defined according to Kabat, at least one or two of the one or more, preferably no more than two, amino acid substitutions are made outside the CDRs defined by Chothia and / or IMGT, most preferably outside the overlap of the CDRs defined according to Kabat and Chothia.

[0043] For example, for amino acid substitutions within the CDRs, respectively the variable heavy and variable light chains and framework amino acid sequences, preferably conservative amino acid substitutions, can be made according to, for example, the Mirsky method. The amino acid substitution is carried out according to the most frequently exchanged amino acids analyzed and described in Mirsky et al., Mol. Biol. Evol. 32 (2014), 806-819 (see Figure 6 on page 813 of Mirsky et al.). Specifically, in VH-CDR1, S may be substituted with T; in VH-CDR3, V may be substituted with E, T may be substituted with S, and / or M may be substituted with V; in VL-CDR1, R may be substituted with K, R may be substituted with E, and / or T may be substituted; in VL-CDR2, S may be substituted with A, and / or A may be substituted with G; and in VL-CDR3, P may be substituted with S. As already mentioned, preferably, the amino acid substitution is carried out according to the above Mirsky method. Amino acid substitutions belonging to the same category are selected in either or preferably both of models LG and AB shown in Figure 6 of [End Page 101] et al. (2014), and the LG model is preferably one that tends to maintain the properties of the amino acid, where the amino acid substitutions are preferably selected so that the physicochemical properties, i.e., hydrophobicity, polarity, or charge properties, of the original amino acid are substantially maintained, or, for example, when two or more amino acid substitutions are made, they are selected so that they compensate each other to all together provide the physicochemical properties of the surface. In a preferred embodiment, the antibody of the present invention comprises variants of the amino acid sequences of the VH and / or VL regions that are at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL regions shown in Figures 1A and 1B.

[0044] Of course, in addition to theoretical considerations, there are also experimental approaches for identifying CDR variants within a reasonable time and effort. For example, Tiller et al., Front Immunol. 8 (2017), 986, describes a simple affinity maturation of antibody variable domains using natural diversity mutagenesis. In fact, already several years ago, Rajpal et al., PNAS 102 (2005), 8466-8471, reported a general method for significantly improving the affinity of antibodies by using combinatorial libraries, and described their method using the anti-TNF-α antibody D2E7 (HUMIRA ©), identifying 38 substitutions in 21 CDR positions that result in higher affinity binding to TNF-α. More recently, Cannon et al., PLOS Computational Biology, https: / / doi.org / 10.1371 / journal.pcbi.1006980, May 1, 2019, describe experimentally guided computational antibody affinity maturation using de novo docking, modeling, and rationally designed in silico affinity maturation together with alanine scanning, which allowed for fine-tuning of a protein-protein docking model and subsequent identification of two single-point mutations that increased the affinity of hybridoma-derived antibody AB1 for its antigen, murine CCL20.

[0045] Thus, while each antibody may be unique and have different characteristics, once a lead candidate is provided, a person skilled in the art, taking into account the teachings of the invention disclosed in this application and the computational design and experimental approaches developed to date, can nevertheless obtain an equivalent anti-Nogo-A antibody that retains the desired characteristics of the antibody, such as those described for the anti-Nogo-A antibodies exemplified in the Examples and specifically defined in the Claims. In this regard, it is well understood that the variant antibody will substantially maintain the binding specificity of the parent antibody, e.g., compete with the parent antibody for binding to Nogo-A, while not competing with one or more, preferably all, of the prior art antibodies already mentioned, i.e., not competing with at least 11C7 and preferably not competing with ozanzumab, which can be assessed according to the competition assay described in Example 5. In particular, antibodies of the invention derived from antibody NG004 do not compete with antibodies 11C7 and ozanzumab. Preferably, however, an antibody of the invention comprises, in one or both of its immunoglobulin chains, one, two, or all three CDRs of the variable regions shown in Figure 1, or one, two, or all three CDRs that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the CDRs of the variable regions shown in Figure 1. Additionally or alternatively, one or more framework regions (FRs) from the FRs are 80% identical to the corresponding FRs shown in Figures 1A and 1B, and preferably 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the framework regions shown in Figures 1A and 1B. In some embodiments, one, two, three, or all four FRs (each at least 90%, 90-95%, and / or 95-99% identical to the FRs shown in Figures 1A and 1B, respectively) are present.

[0046] As is known in the art, the CDR3 of the variable heavy chain (VH-CDR3) appears to primarily determine antigen specificity (see, e.g., Xu and Davis, Immunity 13 (2000), 37-45). In this context, it is the diversity of the heavy chain CDR3 that confers specificity, whereas VH-CDR1 and VH-CDR2 residues are known to be broadly cross-reactive and subject to improvement by somatic hypermutation (see Davis, Semin. Immunol. 16 (2004), 239-243). Thus, in one embodiment, an antibody of the invention having the immunological characteristics of the reference antibody NG004 and capable of competing with Nogo-A for its binding to the respective epitope comprises, in its variable region, at least the VH-CDR3 of the corresponding reference antibody, or a VH-CDR3 whose amino acid sequence is at least 90% identical, preferably 95% identical, or even 96%, 97%, 98%, 99% or 100% identical to the reference VH-CDR3. For example, a variant antibody of a reference antibody may retain the VH-CDR3 of the reference (parent) antibody, but VH-CDR1 and / or VH-CDR2 may contain one or more amino acid substitutions (see above).

[0047] In further additional or alternative embodiments of the invention, anti-Nogo-A antibodies, antigen-binding fragments thereof, synthetic variants or bioengineered variants may be optimized to have suitable binding affinity to the target, as well as pharmacokinetic and stability properties. Thus, at least one amino acid in the CDRs or variable regions that is susceptible to a modification selected from the group consisting of glycosylation, oxidation, deamination, peptide bond cleavage, iso-aspartic acid formation and / or unpaired cysteine ​​is substituted by a mutant amino acid that lacks such an alteration or in which at least one carbohydrate chain is deleted or chemically or enzymatically added to the antibody (see, e.g., 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).

[0048] The immunoglobulin or its encoding cDNA can be further modified. Thus, in a further embodiment, the method of the present invention comprises any one of the steps of producing a chimeric antibody, a murine antibody, a single-chain antibody, a Fab fragment, a bispecific antibody, a fusion antibody, a labeled antibody, or an analog of any one of them. Corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual," 1st Edition, CSH Press, Cold Spring Harbor (1988), and in Edward A. Greenfield, 2nd Edition, Dana-Farber Cancer Institute (Copyright) 2014, ISBN 978-1-936113-81-1. For example, Fab fragments and F(ab')2 fragments can be produced recombinantly or by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region, and the CH1 domain of the heavy chain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures which involve coupling an immunospecific portion of an immunoglobulin to a detection reagent such as a radioisotope.

[0049] Thus, in one embodiment, the antibodies of the present invention may be provided in a format selected from the group consisting of single-chain Fv fragments (scFv), F(ab') fragments, F(ab) fragments and F(ab') fragments, Fd, Fv, single-chain antibodies, and disulfide-linked Fvs (sdFv), and / or in the format of chimeric mouse-human antibodies or murine antibodies.

[0050] However, as illustrated in the examples according to the present invention, preferably, complete IgG antibodies are used, in which the antibody contains a constant domain. The constant domain may be natural, i.e., originally cloned together with the variable domain, or it may be heterologous, e.g., a murine constant domain if animal experiments are envisaged. Preferably, the constant domain is of human origin, having a different IgG subtype, e.g., IgG4 versus IgG1, or a different allotype and allele, respectively, compared to the constant domain of an antibody naturally occurring in humans. The definition of "allotype" requires that antibody reagents are available for serologically determining the allotype. If the determination is made only at the sequence level, the polymorphism must be described as an "allele." This does not preclude establishing an allotype correspondence when the allele-allotype correspondence has been experimentally demonstrated or when the individual sequences are identical to those demonstrated.

[0051] In a preferred embodiment of the invention, the constant domain is heterologous to at least one of the CDRs and the VH and VL chains, respectively, e.g., an immunoglobulin heavy chain constant domain and / or an immunoglobulin light chain constant domain, preferably of the IgG type. Additionally or alternatively, the heterologous portion of the antibody may be a mammalian secretory signal peptide. In other words, in one embodiment, the anti-Nogo-A antibodies and Nogo-A-binding fragments, synthetic and biotechnological derivatives of the invention are (i) polypeptide sequences heterologous to the VH and / or VL regions or fusion proteins comprising at least one CDR, and / or (ii) non-naturally occurring variants of polypeptides derived from immunoglobulins, comprising a heavy chain constant region comprising one or more amino acid deletions, substitutions and / or additions compared to the wild-type polypeptide.

[0052] As mentioned above, there are five immunoglobulin isotypes, of which immunoglobulin G (IgG) is the most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. These regions are responsible for binding to both the IgG-Fc receptor (FcgR) and C1q. As a result, different subclasses have distinct effector functions, both in triggering FcgR-expressing cells to undergo phagocytosis or antibody-dependent cell-mediated cytotoxicity and in activating complement. The Fc region also contains a binding epitope for the neonatal Fc receptor (FcRn), which is involved in the extended half-life, placental transport, and bidirectional transport of IgG across mucosal surfaces. However, FcRn is also expressed on myeloid cells and, together with classical FcgR and complement, is involved in both phagocytosis and antigen presentation. These antibody properties, such as glycosylation, IgG polymorphism, and post-translational modifications, affect IgG function as described in Vidarsson et al. (2014) IgG subclasses and allotypes: from structure to effector function. Front. Immunol. 5:520. 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 are of the IgG type, most preferably the IgG4 class or isotype. Human immunoglobulin G isotype 4 (IgG4) antibodies are promising candidates for antibody therapy when reduced immune effector function is desired.

[0053] In one embodiment of the antibody of the present invention, the Fc portion can be mutated to reduce immune effector function using techniques known in the art. For example, deletion or inactivation of the constant region domain (by point mutation or other means) can reduce Fc receptor binding to transepithelial transporters at the blood-brain barrier of the modified antibody applied to the cerebrospinal fluid / CNS compartment, thereby increasing its Nogo-A protein binding. In other cases, constant region modifications in accordance with the present invention can inhibit complement binding, thereby reducing the serum half-life and nonspecific association of the conjugated cytotoxin. Still other modifications of 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. The resulting physiological profile, bioavailability, and other biochemical effects of the modifications, such as Nogo-A protein binding and neutralization, biodistribution, and serum half-life, can be easily measured and quantified using well-known immunological techniques without undue experimentation. Recombinant human IgG antibodies (hIgG) that completely lack binding to Fcγ receptors (FcγR) and the complement protein C1q, and therefore lack immune effector functions, are useful for various therapeutic applications. The combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) was shown to eliminate FcγRIIa binding and detectable binding to FcγRI, IIa, and IIIa for both IgG1 and IgG4, and the LALA-PG mutation was found to be an improvement over the LALA mutation alone in that it abolished Fc function in both mouse and human IgG. For corresponding reports, see, for example, Saunders (2019) Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life. Front. Immunol. 10:1296. doi:10.3389 / fimmu.2019.01296 and Schlothauer et al., Protein Engineering, Design and Selection 29 (2016), 457-466.

[0054] IgG4 antibodies are dynamic molecules that can undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent bispecific antibodies (bsAbs) with unknown specificity, thus potentially reducing therapeutic efficacy. As illustrated in the Examples, in certain preferred embodiments, the antibodies of the present invention are IgG4 class or -mutation specific. The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro, as demonstrated using a novel quantitative immunoassay in combination with physiological matrix preparations (see Silva et al., J. Biol. Chem. 290 (2015), 5462-5469). As confirmed in Example 12, NG004 IgG4 S228P indeed exhibits reduced reactivity to C1q and behaves similarly to other IgG4 antibodies, such as natalizumab.

[0055] It is a known problem in the art that repeated freeze-thaw cycles can denature antibodies, potentially leading to the formation of aggregates that reduce their binding capacity (freeze-thaw damage) (see, e.g., Abcam's Antibody Storage Guide). Such antibody degradation is particularly detrimental to therapeutic antibodies, as aggregation or degradation can lead not only to reduced antibody activity but also to immunogenic responses (Ishikawa et al., Biol. Pharm. Bull. 33 (2010), 1413-1417). In contrast, the antibodies of the present invention are particularly stable. As demonstrated by size exclusion chromatography (SEC), exposure of the antibodies to repeated freeze-thaw cycles does not result in aggregation or degradation after 20 freeze-thaw cycles (see Example 10 and Figure 9B). Furthermore, it can be shown that exposure of the antibodies of the present invention to different pH values ​​from pH 6 to 8 does not affect the integrity of the antibodies as determined by SEC (see Example 10 and Figure 9A). Without being bound by theory, it is believed that the variable regions, particularly the CDRs and the VH and VL, respectively, confer the necessary integrity and stability to the antibody molecule, since previous observations have shown that the constant domains themselves are not, or are not solely, responsible for stability and / or suitability for formulation at concentrations applicable to administration to human subjects. Thus, the antibodies of the present invention preferably comprise at least the CDRs according to any of the above definitions, preferably according to Kabat, and most preferably substantially the entire amino acid sequence of the VH and VL as shown in Figure 1A or Figure 1B, respectively, but may allow for about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% variation, especially when considering conservative amino acid substitutions.

[0056] The present invention also relates to one or more polynucleotides encoding the VH and VL of the antibody or antigen-binding fragment thereof or immunoglobulin thereof of the invention; preferably the polynucleotide is cDNA.

[0057] In a preferred embodiment of the invention, the polynucleotide is a V-type polynucleotide of an anti-Nogo-A antibody shown in Table II. H Chain or V LThe polynucleotides may comprise, consist essentially of, or consist of nucleic acids having polynucleotide sequences encoding the light and / or heavy chains. In this regard, those skilled in the art will readily appreciate that the polynucleotides encoding the light and / or heavy chains may be encoded by one or more polynucleotides. Thus, in one embodiment, the polynucleotides may be encoded by one or more polynucleotides, such as the V and V sequences of the anti-Nogo-A antibodies shown in Table II. H Chain and V L A strand of nucleic acid comprising, consisting essentially of, or consisting of a nucleic acid having the polynucleotide sequence of the strand.

[0058] [Table 2]

[0059] In one embodiment of the invention, the polynucleotide is linked to heterologous nucleic acid, e.g., expression control sequences such as a promoter, transcriptional and / or translational enhancer sequences, an internal ribosome binding site, a nucleic acid encoding a peptide leader sequence for recombinant expression in a host, etc. Thus, the present invention relates to a polynucleotide encoding a recombinant anti-Nogo-A antibody of human origin, or a Nogo-A-binding fragment, synthetic derivative or biotechnological derivative thereof, which polynucleotide is (i) a VH chain comprising CDR1, CDR2 and CDR3 as defined by Kabat, and / or a VL chain comprising VL CDR1, VL CDR2 and VL CDR3, wherein: (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) a VH chain and / or a VL chain, (a) the VH chain comprises the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) VL comprises the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; wherein preferably the VH chain amino acid sequence and the VL chain amino acid sequence are at least 90% identical to SEQ ID NO: 2 and SEQ ID NO: 7, respectively; or (iii) a VH chain comprising CDR1, CDR2, and CDR3 as defined by Kabat, and / or a VL chain comprising VL CDR1, VL CDR2, and VL CDR3, wherein: (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (iv) a VH chain and / or a VL chain, wherein: (a) the VH chain comprises the amino acid sequence set forth in SEQ ID NO: 12 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) VL comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; Preferably, the VH chain amino acid sequence and the VL chain amino acid sequence are at least 90% identical to SEQ ID NO: 12 and SEQ ID NO: 17, respectively. A polynucleotide encoding

[0060] Furthermore, the present invention relates to a polynucleotide linked to a heterologous nucleic acid, said polynucleotide comprising: (a) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, wherein the polynucleotide binds to Nogo-A when the VH is paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:7; (b) a polynucleotide encoding an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively, wherein the polynucleotide binds to Nogo-A when the VL is paired with a VH comprising the amino acid sequence set forth in SEQ ID NO:2; (c) a polynucleotide comprising: (i) an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and (ii) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; a polynucleotide encoding (d) a polynucleotide encoding an immunoglobulin heavy chain or fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the VH binds to Nogo-A when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 7; (e) a polynucleotide encoding an immunoglobulin light chain or fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 7, wherein the polynucleotide binds to Nogo-A when the VL is paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 2; (f) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH having the amino acid sequence shown in SEQ ID NO: 2, and an immunoglobulin light chain or a fragment thereof comprising a VL having the amino acid sequence shown in SEQ ID NO: 7; (g) any one of the polynucleotides (a) to (f), wherein the CDRs contain one or more, preferably two or less, amino acid substitutions, and / or the variable region sequence is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 7; The polynucleotide is selected from the group consisting of:

[0061] Alternatively, the present invention relates to a polynucleotide linked to a heterologous nucleic acid, said polynucleotide comprising: (a) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, wherein the polynucleotide binds to Nogo-A when the VH is paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 17; (b) a polynucleotide encoding an immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, wherein the VL binds to Nogo-A when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 12; (c) a polynucleotide comprising: (i) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; (ii) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; a polynucleotide encoding (d) a polynucleotide encoding an immunoglobulin heavy chain or fragment thereof comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 12, wherein the VH binds to Nogo-A when paired with a VL comprising the amino acid sequence set forth in SEQ ID NO: 17; (e) a polynucleotide encoding an immunoglobulin light chain or fragment thereof comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 17, wherein the polynucleotide binds to Nogo-A when the VL is paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 12; (f) a polynucleotide encoding an immunoglobulin heavy chain or a fragment thereof comprising a VH having the amino acid sequence set forth in SEQ ID NO: 12, and an immunoglobulin light chain or a fragment thereof comprising a VL having the amino acid sequence set forth in SEQ ID NO: 17; (g) A polynucleotide selected from any one of (a) to (f), wherein the CDRs contain one or more, preferably two or less, amino acid substitutions, and / or the variable region sequence is at least 90% identical to SEQ ID NO: 12 or SEQ ID NO: 17. The polynucleotide is selected from the group consisting of:

[0062] Furthermore, the present invention relates to a vector or vectors comprising one or more of these polynucleotides, preferably wherein the vector is an expression vector and the one or more polynucleotides are operably linked to an expression control sequence.

[0063] The polynucleotides can be modified using methods known in the art, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc., to create antibodies with different amino acid sequences, e.g., to provide amino acid substitutions, deletions, and / or insertions (see, e.g., Molecular Cloning: A Laboratory Manual (4th Edition): Three-volume set; Green and Sambrook (2012) ISBN 10:1936113422 / ISBN 13:9781936113422 Cold Spring Harbor Laboratory Press; latest edition (2014) ISBN 978-1-936113-42-2, and Ausubel et al., eds., Current Protocols in ... in Molecular Biology, John Wiley & Sons, NY (1998) and its latest editions, which are incorporated herein by reference in their entireties), can be produced and manipulated as needed.

[0064] Once a polynucleotide encoding an antibody molecule of the invention, or an antibody heavy or light chain, or a portion thereof (preferably including a heavy or light chain variable domain), is obtained, a vector for producing the antibody molecule can be generated by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the invention provides replicable vectors containing a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may contain nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., WO 86 / 05807 and WO 89 / 01036, and U.S. Pat. No. 5,122,464), and the variable domain of an antibody may be cloned into such a vector for expression of the entire heavy or light chain.

[0065] The terms "vector" or "expression vector" are used herein to refer to vectors used in accordance with the present invention as vehicles for introducing and expressing desired genes in host cells. As known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present invention contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells. Markers may provide prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by cotransformation. Additional elements may be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, and transcriptional promoters, enhancers, and termination signals. For the expression of double-chain antibodies, a single vector encoding both the heavy and light chains or multiple vectors can be coexpressed in the host cell for expression of the entire immunoglobulin molecule, as described in more detail below.

[0066] Host cells can be co-transfected with two expression vectors of the present invention: a first vector encoding heavy chain-derived polypeptides and a second vector encoding light chain-derived polypeptides. The two vectors can contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. In such situations, the light chain is advantageously placed before the heavy chain to avoid an excess of non-toxic heavy chains (see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad. Sci. USA 77 (1980), 2197). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vectors are transfected into host cells by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce antibodies for use in the methods described herein. Accordingly, the present invention also relates to host cells containing one or more polynucleotides of the present invention or one or more vectors of the present invention.

[0067] As used herein, "host cells" refers to cells constructed using recombinant DNA technology and harboring a vector encoding at least one heterologous gene. In describing the process for isolating antibodies from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the antibody, unless clearly specified otherwise. In other words, recovery of polypeptides from "cells" can mean either recovery from spun-down whole cells or recovery from the cell culture containing both the medium and suspended cells.

[0068] 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, the recent development of glycosylation-engineered yeast, insect cell lines, and transgenic plants holds promise for obtaining antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments, including aglycosylated bispecific antibodies, have been successfully produced in bacteria and are progressing into clinical trials. We can expect the first therapeutic antibody products derived from non-mammalian sources within the next few years. For a description of current antibody production systems that can be applied to prepare the human-derived recombinant anti-Nogo-A antibodies or their Nogo-A-binding fragments, synthetic derivatives, or biotechnological derivatives of the present invention, including their utility for various applications, see Frenzel et al. al., Front Immunol. 2013;4:217, published online on July 29, 2013 doi:10.3389 / fimmu.2013.00217, and transient expression of human antibodies in mammalian cells is described in Vazquez-Lombardi et al., Nature protocols 13(2018),99-117; and Hunter et al., Optimization of protein expression in mammalian cells. Current Protocols in Protein Science 95(2019),e77.doi:10.1002 / cpps.77.

[0069] Once the antibody molecules of the invention have been recombinantly expressed, whole antibodies of the invention, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures in the art, for example by chromatography (e.g., ion exchange chromatography, affinity chromatography for specific antigens, in particular Protein A-based affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, e.g., ammonium sulfate precipitation, or any other standard technique for protein purification (see, e.g., 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). Thus, the invention also relates to a method for preparing anti-Nogo-A antibodies and / or fragments thereof or immunoglobulin chains thereof, comprising: (a) culturing a host cell as defined herein above, comprising a polynucleotide or vector as defined herein above, under conditions allowing expression of an anti-Nogo-A antibody, a Nogo-A binding fragment or an immunoglobulin chain thereof; and (b) isolating anti-Nogo-A antibodies, Nogo-A binding fragments or immunoglobulin chains thereof from the culture.

[0070] Furthermore, the present invention also relates to anti-Nogo-A antibodies, Nogo-A binding fragments and immunoglobulin chains thereof, which are encoded by the polynucleotides defined herein above and / or which are obtainable by the recombinant production methods described above.

[0071] In certain embodiments, an antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with antibodies. Exemplary variations are described in more detail below. For example, antibodies or Nogo-A binding fragments thereof, such as the single chain Fv antibody fragments of the invention, may comprise a free linker sequence or may be modified to attach a functional moiety or detectable label (e.g., PEG, a drug, a toxin, or a label, e.g., fluorescent, chemiluminescent, radioactive, enzymatic, nuclear magnetic, heavy metal, tag, flag, etc.) (see, for example, Antibodies A Laboratory Manual 2nd edition, 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA for general techniques, Dean and Palmer, Nat. Chem. Biol. 10 (2014), 512-523 for advances in fluorescent labeling strategies for dynamic cellular imaging, and Falck and Mueller, Antibodies 7 (2018), 4; doi:10.3390 / antib7010004 for enzyme-based labeling strategies for antibody-drug conjugates and antibody mimetics).

[0072] Antibody polypeptides of the present invention can comprise, consist essentially of, or consist of a fusion protein. A fusion protein is, for example, a chimeric molecule comprising an immunoglobulin Nogo-A binding domain with at least one target binding site and at least one heterologous portion, i.e., a portion not naturally linked in nature. The amino acid sequences may be found in separate proteins that are normally combined in the fusion polypeptide, or they may be found in the same protein but arranged in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0073] The term "heterologous," when applied to a polynucleotide or polypeptide, means that the polynucleotide or polypeptide is derived from a different entity than the other entity to which it is being compared. For example, as used herein, a "heterologous polypeptide" fused to an antibody, or antigen-binding fragment, variant, or analog thereof, is derived from a non-immunoglobulin polypeptide of the same species or an immunoglobulin or non-immunoglobulin polypeptide of a different species.

[0074] Recombinant human-derived anti-Nogo-A antibodies or Nogo-A binding fragments, synthetic or biotechnological derivatives thereof may be provided for a variety of applications, optionally as fusion proteins and / or labeled, as described herein above, and according to standard techniques known in the art (e.g., Antibodies A Laboratory Manual 2nd edition,2014 by (See Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.) Current advances in the design, production, 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 tailored functions.

[0075] The present invention relates to a composition comprising the above-mentioned Nogo-A binding molecule of the invention, such as an antibody or a Nogo-A binding fragment, variant or biotechnological derivative thereof, or a polynucleotide, vector or cell of the invention as defined herein above. In one embodiment, the composition of the invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

[0076] Polynucleotides and compositions of the present invention containing such polynucleotides can be used in therapeutic approaches. For example, the therapeutic use of nucleotide sequences encoding antibodies in DNA or mRNA form is summarized in Hoecke and Roose, J. Transl. Med. 17 (2019), 54. These nucleotide sequences can be directly administered to the subject to be treated, allowing the respective antibodies to be produced in situ. Furthermore, Schlake et al., Cellular and Molecular Life Sciences 76 (2019), 301-328, describe in vivo DNA-based antibody expression and corresponding plasmid and viral vectors, such as adeno-associated virus (AAV) and mRNA constructs prepared by in vitro transcription (IVT), for use in therapeutic approaches and passive immunotherapy. In general, RNA vaccination is a field with a wide range of applications, including cancer immunotherapy, neurodegenerative diseases, infectious diseases, tissue regeneration, and protein replacement therapy.

[0077] Thus, polynucleotides of the present invention, including RNA, can be used for intracellular translation as therapeutic agents. Thus, polynucleotides of the present invention, specifically RNA, can be used to produce antibodies of the present invention in target cells. Various approaches for producing suitable RNA are known to those skilled in the art and are commercially available (e.g., kits for in vitro transcription, RNA capping, and the generation of poly(A)-tailed mRNA for intracellular translation). WO 2008 / 083949A2 describes antibody-encoding unmodified and modified RNAs for the expression of corresponding antibodies, as well as transcription and antibody expression methods. WO 2009 / 127230 describes modified (m)RNAs suitable for suppressing and / or avoiding innate immune stimulatory responses. Furthermore, technology has been developed using CELLSCRIPT™ in which RNA contains pseudouridine (Ψ) and / or 5-methylcytidine (m5C) instead of the corresponding U or C canonical nucleosides. Such RNAs have been shown to be less immunogenic and are translated into proteins at much higher levels than corresponding mRNAs that do not contain modified nucleosides. Corresponding techniques are described, for example, in Kariko et al., Immunity 23 (2005), 165-175; Kariko et al., Molecular Therapy 16 (2008), 1833-1840; and Anderson et al., Nucleic Acids Res 38 (2010), 5884-5892. Furthermore, European Patent Application Publication No. 1 604 688 describes stabilized and translation-optimized mRNAs with increased G / C content and optimized codon usage. Further approaches for modifying RNA are described, for example, in Kormann et al., Nature Biotechnology 29 (2011), 154-157 and International Publication No. WO 2007 / 024708.

[0078] Thus, in one embodiment, a polynucleotide of the invention is RNA, which may be mRNA or a derivative thereof, either unmodified or modified as described above and suitable for translation into the corresponding antibody.

[0079] As mentioned above, the present invention relates to a vector comprising a polynucleotide of the present invention. In one embodiment, the vector is a gene transfer vector, such as an adeno-associated virus (AAV) vector. Therapeutic approaches for treating neurodegenerative diseases using AAV vectors are described, for example, in WO 2015 / 035190 and Lui et al., The Journal of Neuroscience 36 (2016), 12425-12435, both of which relate to AAV-vectored anti-tau antibodies. Such constructs can be used to deliver antibody-encoding genes directly to the brain, thus bypassing the blood-brain barrier. Furthermore, WO 2017 / 189963 generally describes novel AAV particles with viral genomes engineered to encode antibodies and antibody-based compositions, as well as methods of using these constructs (e.g., VADs) for the treatment, prevention, diagnosis, and study of diseases, disorders, and / or conditions. The progress and clinical application of AAV in neurodegenerative diseases of the central nervous system are reviewed in Qu et al., Neural Regen Res 14(2019), 931-938.

[0080] AAV vectors are widely used in gene therapy approaches due to several advantageous features. AAV is non-replicating in infected cells and therefore not associated with any known diseases. Furthermore, AAV can be introduced into a wide variety of host cells, does not integrate into the host cell genome, and can infect both quiescent and dividing cells. AAV transforms non-replicating and long-lived cells in vivo, resulting in long-term expression of a protein of interest. Furthermore, AAV can be engineered using cell biology and molecular biology techniques to generate non-toxic particles carrying a payload encoded in the AAV viral genome and deliver them to target tissues or cell sets with minimal or no side effects. Considering the above, the use of AAV to deliver vectored antibodies allows for longer-term efficacy, lower doses of treatment, and more consistent antibody levels throughout the treatment period.

[0081] AAV is a member of the Parvoviridae family and contains a linear, single-stranded DNA genome of less than approximately 5,000 nucleotides. For efficient replication, AAV requires co-infection with a helper virus (i.e., adenovirus or herpesvirus) or expression of helper genes. AAV vectors used to administer therapeutic nucleic acids typically lack approximately 96% of the parent genome, leaving only the interterminal repeats (ITRs), which contain recognition signals for DNA replication and packaging. This eliminates immunological or toxic side effects due to viral gene expression. Furthermore, delivery of specific AAV proteins to producer cells allows AAV vectors containing AAV ITRs to be integrated into specific regions of the cellular genome as needed (see, e.g., U.S. Pat. Nos. 6,342,390 and 6,821,511). Host cells containing the integrated AAV genome do not exhibit changes in cellular growth or morphology (see, e.g., U.S. Pat. No. 4,797,368). AAV vectors can be produced using any AAV serotype known in the art. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or human or non-human primate tissues (reviewed, for example, in Wu et al., Molecular Therapy 14(3), (2006), 316).

[0082] In addition to the nucleic acid sequence encoding the antibody or antigen-binding fragment thereof of the present invention, the AAV vector may include expression control sequences that provide for expression of the nucleic acid sequence in a host cell, such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA. (1990).

[0083] Thus, the present invention relates to a gene transfer vector comprising an isolated nucleic acid sequence encoding an antibody of the present invention. The gene transfer vector may be an adeno-associated virus (AAV) vector as described above.

[0084] The present invention also provides pharmaceutical and diagnostic compositions, respectively, in the form of a pack or kit comprising one or more containers containing one or more of the above-mentioned components, e.g., an anti-Nogo-A antibody, Nogo-A-binding fragment, biotechnological derivative or variant thereof, polynucleotide, vector, or cell of the present invention. Such containers may be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, indicating approval by the agency of the manufacture, use, or sale for human administration. Additionally or alternatively, the kit may include reagents and / or instructions for use in a suitable immune-based diagnostic assay. The compositions, e.g., kits, of the present invention are, of course, particularly suitable for risk assessment, diagnosis, prevention, and treatment of diseases or disorders associated with the presence of Nogo-A, and are particularly applicable to the treatment of disorders generally associated with Nogo-A, as discussed hereinabove.

[0085] 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 (See, e.g., "Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472"). Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. With regard to RNA-based compositions, WO 2020 / 089342, WO 2019 / 207060, and WO 2018 / 232355 describe lipid-based and polymer-based formulations, respectively, for efficient administration of RNA to subjects. Furthermore, encapsulation of RNA in neutral lipopolyplexes (LPPs) is described in Perche et al., Molecular Therapy: Nucleic Acids 17 (2019). Romani et al., Scientific Reports 7 (2017), 10863, also describes an approach for intravenous administration of RNA-lipoplexes. These pharmaceutical compositions can be administered to subjects at appropriate doses. Administration of suitable compositions can be achieved by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, intravitreal, topical, or intradermal administration, or by spinal or brain delivery. Aerosol formulations, such as nasal spray formulations, include purified aqueous or other solutions of the active agent along with preservatives and isotonicity agents. Such formulations are preferably adjusted to a pH and isotonicity compatible with the nasal mucosa.

[0086] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient will depend on many factors, such as the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly.

[0087] Due to its growth-restricting properties, Nogo-A may have adverse effects on nervous system injuries and diseases. Therefore, Nogo-A has been implicated in various CNS injuries and diseases, correlating with its various neurobiological roles. In principle, Nogo-A-related diseases are understood as nervous system diseases or trauma related to nerve and / or vascular repair. Nogo-A inhibition is thought to have beneficial effects in various diseases of the peripheral (PNS) and central (CNS) nervous systems, more specifically in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Lewy-like pathology or other dementias in general, traumatic brain injury, spinal cord injury, diseases after traumatic skull, brain, or spinal cord injury, stroke, or demyelinating diseases. Such demyelinating diseases include, but are not limited to, multiple sclerosis, monophasic demyelination, encephalomyelitis, multifocal leukoencephalopathy, panencephalitis, Marchiafava-Bignami disease, pontine myelinolysis, adrenoleukodystrophy, Pelizaeus-Merzbach disease, spongiform degeneration, Alexander disease, Canavan disease, metachromatic leukodystrophy, and Krabbe disease.

[0088] Furthermore, degenerative eye disorders may involve direct or indirect degeneration of retinal or corneal cells, such as general ischemic retinopathy, anterior ischemic optic neuropathy, all forms of optic neuritis, wet and atrophic age-related macular degeneration (AMD), diabetic retinopathy, diabetic macular edema, cystoid macular edema (CME), retinitis pigmentosa, Stargardt's disease, Best vitreoretinal degeneration, Leber's congenital amaurosis and other hereditary retinal degenerations, pathological myopia, retinopathy of prematurity, and Leber's hereditary optic neuropathy, sequelae of corneal transplants or refractive corneal surgery, and herpes simplex keratitis. Furthermore, Nogo-A has been found to play a role in psychiatric conditions, particularly schizophrenia and depression.

[0089] In vivo experiments confirmed that treatment with the antibodies of the invention resulted in better recovery of locomotor tasks requiring fine motor control in a mouse stroke model, as demonstrated by irregular horizontal ladder walking (see Example 11 and Figure 10).

[0090] The present invention therefore also relates to a method for treating a Nogo-A related disease or disorder, preferably a disease of the PNS or CNS, including those listed above, which method comprises administering to a subject in need thereof a therapeutically effective amount of any one of the above-mentioned Nogo-A binding molecules, antibodies, polynucleotides, vectors or cells of the invention. In principle, the anti-Nogo-A antibodies of the present invention are suitable for the treatment of the same diseases and disorders as disclosed in the references relating to conventional anti-Nogo-A antibodies cited herein in the "Background" section above.

[0091] In further embodiments, simultaneous or sequential administration of other agents useful for treating PNS or CNS diseases, disorders, or conditions associated with Nogo-A may be desirable. For example, the antibodies of the invention, or Nogo-A-binding fragments, variants, or biotechnological derivatives thereof, can be administered in combination with anti-inflammatory agents such as corticosteroids, neurotrophic factors such as nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), or other drugs for neurodegenerative diseases, such as Exelon™ (rivastigmine) or levodopa (L-DOPA (3,4-dihydroxy-L-phenylalanine)), for example, as a means of blocking neuronal damage and further inhibition of axonal regeneration after, but not limited to, stroke or spinal cord injury. Other suitable combination partners for the treatment of stroke are alteplase and desmoteplase (e.g., the DSPA disclosed in WO 90 / 09438). In one embodiment, the invention provides a combination comprising an antibody or Nogo-A binding fragment of the invention and desmoteplase, as well as a pharmaceutical composition comprising said combination, particularly for the treatment of stroke. As used herein, two agents are said to be administered in combination when they are administered simultaneously or independently in such a way that they act simultaneously.

[0092] The structures of active ingredients identified by code numbers, generic names or trade names can be obtained from the actual version of the standard compendium "Merck Index", or from databases such as the International Patents (e.g. IMS World Publications) provided by IMS Health or other databases.

[0093] In another example, cells expressing an antibody of the invention, or a Nogo-A-binding fragment, variant, or derivative thereof, can be transplanted into the site of spinal cord injury to promote axonal growth throughout the injury site. Such transplanted cells provide a means for restoring spinal cord function after injury or trauma. Such cells can include olfactory ensheathing cells and stem cells of different lineages from fetal nerve or tissue grafts.

[0094] Throughout the text of this specification, several documents are cited. The contents of all cited references (including literature references cited throughout this application, including the Background section, issued patents, published patent applications, and manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference, without any admission that the cited documents are in fact prior art with respect to this invention.

[0095] A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. [Example]

[0096] Example 1: Isolation and identification of anti-Nogo-A antibodies Neurimmune, originally described in WO 2008 / 081008 Utilizing AG's proprietary technology platform, Reverse Translational Medicine™ (RTM™) technology, human-derived antibodies targeting Nogo-A were identified that were modified, improved, and specifically adapted to target Nogo-A.

[0097] Example 2: Determination of antibody sequence and recombinant expression The amino acid sequences of the variable regions of the anti-Nogo-A antibodies identified above were determined based on their mRNA and cDNA sequences obtained from human memory B cells (see Figures 1A and 1B, respectively). Recombinant expression of fully human IgG1 antibodies with human or mouse constant domains was performed essentially as described in the Examples of WO 2008 / 081008, e.g., in the Methods section on pages 99 and 100.

[0098] Framework and complementarity determining regions were determined by comparison with reference antibody sequences available in databases such as Abysis (http: / / www.bioinf.org.uk / abysis / ) and annotated using the Kabat numbering scheme (http: / / www.bioinf.org.uk / abs / ).

[0099] Example 3: Binding Properties The neurite outgrowth inhibitor Nogo-A contains three inhibitory domains: two shared with the splice variant Nogo-B (Nogo-66 located between the two transmembrane domains and the N-terminal tip of the NIR domain) and one shared with the splice variant Nogo-C (Nogo-66). The unique domain highly inhibitory to neurite outgrowth in Nogo-A is located in exon 3 of Nogo-A and is called the delta 20 domain (d20; human amino acid positions 566–748) (Oertle et al., J. Neurosci. 23 (2003), 5393–5406). To confirm the binding properties of the antibodies NG004 and NG034 and to monitor cross-reactivity with other species, such as rats, we performed ELISAs using fragments containing the d20 domain and several additional amino acids at the C- and N-termini of the inhibitory domain (human amino acid positions 543–866). This fragment, called rat or human d20plus, was recombinantly produced in Escherichia coli. To determine whether the antibodies bind strongly to the d20plus region and to compare the binding characteristics of the subject antibodies NG004 and NG034 with those of previously known antibodies (11C7 and ozanzumab), ELISA was used and EC 50 The values ​​were compared.

[0100] ELISA was performed according to standard protocols (Engvall & Perlmann, J. Immunol. 109 (1972), 129-135; Engvall & Perlmann, Immunochemistry 8 (1971), 871-874). Briefly, ELISA plates were coated with 3 μg / ml of either rat- or human-derived d20plus, blocked with 5% milk powder (Lapilite, Migros), and probed with NG004. Each plate contained serial dilutions of 11C7 and / or ozanzumab as internal standards. Finally, the plates are incubated with the corresponding secondary antibodies (11C7 with anti-mouse HRP (Invitrogen, A16078), NG004 and ozanzumab with anti-human HRP (Sigma, A0170-1ML). The plates are developed with TMB substrate (ThermoFisher) and stopped with 1 M HCl. Readout is performed on a Tecan Sparc plate reader.

[0101] As shown in Figure 2A, NG004 inhibited the EC 50 Nogo-A binds to the human d20plus domain at physiological pH with high affinity in the low nM range of 0.26 nM. The EC50 values ​​for antibody 11C7 are 0.14 nM and for ozanzumab are 0.20 nM. The corresponding rat peptide d20plus binds only weakly to NG004 (Figure 2B). These data confirm that the delta20 domain is the active binding site within the Nogo-A protein.

[0102] As shown in Figures 2C and 2D, NG034 exhibited EC α-amyloid activity against both the human d20 plus region and the rat d20 plus region at physiological pH. 50 However, it binds with high affinity in the low nM range of 0.298 nM for the human form and 0.229 nM for the rat form.

[0103] Furthermore, NG004 and NG034 have been shown to positively stain rat corpus callosum oligodendrocytes (unfixed) (Figure 2E), rat corpus callosum spinal cord (fixed), and fixed human MO3.13, rat NS-1 cells, and oligodendrocytes and motor neurons in rat CNS tissue, with staining patterns similar to those of, for example, ozanzumab.

[0104] Example 4: Evaluation of the binding epitope of antibody NG004 Epitope mapping of NG004 was performed using overlapping peptide scanning. The sequence of the d20plus region of Nogo-A (amino acids 543-866 of human Nogo-A) was synthesized as linear 15-mer peptides with an 11-amino acid overlap between individual peptides. The peptides were spotted onto a nitrocellulose membrane (JPT Peptide Technologies, Berlin, Germany). The membrane was activated in methanol for 5 min and washed in TBS for 10 min at room temperature. Nonspecific binding sites were blocked with Roti®-Block (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for 2 h at room temperature. NG004 (1 μg / ml) was incubated in Roti®-Block for 3 h at room temperature. Primary antibody binding was examined using an HRP-conjugated donkey anti-human IgG secondary antibody. Blots were developed and evaluated using ECL and an ImageQuant350 detector (GE Healthcare, Otelfingen, Switzerland).

[0105] Antibody NG004 recognizes spots 34, 35, and 36 (Figure 3, white boxes), which correspond to the sequence 141-INAALQE-147 within the d20plus region of Nogo-A. Further alanine and truncation scans were performed to confirm the identified minimal epitope.

[0106] Example 5: Competition Assay The assay was based on the method presented by Kwak & Yoon, J. Immunol. Methods 191 (1996), 49-54. Specifically, the antigen human d20+ was coated, followed by blocking with 5% milk powder in TBS-0.1% Tween 20. After blocking, the competing antibodies 11C7 and ozanzumab were used to measure the EC of specific antibodies. 50 After washing, either the mouse IgG1 or human IgG4 isotype of NG004 or NG034 was added to the wells in serial dilutions starting at 30 μg / ml (200 nM). Three-fold serial dilutions were performed over 12 dilutions. If competition with a competing antibody occurs, binding of NG004 or NG034 is reduced, resulting in an EC 50 This is shown as a shift in the absorbance values ​​and / or a decrease in the absorbance values ​​of the high concentration plateau.

[0107] NG004 does not show competitive binding to Nogo-A with the antibodies ozanzumab (Figure 4A) and 11C7 (Figure 4B). NG034 does not show competitive binding to Nogo-A with the antibodies 11C7 and NG004 (Figure 4C).

[0108] Example 6: Target engagement in in vivo models Intact adult rats (Long Evans, Janvier) were administered antibodies intrathecally via an osmotic minipump (Alzet 2ML1 pump) at a pumping rate of 10 μl / h onto the lumbar spinal cord for 7 days. After this period, the animals were sacrificed, and their tissues were processed to assess the effects of the antibodies on biomarkers. Specifically, the animals were anesthetized and transcardially perfused with saline followed by 4% formalin. CNS tissue samples were then embedded in OCT mounting medium, frozen, and sectioned on a cryostat. The effects of injected antibodies NG004.m1, 11C7 (positive control), and isotype control anti-BrdU (AbD Serotec) on selected biomarkers, namely Nogo-A, Nogo-B, and NgR1, were examined.

[0109] Prism 7.0 (GraphPad Software Inc.) and R (R version 3.4.1) were used for statistical analysis. For statistical testing within groups over time, a standard one-way ANOVA was used, followed by Dunnett's multiple comparison test. For detecting differences between and within groups over time, and for comparing three or more groups over time, a two-way ANOVA with repeated measures was used, followed by Tukey's multiple comparison test. The significance threshold for all experiments was set at *P<0.05. Smaller P values ​​are represented as **P<0.01 and ***P<0.001. For bar graphs, all data are plotted as mean ± SEM (standard error of the mean). For box plot graphs, data are represented as median ± 25th percentile (box) and min / max (whiskers). In all graphs, dots represent individual animals.

[0110] Intrathecal treatment of rats with 2 mg and 4 mg of NG004 for 1 week, respectively, resulted in downregulation of endogenous Nogo-A protein levels in the CNS as assessed by immunofluorescence staining (Figure 5A). Antibody 11C7 was used as a positive control. In contrast, NG004 and 11C7 upregulate endogenous Nogo-B protein levels in the CNS (Figure 5B). The CA3 region of the hippocampus of rats infused with the anti-Nogo-A antibodies NG004 and 11C7 for 7 days showed significantly higher NgR1 fluorescence intensity compared to rats treated with the control anti-BrdU antibody. Thus, significant upregulation of NgR1 is observed (Figure 5C).

[0111] Example 7: LTP assay Neutralization of Nogo-A with the antibody 11C7 has been shown to significantly increase long-term synaptic plasticity (long-term potentiation, LTP) in the mouse hippocampus (Delekate et al., PNAS 108 (2011), 2569-2574). NG004 was analyzed for its ability to increase LTP according to published protocols (Delekate et al. (2011) supra).

[0112] As shown in Figure 6B, NG004 exhibits similar ex vivo activity as the positive control 11C7 (compare Figure 6A and Figure 6B). Furthermore, higher doses of NG004 (25 μg / ml) increase the magnitude and onset of action.

[0113] Example 8: In vitro neurite outgrowth assay To evaluate the biological activity of anti-Nogo-A antibodies, we performed a neurite outgrowth inhibition assay. Treatment of cultured neurons with crude cerebrospinal detergent extract (a Nogo-A-containing extract) inhibits neurite outgrowth. Previous studies have shown that this inhibitory activity can be neutralized by approximately 20% with specific antibodies against Nogo-A, such as 11C7, ATI355, or ozanzumab (Oertle et al. (2003), supra; Liebscher et al. (2005), supra; Weinmann et al., Mol. Cell Neurosci. 32 (2006), 161-173). The assay was performed according to the protocol established by Rubin et al., Europ. J. Neurosci. 7 (1995), 2524-2529. Neurite outgrowth of primary neurons or neuroblastoma cells was shown to be inhibited by rat spinal cord extract or non-human primate CNS extract (CNSE) (containing Nogo-A), and this inhibition was partially reversed / neutralized by functionally active anti-Nogo-A antibodies. The biological activities of NG004 and NG034 were tested in comparison with the positive control anti-Nogo-A antibodies 11C7 and ATI355.

[0114] The N1E-115 cell line was established in 1971 by T. Amano, E. Richelson, and M. Nirenberg by cloning the spontaneous mouse neuroblastoma tumor C-1300. N1E-115 cells are manufactured by the American Type The cells were supplied by the American College of Cardiovascular Medicine (ATCC) (Order Number: ATCC® CRL-2263). For differentiation, adherent N1E-115 cells were grown in 48-well plates in differentiation medium (Neurobasal® medium supplemented with 2% L-glutamine). The cells were harvested and resuspended in serum-free differentiation medium to a density of 2.2 × 10 4 Then, 450 μl of the cell suspension per well is seeded into a 48-well plate to obtain a final density of 1 × 10 cells / ml. 4 Cells were incubated at 37°C in a humidified incubator with 5% CO2 before the addition of inhibitory extracts and test antibodies. and incubated for 24 hours.

[0115] To ensure comparability of independent assays, the half maximal inhibition (HMI) of CNS extract was measured each time a new preparation of CNS extract was performed. 50 It was necessary to determine the half-maximal inhibition (HMI50) value. 50 The procedure for determining values ​​was as follows, using three wells per concentration. Increasing concentrations of CNS extract (5 μg / ml, 10 μg / ml, 12.5 μg / ml, 15 μg / ml, 20 μg / ml, 40 μg / ml) were added to N1E-115 cells premixed in PBS to a final volume of 50 μl per well. After 24 hours, cells were fixed and Coomassie stained for analysis. Coomassie-stained cells were imaged using a semi-automated IN Cell Analyzer 2500HS, acquiring eight 10x brightfield images of predefined locations in each well, four of which were scanned with HMI. 50 The HMI was analyzed to determine the value. 50Values ​​were estimated visually based on morphological criteria: for the solvent control (PBS), approximately 80% of N1E-115 cells exhibited moderately long neurites; for example, 12.5 μg / ml of CNS extract reduced the number of neurite-bearing cells to 60%, for example, 20 μg / ml of CNS extract reduced the number of neurite-bearing cells to 40%, and for example, 40 μg / ml of CNS extract reduced the number of neurite-bearing cells to nearly 0%. Based on these morphological criteria, HMI 50 A value can be defined as 50% of cells exhibiting neurite-bearing cell morphology compared to the solvent control condition. 50 The values ​​were constant in each experiment using the same source of CNS extract. When a fresh CNS extract was prepared, the HMI 50 had to be decided again.

[0116] N1E-115 cells were treated with CNS extracts and the tested antibodies (NG004.h4.m1 backbone human IgG4S228P and NG004.m1 backbone mouse IgG1) and incubated for 24 hours before fixation, Coomassie staining, and image acquisition. TIFF images were analyzed using the built-in grid and cell counter plugins in Fiji (ImageJ software). Pixels were expressed in μm based on the objective magnification. 2 Converted to Count Frame The grid is divided into two parts, one for each point size, with a constant area (21708.8 μm) between the lines of the counting frame grid. 2) were overlaid on the image. Cell bodies (Counter 1) were marked for each image using a computer mouse and counted using the Fiji Cell Counter software plugin. Similarly, intersections of neurites (processes longer than the cell body diameter) with grid lines (Counter 2) were marked. To accurately quantify neurite outgrowth, specific cutoffs were set: (a) if the cell body touched the outer edge of the image frame, it was not counted; (b) if the process was longer than the cell body diameter, it was considered a neurite; (c) intersections with the outer edge of the counting frame were not counted; and (d) dead cells were excluded from the count. Potentially dead cells were visually determined by their small circular morphology (Ronn et al., J. Neurosci. Methods 100 (2000), 25-32). The ratio of the resulting number of intersections to the number of cells was then calculated using the following formula: average neurite outgrowth per cell = total number of intersections / total number of cells.

[0117] For each experimental condition, three wells were replicated per experiment, and four images from each of three independent experiments were analyzed. Data plotting and statistical analysis were performed using GraphPad Prism 7.03 software. Data were statistically analyzed using post-hoc one-way ANOVA.

[0118] As shown in Figures 7A and 7B, treatment of differentiated, rat CNS extract-treated N1E-115 cells with antibody NG004 stimulates neurite outgrowth, i.e., reverses Nogo-A-induced inhibition of neurite outgrowth. The IC value of NG004 for this effect was 0.01. 50The values ​​were 11.16 nM and 19.34 nM for the positive control antibody 11C7. Furthermore, Figures 7C and 7D show that the anti-Nogo-A antibodies NG004 and NG034 exhibited similar functional activity as the internal reference antibody ATI355 for promoting neurite outgrowth in the presence of growth-inhibitory primate CNS extracts. The anti-Nogo-A antibodies NG004 and NG034 neutralized crude nonhuman primate CNS extract (containing Nogo-A)-mediated neurite outgrowth inhibition, demonstrating evidence of biological activity in a species phylogenetically close to humans. The isotype control antibody 3.1 (recombinant human anti-IAV HA antibody Fab fragment mAb 3.1, Wyrzuckia et al., J. Virol. 88 (2014), 7083-7092) was used as a negative control, and the anti-Nogo-A antibody ATI355 was used as a positive control.

[0119] Example 9: Angiogenesis in an in vivo mouse model of stroke In addition to inhibiting neurite outgrowth, Nogo-A has also been shown to act as a negative regulator of angiogenesis in the developing CNS (Waelchli et al., Proc Natl Acad Sci 110 (2013), E1943-52). Therefore, we investigated the potential of a monoclonal anti-Nogo-A antibody (NG004) to increase penumbra angiogenesis after stroke injury in a mouse model of stroke (Rust et al., PNAS 116 (2019), 14270-14279 and Watson et al., Ann. Neurol. 17 (1985), 497-504), comparing it with the established anti-Nogo-A antibody 11C7 or the control antibody FG12 / B5 (Muranova et al., Acta Crystallogr. D. Biol. Crystallogr. 60 (2004), 172-174). Antibodies were administered to adult stroke-affected mice for 14 days via osmotic minipumps implanted in the cerebral ventricles. After 21 days, the animals were sacrificed, and their tissues were processed to evaluate the effects of antibodies (NG004, 11C7, and FG12 / B5) on the vascular network within the penumbra, as well as on vascular area fraction, the number of vascular branches, vascular length and diameter, and intervascular distance after stroke. Adult female mice (10 weeks old) underwent photothrombotic stroke of the right motor cortex according to established protocols (Wahl et al., Science 50 (2014), 1250-1255, and Bachmann et al., J. Neurosci. 34 (2014), 3378-3389). To label proliferating vascular endothelial cells, mice were injected intraperitoneally with 5-ethynyl-2'-deoxyuridine (EdU, 50 mg / kg body weight, ThermoFisher) three times consecutively on days 6, 7, and 8 after stroke. EdU incorporation was detected in 40 μm free-floating coronal sections 21 days after stroke using the Click-iT EdU Alexa Fluor 647 Imaging Kit (ThermoFisher).For sustained CNS delivery, antibodies were loaded into an osmotic Alzet minipump model 1002 (0.25 μl / h pump flow rate, Alza Corporation, Palo Alto, CA, USA) with a 32 ga catheter (CR3218, ReCathCo, LLC, 2853-106 Oxford Boulevard, Allison Park, PA 15101) and administered into the contralesional cerebral lateral ventricle according to published protocols (Ineichen et al., Nature Protocols 12 (2017), 104-131). Each animal received one of the assigned osmotic pumps loaded with the IgG1 mouse monoclonal antibody 11C7 (positive control), FG12 / B5 (negative control), or IgG1 chimeric monoclonal antibody NG004. All antibodies were at a concentration of 7 mg / ml. To assess overall health, animals were weighed daily and interactive neuro-scores were performed according to a previously published protocol (Shelton et al., J. Neuros.). ci. Methods 168 (2008), 431-442). No statistical differences were observed between groups, but animals receiving NG004 tended to recover better during the first few days. After 21 days, animals were sacrificed and transcardially perfused to obtain brain sections according to standard protocols (Rust et al., Proc. Natl. Acad. Sci. USA 116 (2019), 14270-14279). To evaluate the effects of the injected antibodies NG004, 11C7, and FG12 / B5 on angiogenesis within the penumbra, various biologically relevant vascular parameters were evaluated, including vascular area fraction, vessel length, vessel branching, vessel distance, and distance variability. Furthermore, the formation of newly formed blood vessels was assessed by the incorporation of nucleotide analogs and the mitotic marker EdU into the nuclei of CD-31-positive vascular endothelial cells, determined after immunohistochemical staining with anti-CD31 antibody (rat, 1:50, BD Biosciences #550274).

[0120] Newly generated blood vessels were identified by quantifying the amount of CD31 / EdU double-positive cells within the ischemic border zone. Images were analyzed with ImageJ (FIJI). Images were converted to 8-bit format and manually thresholded using the Adaptive Threshold plugin to obtain binarized images. A median value of 0.5 pixels was applied to remove noise. Regions of interest (ROIs) were manually selected and analyzed for all parameters: 1) Area fraction: The percentage of pixels within the ROI that were highlighted and not zero. 2) Vessel length: Images were skeletonized and analyzed with the Skeleton length tool plugin—the lengths of all structures within the ROI were summed. 3) The number of branches was calculated as: The analysis was performed using the Analyze Skeleton tool. 4) The distance and variability of blood vessels were calculated using the NND tool, which calculates the minimum distance between single blood vessels. From these, the mean and standard deviation were calculated to obtain information on the average distance between blood vessels and the variability of their distribution within the ROI. Values ​​for blood vessel length and branch number were normalized to the total area of ​​the region of interest. Statistical analysis was performed using Prism 7.0 (GraphPad Software Inc.) and R (R version 3.4.1). For statistical testing within groups over time, a standard one-way ANOVA followed by Dunnett's multiple comparison test was used. For detecting differences between and within groups over time, and for comparing three or more groups over time, a two-way ANOVA with repeated measures followed by Tukey's multiple comparison test was used. Spearman's correlation was applied to analyze the correlation between behavioral recovery and out-sprouting fibers. In all experiments, the significance threshold was set at *P<0.05. Smaller P values ​​are represented as **P<0.01 and ***P<0.001. In bar graphs, all data are plotted as mean ± SEM (standard error of the mean). In box plot graphs, data are represented as median ± 25th percentile (box) and min / max (whiskers). In all graphs, dots represent individual animals.

[0121] As shown in Figure 8, post-stroke brain tissue from animals treated with NG004 and 11C7 showed a more highly developed vascular bed in the ischemic penumbra compared to controls 21 days after stroke, as evidenced by an increase in the total area fraction occupied by blood vessels (0.12 ± 0.03 for NG004, 0.115 ± 0.03 for 11C7, and 0.07 ± 0.01 for controls), and an increase in the area fraction occupied by blood vessels (1 mm 2 increased number of branches per mm (379.18 ± 96.62 in NG004, 349.4 ± 71.96 in 11C7, and 156.79 ± 31.82 in the control); 2 The difference in vascular length (mm) per ischemic zone was shown to be 21.90 ± 3.83 for NG004, 20.03 ± 1.69 for 11C7, and 12.89 ± 2.82 for controls (Figure 8A-8C). No differences were detected between 11C7 and NG004 in any of the vascular parameters. Importantly, there was no detectable difference in stroke size between all groups (data not shown). It was hypothesized that the more highly developed vascular bed in the ischemic border zone is generated through newly formed blood vessels. Therefore, the nucleotide analog EdU (50 mg / kg body weight) was injected systemically daily 6–8 days after injury, which is the peak time of angiogenesis. Newly formed vascular endothelial cells (CD31+) in the ischemic border zone were counted. 1 mm in both groups treated with anti-Nogo-A antibody (NG004: 55.20 ± 12.7, 11C7: 57.30 ± 19.57) compared with the control (28.51 ± 8.8). 2 An increase in the number of CD31 / EdU+ cells per 1000 μg / ml was observed (Figure 8D). In summary, both groups treated with anti-Nogo-A antibodies demonstrated indistinguishable enhancement of vascular repair compared with control antibody-treated animals 3 weeks after injury. We also found that the number of newly formed vascular endothelial cells increased to a similar extent in both NG004- and 11C7-treated animals. Thus, NG004 is a potent anti-Nogo-A antibody for vascular repair after stroke.

[0122] Example 10: Antibody Integrity and Stability To analyze the stability and integrity of the antibodies, size exclusion chromatography (SEC) was performed according to standard protocols (Porath & Flodin, Nature 183 (1959), 1657-1659). Briefly, the antibodies were dialyzed against different buffers. After dialysis, the antibodies were transferred to test tubes and incubated at 40°C and 4°C for up to 9 weeks. At weeks 1, 2, 4, and 9, samples were drawn and injected into a 100 μl loop (Amersham) of a Superdex™ 200 Increase column at a flow rate of 0.75 ml / min. Dulbecco's PBS, pH 7.4, was used as the running buffer.

[0123] As shown in Figures 9A and 9B, antibody NG004 is highly stable at different pH values ​​(pH 6, 7.4, 8) and in artificial CSF, as well as after repeated freeze-thaw cycles. Furthermore, no degradation or aggregation was observed, and affinity was maintained (data not shown).

[0124] Example 11: Functional recovery of locomotor tasks after treatment with NG004 The preclinical efficacy of the human monoclonal anti-Nogo-A antibody NG004 was evaluated in comparison with the established anti-Nogo-A antibody 11C7 or a control antibody ("anti-BrdU") for motor function recovery in a rat model of unilateral photothrombotic stroke, specifically for the functional recovery of skilled forelimb function after intrathecal application.

[0125] To address functional recovery, well-acclimated and well-handled young adult female Long-Evans rats were trained in a fine motor task (horizontal ladder test) and their baseline behavioral performance was recorded over three sessions. Subsequently, they underwent photothrombotic strokes directed at the sensorimotor cortex, and the stroke-affected rats received intrathecal administration of antibodies (11C7, NG004 (IgG1 chimeric monoclonal antibody NG004), and anti-BrdU) via an osmotic pump for 14 days. The animals' behavioral performance was recorded weekly after stroke induction (day 4 post-injury) and for up to 9 weeks (days 7, 14, 21, 28, 35, 42, 49, 56, and 63 post-injury).

[0126] Forty rats were divided into the following groups: 1. NG004_4mg: Ten rats were administered a cumulative dose of 4mg of NG004 for 14 days. 2. NG004_8mg: 10 rats were administered a cumulative dose of 8mg of NG004 for 14 days. 3. 11C7: 10 rats were administered a cumulative dose of 4 mg of 11C7 over 14 days (positive control). 4. Anti-BrdU: 10 rats were administered a mouse monoclonal antibody against BrdU at a cumulative dose of 4 mg over 14 days (negative control). Here, four animals had to be sacrificed (two animals from group 1 and one animal each from groups 2 and 4).

[0127] Animals were housed in individually ventilated cages (type IV) in groups of three with ad libitum access to food and water and a 12-h dark / light cycle. After arrival from a commercial supplier (Janvier.Labs, Le Genest-Saint-Isle, France), 40 female Long Evans rats (age: 12-16 weeks, weight: 200-250 g) were acclimated to the animal facility for one week. Experimenters then handled the animals according to standard procedures for one week prior to the start of the experiment to reduce stress levels.

[0128] The irregular horizontal ladder walking test is a motor and coordination test for assessing skilled walking by determining the clear placement of the forepaws on irregularly spaced ladder rungs (see Maier et al., J Neurosci. 28 (2008), 9386-403). The horizontal ladder walking test apparatus consists of transparent Plexiglas sidewalls and metal rungs (3 mm in diameter), which can be inserted to form a ladder path with a total length of 1 m and a minimum distance of 1 cm between rungs. The rungs are irregularly spaced with a maximum distance of 3 cm to allow the cerebral cortex to reassess foot placement. Baseline horizontal ladder performance was recorded for three consecutive days. Three runs were recorded for each session, and high-speed video recordings of all runs were analyzed for foot placement on the rungs.

[0129] All animals underwent unilateral phototrobotic strokes to lesion the sensorimotor cortex of their selected paw, as previously described in Lindau et al., Brain 137 (2014), 739-756, and Watson et al., Annals of Neurology 17 (1985), 497-504. The animals recovered adequately from the injury. Although the affected forelimb showed signs of paralysis, the animals were able to walk, climb, eat, and groom. For continuous CNS delivery, catheter-equipped osmotic pumps filled with the indicated antibodies were prepared and delivered into the lumbar cavity immediately after the phototrobotic stroke procedure. After 14 days, the pumps were removed, and behavioral testing began. For analysis of the horizontal ladder data, the success rate of correct forelimb / paw placement was calculated as a percentage of the total number of steps taken by the corresponding limb.

[0130] Statistical analysis was performed using Prism 7.0 (GraphPad Software Inc.). For statistical testing within groups over time, two-way ANOVA followed by the LSD (least significant difference) Fisher's test was used. To detect differences between groups at specific time points, an unpaired one-tailed t-test was used. Spearman correlation was applied for correlation analysis between behavioral recovery and CST cervical spinal cord sprouting. In all experiments, the significance threshold was set at *P<0.05. Smaller P values ​​are represented as **P<0.01 and ***P<0.001. In bar graphs, all data are plotted as the mean ± SEM (standard error of the mean). In all graphs, dots represent individual animals.

[0131] Animals treated with anti-Nogo-A showed improved functional recovery in the horizontal ladder task compared to animals treated with a control antibody.

[0132] At day 4 post-injury, all animals showed a comparable decline in success rate (to 34.43% ± 8.08% for anti-BrdU, 38.37% ± 4.87% for anti-Nogo-A-treated 11C7, 34.85% ± 6.12% for NG004 4 mg / ml, and 33.75% ± 5.25% for NG004 8 mg / ml). From day 14 onwards, the performance of animals treated with anti-Nogo-A antibody NG004 8 mg / ml and 11C7 steadily improved, reaching significance at day 63 post-injury when compared with animals treated with anti-BrdU (see Figure 10). Thus, the results indicated that treatment with anti-Nogo-A antibody NG004 resulted in better recovery of locomotor tasks requiring fine motor control, as indicated by irregular horizontal ladder crossing.

[0133] Example 12: Anti-Nogo-A antibody NG004 has reduced complement-dependent CDC A C1q-binding ELISA assay was performed to analyze the Fc characteristics of anti-Nogo-A antibodies for complement-dependent cytotoxicity (CDC) activity and measure the deposition of human C1q (Sigma C1740-5mg). C1q deposition was measured on 1 μg / ml antibody-coated polystyrene plates with eight different concentrations of C1q (1.2–20 μg / ml) in TBS-0.1% (v / v) Tween 20, 0.15 mM CaCl2, and 1 mM MgCl2 at 37°C for 1 h. The assay was evaluated by incubation. A sheep anti-human C1q polyclonal antibody (biorad2221-5004P) was used for detection. Two clinical antibodies with known mechanisms of action were used as controls: rituximab (human IgG1) was used as a positive control, and natalizumab (human IgG4) was used as a negative control. The internal positive control for NG004 IgG4 S228P was NG004, an IgG1 isotype. After 10 minutes, the colorimetric TMB reaction was stopped with 1 M HCl, and the OD at 450 nm was measured using a TECAN Spark plate reader.

[0134] NG004 IgG4 S228P anti-Nogo-A antibody and natalizumab showed reduced C1q binding activity compared to the IgG1 isotype (see Figure 11). These results indicate that NG004 IgG4 S228P behaves similarly to other IgG4s, with reduced CDC.

Claims

1. A recombinant monoclonal anti-Nogo-A antibody or antigen-binding fragment thereof comprising a variable heavy (VH) chain and a variable light (VL) chain, (a) the VH is VH complementarity-determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 13; a VH complementarity-determining region 2 (VH-CDR2) comprising the amino acid sequence of SEQ ID NO: 14; and a VH complementarity-determining region 3 (VH-CDR3) comprising the amino acid sequence of SEQ ID NO: 15; (b) the VL is VL complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 18; a VL complementarity-determining region 2 (VL-CDR2) comprising the amino acid sequence of SEQ ID NO: 19; and a VL complementarity-determining region 3 (VL-CDR3) comprising the amino acid sequence of SEQ ID NO: 20; An antibody or antigen-binding fragment thereof.

2. (a) the VH comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof, wherein the variant comprises one or more amino acid substitutions, and / or the VL comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof, wherein the variant comprises one or more amino acid substitutions, or (b) The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and / or the VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

17.

3. An antibody or its antigen-binding fragment described in claim 1 or 2, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 12, or the VL comprises the amino acid sequence shown in SEQ ID NO:

17.

4. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, wherein the VH comprises the amino acid sequence of SEQ ID NO: 12 and the VL comprises the amino acid sequence of SEQ ID NO:

17.

5. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 4, further comprising an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region, optionally wherein the immunoglobulin heavy chain constant region is of an IgG type, an IgG4 type, or an IgG4 type isotype.

6. An antibody or antigen-binding fragment thereof described in claim 5, wherein the immunoglobulin heavy chain constant region is of IgG4 type and contains an S228P mutation.

7. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 6, comprising a mutant Fc region having reduced effector function compared to the Fc region of wild-type IgG.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is selected from the group consisting of a single-chain Fv fragment (scFv), an F(ab') fragment, an F(ab) fragment, an F(ab') 2 fragment, Fd, Fv, a single-chain antibody, and a disulfide-linked Fv (sdFv).

9. An antibody or an antigen-binding fragment thereof described in any one of claims 1 to 8, wherein the antibody is a mouse-human chimeric antibody or a human-derived antibody. (i) the antibody or antigen-binding fragment thereof binds to the Nogo-A-Δ20 domain, which is the region between amino acid positions 543 and 866 of human Nogo-A, and is capable of inducing neurite outgrowth and / or angiogenesis in the stroke penumbra in a dose-dependent manner; and / or (ii) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof binds to a peptide comprising or consisting of the amino acid sequence of SEQ ID NO:

21.

11. One or more nucleic acids encoding an antibody or antigen-binding fragment thereof described in any one of claims 1 to 10.

12. One or more nucleic acids described in claim 11, each of which encodes a VH, a VL, or both.

13. A nucleic acid described in claim 11 or 12, wherein the one or more nucleic acids are cDNA and / or are operably linked to a heterologous nucleic acid.

14. One or more vectors comprising one or more nucleic acids according to any one of claims 11 to 13.

15. A host cell comprising one or more nucleic acids described in any one of claims 11 to 13 or one or more vectors described in claim 14.

16. A method for preparing an anti-Nogo-A antibody or antigen-binding fragment thereof, comprising: (a) culturing the cells of claim 15; and (b) isolating said antibody or antigen-binding fragment thereof from said culture.

17. An anti-Nogo-A antibody or an antigen-binding fragment thereof obtained by the method of claim 16.

18. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10 and 17, (i) detectably labeled with an enzyme, radioisotope, fluorescent compound, chemiluminescent compound, bioluminescent compound, tag, flag, or heavy metal; (ii) bound to a drug, or (iii) containing polyethylene glycol; An antibody or antigen-binding fragment thereof.

19. A composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, 17, and 18, one or more nucleic acids described in any one of claims 11 to 13, one or more vectors described in claim 14, or a host cell described in claim 15, optionally wherein the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier.

20. The composition described in claim 19, wherein the composition is a diagnostic composition and comprises reagents conventionally used in immune-based diagnostic methods.

21. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, 17, and 18, one or more nucleic acids described in any one of claims 11 to 13, one or more vectors described in claim 14, a host cell described in claim 15, or a composition described in claim 19 in the treatment of a disease or injury of the peripheral (PNS), central (CNS) nervous system, and / or retina, optionally including where the disease or injury is spinal cord injury, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Lewy-like pathology, dementia, cranial trauma, brain trauma, spinal cord trauma, stroke, traumatic brain injury, demyelinating disease, ophthalmic disease, diabetic retinopathy, diabetic macular edema, or wet and dry age-related macular degeneration (AMD).

22. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 10, 17, and 18, one or more nucleic acids described in any one of claims 11 to 13, one or more vectors described in claim 14, a host cell described in claim 15, or a composition described in claim 20 for in vivo detection of Nogo-A in a subject.

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