Anti-UNC5C antibody

Abstract

Antibodies and fragments thereof that bind to UNC5C are described. Compositions comprising these antibodies, as well as methods of treatment and therapeutic uses of the antibodies, are also described.

Description

[Technical Field]

[0001] The present invention relates to antibodies and fragments thereof capable of binding to UNC5C, particularly, but not exclusively, to novel therapeutic antibodies. Methods for using anti-UNC5C antibodies in the treatment of neurodegenerative diseases are also described. [Background technology]

[0002] The World Health Organisation (WHO) estimates that dementia is now the seventh leading cause of death among all diseases and one of the leading causes of disability and dependency among older people worldwide.

[0003] Alzheimer's disease (AD) is the most common cause of dementia, affecting 1 in 14 individuals over the age of 65 (Prince, M et al. (2014)). AD is a progressive neurodegenerative disorder accompanied by brain atrophy, leading to progressive cognitive impairment, including short-term memory difficulties, expressive speech, visuospatial processing, and executive (mental agility) processing deficits (Knopman, DS, et al. (2021)). New therapies for AD are actively sought to modify the disease course. Current candidates targeting beta-amyloid, tau, and innate immunity in the brain have, in some cases, demonstrated pharmacodynamic effects on pathological mechanisms in clinical trials, but have yet to demonstrate convincing disease modification in late-stage clinical trials to date (Golde, TE (2022)).

[0004] Frontotemporal dementia (FTD) is a common form of dementia in people under 65 years of age, and its prevalence is expected to increase as the population ages (Rosso SM, et al. 2003). FTD is an umbrella term for a group of neurodegenerative disorders characterized by progressive deficits in behavior, executive function, and language (Bang J, et al. 2015). Our understanding of the pathophysiology has improved considerably over the past decade, although development of disease-modifying drugs has been suggested to be ongoing for many years (Panza, F., et al. (2020)).

[0005] Both AD and FTD are part of a spectrum of dementias with related etiologies and pathologies that also include Parkinson's disease (PD), vascular dementia, and chronic traumatic encephalopathy (CTE).

[0006] Amyotrophic lateral sclerosis (ALS) is a subtype of motor neuron disease. It is a neurodegenerative disorder that primarily affects the motor neuron system, resulting in muscle weakness and ultimately paralysis (Hardiman, O., et al. (2017)). Currently, there is no effective treatment for ALS. ALS and FTD overlap clinically, radiologically, pathologically, and genetically, and 10–15% of ALS patients are also diagnosed with FTD (Phukan J, et al. Lancet Neurol. 2007;6(11):994–100). Due to the mechanistic overlap between FTD and ALS, both diseases are considered two extremes of a disease spectrum, with primarily cognitive symptoms at one end and motor neuron dysfunction at the other (Burrell JR et al. 2011).

[0007] There remains a need for improved disease-modifying therapies in neurodegenerative diseases such as AD, FTD, and ALS. Summary of the Invention [Means for solving the problem]

[0008] The present invention relates to novel antibodies against UNC5C. By studying the immune responses of individuals who exhibit resilience to neurodegeneration despite increased disease risk, the inventors identified a convergent heavy chain variable (VH) domain sequence present in multiple resilient individuals and therefore likely to have a protective function against neurodegeneration. This VH was paired with a light chain variable (VL) domain, and target deconvolution of the resulting antibody, ATL_5262, revealed UNC5C as its target. Further testing of this antibody showed that it competes with netrin-1, the ligand of UNC5C, for binding to UNC5C. The inventors further identified candidate antibodies derived from ATL_5262 by modifying the heavy chain sequence. These modifications were performed to further improve properties, including, but not limited to, reduced immunogenicity, improved stability, improved binding capacity, and improved pharmacokinetic properties. These novel antibodies with improved properties are referred to herein as ATL_0005262, ATL_0005998, ATL_0006001, ATL_0006002, ATL_0006003, ATL_0006036, ATL_0006039, ATL_0006177, ATL_0006178, ATL_0006187, AT Included are antibodies comprising the heavy chains of the antibodies designated herein as ATL_0006291, ATL_0006530, ATL_0006531, ATL_0006532, ATL_0006533, ATL_0006534, ATL_0006535, ATL_0006536, ATL_0006537, ATL_0006538, and ATL_0006539. The heavy chains of the antibodies designated herein as ATL_0005262, ATL_0006002, ATL_0006036, ATL_0006039, ATL_0006177, and ATL_0006178 have shown particular promise. The antibodies described herein are expected to bind to UNC5C and thereby ameliorate or reverse neurodegeneration in neurodegenerative diseases such as, for example, FTD, AD, and ALS.In particular, it is pertinent to note that the heavy chains and antibodies derived therefrom described herein were identified based on convergence in resilient individuals in a cohort of patients who would otherwise be at risk for developing a neurodegenerative disease. Because such convergence is highly unlikely to occur by chance, the antibodies are, to a degree, "biologically validated" as having potential therapeutic function.

[0009] In a first aspect, the present disclosure provides an isolated antibody or antibody fragment thereof that specifically binds to an UNC5C protein or a fragment thereof, wherein the antibody mimics and / or competes with netrin-1 for binding to UNC5C, and optionally, the competition with netrin-1 is determined by ELISA.

[0010] Embodiments of the first aspect may have any one or more of the following optional features.

[0011] The isolated antibody may bind to human and / or mouse UNC5C, for example, the antibody may bind to human UNC5C with an EC50 of at most 9.81E-08M, or at most 1E-08M, or at most 5.5E-09M, or at most 9.7E-09M, as assessed by ELISA (such as binding to plated rhUNC5C).

[0012] The antibody can reduce UNC5C-mediated apoptosis. For example, the antibody can reduce UNC5C-mediated neuronal apoptosis. The antibody can reduce (UNC5C-mediated) apoptosis in induced pluripotent stem cell (iPSC)-derived neurons. The antibody can reduce (UNC5C-mediated) apoptosis in neuroblastoma cells, such as SH-SY5Y cells. Apoptosis can be measured by measuring caspase 3 / 7.

[0013] The antibody may selectively bind to UNC5C over one or more other netrin receptors, for example, the antibody may selectively bind to UNC5C over neogenin, DCC, and one or more (or all) of DSCAM, UNC5A, UNC5B, UNC5. The antibody may bind to UNC5C according to SEQ ID NO: 99 and to the UNC5C T835M variant according to SEQ ID NO: 110.

[0014] The antibody may reduce beta-amyloid toxicity in neurons, for example, the antibody may reduce cell death of beta-amyloid-treated neurons in vitro and / or reduce caspase 3 / 7 signaling in response to beta-amyloid-induced cytotoxicity. The antibody may enhance synaptic health. Enhanced synaptic health may be assessed by (i) increased neuronal expression of one or more synaptic proteins selected from PSD95, synaptophysin, synapsin-1, synaptotagmin-1, neurofilament-L, and GAP-43, and / or (ii) increased neuronal growth and / or branching, or (iii) increased neuronal firing synchrony and / or firing rate; for example, the antibody may increase the synchrony index and firing rate of motor neurons carrying the TDP43 Q331K mutation.

[0015] The antibody may improve the function and / or survival of dopaminergic neurons. Improved dopaminergic neuron function and / or survival can be assessed by at least partial rescue by the antibody of the effects of UNC5C or netrin-1 haploinsufficiency on dopaminergic neuron innervation to the medial prefrontal cortex and / or dopamine content in the medial prefrontal cortex. The antibody may reduce neurodegeneration of dopaminergic neurons. This can be assessed by measuring neurodegeneration upon exposure to a neurotoxin such as 6-hydroxydopamine (6-OHDA).

[0016] The antibody can bind to the extracellular region of UNC5C. In particular, the antibody can bind to an epitope in the N-terminal immunoglobulin domain (also referred to as the "first immunoglobulin domain") of UNC5C. The antibody can bind to one or more residues of UNC5C involved in the binding of netrin-1 to UNC5C. The antibody may bind to an epitope in UNC5C that includes one or more or all of the following residues of UNC5C when numbered according to SEQ ID NO:99: Thr89, Gln90, Gln102, Lys103, Val107, Asp108, Glu109, Arg110, Val111, Ile118, Arg120, and optionally the antibody binds to residues Thr89, Gln90, Gln102, Lys103, Val107, Glu109, Ile118, and Arg120 of UNC5C when numbered according to SEQ ID NO:99.

[0017] The antibody may comprise a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20, CDRH2 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 23, or a set of CDRs that contain one or two amino acid mutations, optionally substitutions, compared to the above set of CDRs. Thus, the antibody may have a VH that comprises CDRH1, CDRH2, and CDRH3 or ATL_5262 or ATL6178, and variants thereof (e.g., ATL_6033, 6034, 6035, 6036, 6037, 6038, 6039).

[0018] The antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 51, HFWR3 of SEQ ID NO: 55, and HFWR4 of SEQ ID NO: 62, or framework sequences having one to five mutations, e.g., substitutions, compared to the above framework sequences. The mutations in the framework sequences may be selected from the following positions according to standard IMGT numbering: HFWR2: positions 40 and 49; HFWR3: positions 80, 82, and 86; and / or substitutions in the framework sequences may be selected from the following positions according to standard IMGT numbering: HFWR2: position 40: T40S, position 49: R49G; HFWR3: position 80: I80V, position 82: T82K, and position 86: H86Q. The mutations in the framework sequences may not include the mutation at position 67 according to standard IMGT numbering.

[0019] The antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 51, 52, 53, or 54, HFWR3 of SEQ ID NO: 55, 56, 57, 58, or 61, and HFWR4 of SEQ ID NO: 62. For example, the antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 52, HFWR3 of SEQ ID NO: 61, and HFWR4 of SEQ ID NO: 62.

[0020] The antibody may have a heavy chain variable domain (VH) comprising a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH) that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH). For example, the antibody has a heavy chain variable domain (VH) comprising a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 91 (ATL_6177 VH) and SEQ ID NO: 93 (ATL_6178 VH). The antibody may have a heavy chain variable domain (VH) comprising a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH) that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH). For example, the antibody has a heavy chain variable domain (VH) comprising a sequence that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 91 (ATL_6177 VH) and SEQ ID NO: 93 (ATL_6178 VH).

[0021] The antibody may have a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3 in a germline framework, except that position 67 is a Q in standard IMGT numbering.

[0022] The antibody may comprise a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 115 or 116, CDRH2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 119 or 120, optionally the antibody comprises a heavy chain variable domain (VH) having the CDRs of ATL_6187 VH (SEQ ID NOs: 115, 117 and 119) or the CDRs of ATL_6191 VH (SEQ ID NOs: 116, 118 and 120).

[0023] The antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 52 or 195, HFWR3 of SEQ ID NO: 196 or 197, or HFWR4 of SEQ ID NO: 198 or 62, or a framework sequence containing 1 to 5 mutations, such as substitutions, compared to these framework sequences. For example, the antibody may comprise a heavy chain variable domain (VH) having the HFWR of ATL_6187 VH (SEQ ID NOs: 50, 52, 196, 198) or the HFWR of ATL_6191 VH (SEQ ID NOs: 50, 195, 197, 62). The heavy chain variable domain (VH) may comprise a selected sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 112 (ATL_0006187 VH) and 113 (ATL_0006191 VH).

[0024] 1. An isolated antibody according to any of the preceding claims, wherein the antibody comprises a light chain variable domain (VL) having the following CDRs: CDRL1 comprising the amino acid sequence of SEQ ID NO: 24, 27, 30, 31 or 32, CDRL2 comprising the amino acid sequence of SEQ ID NO: 34, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 40, 43, 46, 47, or a set of CDRs comprising one, two or three amino acid mutations, e.g., substitutions, compared to the above sets of CDRs. Thus, the antibody may have a VL having any of the CDRs of ATL_5262, 6033, 6034, 6035, 6036, 6037, 6038, 6039, 6177, 6178, 5998, 6001, 6002, and 6003, and homologs 6187 and 6191.

[0025] The isolated antibody may have a light chain variable domain (VL) having the following framework sequences: LFWR1 of SEQ ID NOs: 63 and 66; LFWR2 of SEQ ID NOs: 71, 73, and 75; LFWR3 of SEQ ID NOs: 77 and 80; and LFWR4 of SEQ ID NOs: 85, 87, and 89; or a set of FWRs containing 1 to 5 amino acid substitutions compared to the above set of FWRs. Thus, the isolated antibody may have a VL having an LFWR of any of ATL_5262, 6033, 6034, 6035, 6036, 6037, 6038, 6039, 6177, 6178, 5998, 6001, 6002, and 6003, and homologs ATL6187 and 6191.

[0026] The antibody may have a light chain variable domain (VL) comprising a selected sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 9 (ATL_5262 VL), 12 (ATL_5998 VL), 15 (ATL_6001 VL), 16 (ATL_6002 VL), and 17 (ATL_6003 VL). For example, the antibody may have a light chain variable domain (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 9 (ATL_5262 VL), SEQ ID NO: 16 (ATL_6002 VL), and SEQ ID NO: 17 (ATL_6003 VL).

[0027] One or more (or all) of VH residues Tyr55, Gly63, Thr64, Thr65, Asn66, Ser74, Arg108, Met110 may interact with one or more residues of UNC5C, and / or one or more (or all) of VL residues Tyr38, Ser107, Tyr108, ​​Ser109, Thr114 may interact with one or more residues of UNC5C. For example, an antibody may comprise a VH having (i) Tyr at position 55, Gly at position 63, Thr at position 64, Thr at position 65, Asn at position 66, Ser at position 74, Arg at position 108, and Met at position 110, or (ii) a sequence with conservative amino acid substitutions at one or more of these positions. The antibody may comprise a VL having a sequence with conservative amino acid substitutions at positions 38: Tyr, 107: Ser, 108: Tyr, 109: Ser, 114: Thr, or one or more of these positions.

[0028] The antibody comprises a VH having at least 80% sequence identity with the VH of SEQ ID NO: 1 (ATL_5262 VH) and a VH of SEQ ID NO: 9 (ATL_5262and / or (b) VH residues 55, 66, 108, and 110 are Tyr, Asn, Arg, and Met, respectively, and optionally, Tyr55, Asn66, Arg108, and / or Met110 interact with Glu109 of UNC5C; and / or (c) VH residue 108 is Arg, and / or residue 110 is Met, and optionally, Arg g108 and / or Met110 interact with Ile118 of UNC5C, and / or (d) VH residues 55, 64, 65 are Tyr, Thr, and Thr, respectively, optionally, Tyr55, Thr64, and / or Thr65 interact with Arg120 of UNC5C, and / or (e) VH residue 64 is Thr, optionally, Thr64 interacts with Thr89 and / or Arg120 of UNC5C, and / or (f) VH residue 63 is Gly, optionally, Gly63 interacts with Gln90 of UNC5C. and / or (g) VH residue 65 is Thr, optionally wherein Thr65 interacts with Gln102 and / or Arg120 of UNC5C, and / or (h) VH residue 74 is Ser, optionally wherein Ser74 interacts with Lys103 of UNC5C, and / or (i) VL residue 114 is Thr, optionally wherein Thr114 interacts with Val107 and / or Glu109 and / or Arg110 of UNC5C, and / or (j) VL residue 109 is Ser, optionally wherein Ser10 and / or (k) VL residue 108 is Tyr, optionally where Tyr108 interacts with Arg110 and / or Val111 of UNC5C, and / or (l) VL residue 107 is Ser, optionally where Ser107 interacts with Val111 of UNC5C, and / or (m) VL residue 38 is Tyr, optionally where Tyr38 interacts with Val111 of UNC5C, wherein the UNC5C residues are numbered according to SEQ ID NO: 99.

[0029] The antibody may comprise a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequences of SEQ ID NOs: 142-149, CDRH2 comprising the amino acids of SEQ ID NOs: 150-158, and CDRH3 comprising the amino acids of SEQ ID NOs: 159-168. For example, the antibody may comprise a VH having the CDRs of any of antibodies ATL_6530 (SEQ ID NOs: 142, 150, 159); ATL_6531 (SEQ ID NOs: 143, 151, 160), ATL_6532 (SEQ ID NOs: 144, 152, 161), ATL_6533 (SEQ ID NOs: 145, 153, 162), ATL_6534 (SEQ ID NOs: 144, 152, 163), ATL_6535 (SEQ ID NOs: 143, 154, 164), ATL_6536 (SEQ ID NOs: 146, 155, 165), ATL_6537 (SEQ ID NOs: 147, 156, 166), ATL_6538 (SEQ ID NOs: 148, 157, 167), and ATL_6539 (SEQ ID NOs: 149, 158, 168).

[0030] The antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NOs: 199, 202, 205, 209, 212, 213, 216, 220, 224, 227; HFWR2 of SEQ ID NOs: 200, 203, 206, 210, 214, 217, 221, 225, 228; HFWR3 of SEQ ID NOs: 201, 204, 207, 211, 215, 218, 222, 226, 229; HFWR4 of SEQ ID NOs: 62, 208, 219, 223. For example, the antibodies include antibodies ATL_6530 (SEQ ID NOs: 199, 200, 201, 62); ATL_6531 (SEQ ID NOs: 202, 203, 204, 62); ATL_6532 (SEQ ID NOs: 205, 206, 207, 208); ATL_6533 (SEQ ID NOs: 209, 210, 211, 208); ATL_6534 (SEQ ID NOs: 212, 206, 207, 208); ATL_6535 (SEQ ID NOs: 213, 214, 215, 208); ATL_6536 (SEQ ID NOs: ATL_6537 (SEQ ID NOs: 220, 221, 222, 223); ATL_6538 (SEQ ID NOs: 224, 225, 226, 208); ATL_6539 (SEQ ID NOs: 227, 228, 229, 208), for example, a heavy chain variable domain (VH) having the CDR and / or framework sequence of ATL_6532 or ATL6533.

[0031] The antibody may have a heavy chain variable domain (VH) comprising a selected sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 122 (ATL_0006530 VH), 123 (ATL_0006531 VH), 124 (ATL_0006532 VH), 125 (ATL_0006533 VH), 126 (ATL_0006534 VH), 127 (ATL_0006535 VH), 128 (ATL_0006536 VH), 129 (ATL_0006537 VH), 130 (ATL_0006538 VH). For example, the antibody may have a heavy chain variable domain (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 124 (ATL_6532 VH), SEQ ID NO: 125 (ATL_6533 VH).

[0032] The antibody may comprise a light chain variable domain (VL) having the following CDRs: CDRL1 comprising the amino acid sequences of SEQ ID NOs: 169 to 176, CDRL2 comprising the amino acid sequences of SEQ ID NOs: 177 to 183, and CDRL3 comprising the amino acid sequences of SEQ ID NOs: 184 to 193. For example, the antibody may comprise a VL having the CDRs of any of antibodies ATL_6530 (SEQ ID NOs: 169, 177, 184), ATL_6531 (SEQ ID NOs: 170, 178, 185), ATL_6532 (SEQ ID NOs: 171, 177, 186), ATL_6533 (SEQ ID NOs: 169, 177, 187), ATL_6534 (SEQ ID NOs: 169, 177, 188), ATL_6535 (SEQ ID NOs: 172, 179, 189), ATL_6536 (SEQ ID NOs: 173, 180, 190), ATL_6537 (SEQ ID NOs: 174, 181, 191), ATL_6538 (SEQ ID NOs: 175, 182, 192), and ATL_6539 (SEQ ID NOs: 176, 183, 193).

[0033] The antibodies may further comprise a light chain variable domain (VL) having the following framework sequences: LFWR1 of SEQ ID NOs: 230-237 and 238, LFWR2 of SEQ ID NOs: 239-246, LFWR3 of SEQ ID NOs: 247-255, and LFWR4 of SEQ ID NOs: 256-258. For example, the antibodies may comprise antibodies ATL_6530 (SEQ ID NOs: 230, 238, 247, 256); ATL_6531 (SEQ ID NOs: 231, 239, 248, 257); ATL_6532 (SEQ ID NOs: 232, 240, 249, 258); ATL_6533 (SEQ ID NOs: 230, 241, 247, 256); ATL_6534 (SEQ ID NOs: 233, 238, 250, 256); ATL_65 ATL_6536 (sequence numbers 68, 243, 252, 257); ATL_6537 (sequence numbers 235, 244, 253, 256); ATL_6538 (sequence numbers 236, 245, 254, 256); ATL_6539 (sequence numbers 237, 246, 255, 256).

[0034] The light chain variable domain (VL) may comprise a sequence selected from SEQ ID NOs: 132-141 (ATL6530-6539 VL) having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity. For example, the antibody may have a light chain variable domain (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 134 (ATL_0006532 VH) or SEQ ID NO: 135 (ATL_0006532).

[0035] The antibody may comprise an scFv antibody molecule, a nanobody, an antibody constant region, or a whole antibody.

[0036] The antibody may be a monoclonal antibody, and / or the antibody is a whole antibody, and / or the antibody is an IgG1 or a variant thereof, optionally the antibody is the IgG1 variant L234A / L235A (LALA).

[0037] According to a second aspect, an isolated VH domain of an antibody according to any embodiment of the first aspect or any antibody described herein is also described. Accordingly, an antibody fragment according to any of the aforementioned aspects may comprise an isolated VH domain having the features of any embodiment of the aforementioned aspects. Also described herein are isolated antibody VL domains of the antibodies described herein. Accordingly, an antibody fragment according to any of the aforementioned aspects may comprise an isolated VL domain having the features of any embodiment of the antibody described herein.

[0038] According to a third aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding an antibody or antibody fragment thereof comprising a VH domain or a VL domain described herein.

[0039] According to a fourth aspect, the present disclosure provides a vector or set of vectors comprising a nucleic acid according to the previous aspect.

[0040] According to a fifth aspect, the present disclosure provides a host cell comprising a vector or set of vectors according to the sixth aspect, or a host cell transformed in vitro with nucleic acid of the fifth aspect.

[0041] According to a sixth aspect, the present disclosure provides a composition comprising an antibody or fragment thereof comprising an antibody VH domain or antibody VL domain described herein, and at least one additional component. The composition may comprise a pharmaceutically acceptable excipient, vehicle, or carrier.

[0042] According to a seventh aspect, the present disclosure provides a method of producing an antibody or fragment thereof, the method comprising culturing a host cell according to the fifth aspect under conditions for production of the antibody or fragment thereof. The method may further comprise isolating and / or purifying the antibody or fragment thereof. The method may further comprise formulating the antibody or fragment thereof into a composition comprising at least one additional component.

[0043] According to an eighth aspect, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of an isolated antibody or antibody fragment described herein.

[0044] According to a ninth aspect, the present disclosure provides a method for reducing apoptosis in a cell expressing UNC5C, the method comprising contacting the cell with an antibody or antibody fragment described herein. The cell may be a neuron. The contacting may be performed in vitro.

[0045] According to a tenth aspect, the present disclosure provides the use of an antibody or fragment thereof that binds to UNC5C in the manufacture of a medicament for the treatment of a neurodegenerative disease or disorder. The antibody or fragment thereof may be according to any embodiment described herein.

[0046] According to an eleventh aspect, the present disclosure provides a method for treating a neurodegenerative disease or disorder, the method comprising administering a therapeutically effective amount of an antibody or fragment thereof that binds to UNC5C to a patient having or at risk of developing the disease or disorder. The antibody or fragment thereof may be an antibody described herein.

[0047] According to a twelfth aspect, the present disclosure provides an antibody or fragment thereof that binds to UNC5C for use in treating a neurodegenerative disease or disorder. The antibody or fragment thereof may be an antibody or fragment thereof described herein. The neurodegenerative disorder according to any aspect, such as any of the twelfth, thirteenth or fourteenth aspects, is selected from the group consisting of frontotemporal dementia (FTD), Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), human immunodeficiency virus (HIV)-induced encephalitis, chronic traumatic encephalopathy (CTE), vascular dementia, prion diseases, Lewy body diseases, spinal muscular atrophy (SMA), motor neuron diseases (MND) such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar ataxias (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease, and rheumatoid arthritis. The neurodegenerative disease may be selected from FTD, AD, HD, and PD.

[0048] The present invention includes combinations of the described embodiments and preferred features except where such combinations are expressly not permitted or explicitly avoided.

[0049] Accordingly, an embodiment of the first aspect also describes an isolated antibody or antibody fragment thereof comprising a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20, CDRH2 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 23. The antibody may comprise a heavy chain variable domain (VH) having CDRs with one or two amino acid substitutions compared to the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20, CDRH2 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 23.

[0050] According to a further aspect, an isolated antibody or antibody fragment thereof that specifically binds to an UNC5C protein or a fragment thereof is described, wherein the antibody comprises a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20, CDRH2 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 23, or a set of CDRs that comprise one or two amino acid substitutions compared to the above set of CDRs. Thus, the antibody may have a VH comprising CDRH1, CDRH2, and CDRH3, or ATL_5262 or ATL6178, and variants thereof (e.g., ATL_6033, 6034, 6035, 6036, 6037, 6038, 6039). The antibody may mimic and / or compete with netrin-1 for binding to UNC5C. The antibody may have any of the characteristics described above in relation to the first aspect.

[0051] According to a further aspect, there is provided an isolated antibody or antibody fragment thereof that specifically binds to an UNC5C protein or a fragment thereof, the antibody comprising a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 115 or 116, CDRH2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 119 or 120. The antibody may comprise a heavy chain variable domain (VH) having the CDRs of ATL_6187 VH (SEQ ID NOs: 115, 117, and 119) or ATL_6191 VH (SEQ ID NOs: 116, 118, and 120). The antibody may mimic and / or compete with netrin-1 for binding to UNC5C. The antibody may have any of the characteristics described above in relation to the first aspect.

[0052] According to a further aspect, an isolated antibody or antibody fragment thereof that specifically binds to an UNC5C protein or a fragment thereof is described, wherein the antibody comprises a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequences of SEQ ID NOs: 142 to 149, CDRH2 comprising the amino acids of SEQ ID NOs: 150 to 158, and CDRH3 comprising the amino acids of SEQ ID NOs: 174 to 183. The antibody may comprise a VH having any of the CDRs of antibodies ATL_6530 (SEQ ID NOs: 142, 150, 159); ATL_6531 (SEQ ID NOs: 143, 151, 160); ATL_6532 (SEQ ID NOs: 144, 152, 161); ATL_6533 (SEQ ID NOs: 145, 153, 162); ATL_6534 (SEQ ID NOs: 144, 152, 163); ATL_6535 (SEQ ID NOs: 143, 154, 164); ATL_6536 (SEQ ID NOs: 146, 155, 165); ATL_6537 (SEQ ID NOs: 147, 156, 166); ATL_6538 (SEQ ID NOs: 148, 157, 167); ATL_6539 (SEQ ID NOs: 149, 158, 168). The antibody may mimic and / or compete with netrin-1 for binding to UNC5C. The antibody may have any of the characteristics described above in relation to the first aspect.

[0053] According to a third aspect, there is provided an isolated antibody or antibody fragment thereof that specifically binds to UNC5C protein or a fragment thereof for use in treating a neurodegenerative disorder. The antibody may mimic and / or compete with netrin-1 for binding to UNC5C. The antibody may comprise a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO:20, CDRH2 comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22, and CDRH3 comprising the amino acid sequence of SEQ ID NO:23. The antibody may comprise a heavy chain variable domain (VH) having CDRs with one or two amino acid substitutions compared to the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO:20, CDRH2 comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22, and CDRH3 comprising the amino acid sequence of SEQ ID NO:23. The antibody may comprise a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 115 or 116, CDRH2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 119 or 120. The antibody may comprise a heavy chain variable domain (VH) having the following CDRs: CDRH1 comprising the amino acid sequences of SEQ ID NOs: 142 to 149, CDRH2 comprising the amino acids of SEQ ID NOs: 150 to 158, and CDRH3 comprising the amino acids of SEQ ID NOs: 174 to 183.

[0054] Embodiments of any of the above aspects may have any one or more of the following optional features.

[0055] The antibody may bind to the same binding site on UNC5C as netrin-1. The antibody may have an epitope that at least partially overlaps with the binding site of netrin-1. The antibody of any embodiment may comprise a heavy chain variable domain (VH) having CDRs having at least 70%, 75%, 80%, 85%, or 90% sequence identity with the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20, CDRH2 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 23. The antibody may have a CDRH having the same length as those CDRHs. The antibody of any embodiment may comprise a heavy chain variable domain (VH) having the heavy chain CDRs of antibody ATL_5262, ATL_0006036, ATL_0006039, ATL_0006177, or ATL_0006178, or a set of CDRs having one, two, or three amino acid substitutions, or having at least 70%, 75%, 80%, 85%, or 90% sequence identity with these CDRs. The one or two substitutions may comprise or consist of the following residues within the CDRs: 58 of CDRH2, using standard IMGT numbering. The substitution may be N58Y. The antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 51, HFWR3 of SEQ ID NO: 55, and HFWR4 of SEQ ID NO: 62, or framework sequences having one to five substitutions compared to the above framework sequences. Substitutions in the framework sequences may be selected from the following positions according to the standard IMGT numbering: HFWR2: positions 40 and 49; HFWR3: positions 80, 82, and 86. Substitutions in the framework sequences may be selected from the following positions according to the standard IMGT numbering: HFWR2: position 40: T40S, position 49: R49G; HFWR3: position 80: I80V, position 82: T82K, and position 86: H86Q. Substitutions in the framework sequences may not include a substitution at position 67 according to the standard IMGT numbering. In other words, the Q at position 67 may be maintained. The present inventors have found that maintaining a Q at position 67 of HFWR3 improves binding affinity to UNC5C.The present inventors found that deleting the N at position 58 in the CDRH2 sequence can eliminate a potential sequence bias (a position that may be problematic during antibody production). The present inventors found that deleting the T at position 82 in HFWR3 reduces the risk of aspartic acid isomerization. Furthermore, replacing this T with a K improved the antibody's binding affinity to UNC5C. The present inventors found that substitutions at positions 40, 49 (in HFWR2), 80, and 86 (in HFWR3), particularly those with residues in the corresponding germline sequences, can reduce immunogenicity and improve antibody expression and safety. The antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NO: 50; HFWR2 of SEQ ID NO: 51, 52, 53, or 54; HFWR3 of SEQ ID NO: 55, 56, 57, 58, or 61; and HFWR4 of SEQ ID NO: 62. The antibody may have a heavy chain variable domain (VH) having the following framework sequences: HFWR1 of SEQ ID NO:50, HFWR2 of SEQ ID NO:52, HFWR3 of SEQ ID NO:61, and HFWR4 of SEQ ID NO:62.

[0056] The antibody may have a heavy chain variable domain (VH) comprising a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH) that has at least 95% sequence identity to a sequence selected from SEQ ID NO: 91 (ATL_6177 VH) and SEQ ID NO: 93 (ATL_6178 VH). ATL_6177 and ATL_6178 are expected to have improved stability and binding capacity compared to other antibodies described herein due to the particular sequences of the VHs of these antibodies (SEQ ID NOs: 91 and 93 in Table 1, or as set forth in Figure 22A). The ATL_5262 VH is as set forth in Table 1 or as set forth in Figure 21A. As further described below and in Figure 21A, the ATL_5262 VH has the same sequence as ATL_0005996_VH, ATL_0005997_VH, ATL_0005998_VH, ATL_0005999_VH, ATL_0006000_VH, ATL_0006001_VH, ATL_0006002_VH, ATL_0006003_VH, ATL_0006004_VH, and ATL_0006005_VH. The antibody may comprise a heavy chain variable domain having a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH). The antibody may have a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3 in a germline framework, with the proviso that position 67 is Q according to standard IMGT numbering.

[0057] The antibody may bind to human and / or mouse UNC5C. The antibody may bind to human UNC5C with an EC50 of at most 9.81E-08M, or at most 1E-08M, or at most 5.5E-09M, as assessed by ELISA (e.g., binding of plated rhUNC5C). The antibody may reduce UNC5C-mediated apoptosis. The antibody may reduce UNC5C-mediated neuronal apoptosis. The antibody may reduce apoptosis in induced pluripotent stem cell (iPSC)-derived neurons. The reduction in apoptosis may be verified in the caspase 3 / 7 assay described herein by comparison with a suitable control (e.g., vehicle only, a control antibody that does not bind to UNC5C, etc.). The antibody may affect synaptic health. For example, the antibody may increase synaptic plasticity. Thus, the antibody may increase one or more measures of synaptic health (e.g., including, but not limited to, plasticity), such as the number of processes per cell, the length and / or branching of neurite outgrowth.

[0058] Antibodies according to the present disclosure may improve neuronal survival and / or synaptic function. For example, antibodies of the present disclosure may reduce amyloid toxicity. Antibodies of the present disclosure may reduce cell death of amyloid-treated neurons in vitro. Cell death may be measured by measuring caspase 3 / 7 signaling in response to beta-amyloid-induced cytotoxicity. Antibodies of the present disclosure may increase the expression of one or more synaptic proteins, such as GAP-43. Antibodies of the present disclosure may at least partially rescue neuronal cell death and axonal damage caused by netrin-1 depletion. Antibodies of the present disclosure may improve neuronal connectivity and / or firing rate and / or synchrony in motor neurons. Antibodies of the present disclosure may improve neuronal connectivity and / or firing rate and / or synchrony in motor neurons of subjects with ALS. Antibodies of the present disclosure may improve the function and / or survival of dopaminergic neurons. For example, antibodies of the present disclosure may at least partially rescue the effects of UNC5C or netrin-1 haploinsufficiency on dopaminergic neuron innervation to the medial prefrontal cortex and / or dopamine content in the medial prefrontal cortex. This can be assessed by determining the increased response to amphetamine in netrin-1 haploinsufficient mice treated with antibodies of the present disclosure. Antibodies of the present disclosure may reduce neurodegeneration of dopaminergic neurons. This can be assessed by measuring neurodegeneration upon exposure to a neurotoxin such as 6-hydroxydopamine (6-OHDA), as evidenced by reduced contralateral limb use. Thus, also described herein are methods for improving the survival and / or function (including synaptic function) of motor neurons and / or dopaminergic neurons in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody of the present disclosure. Similarly, also described herein are methods for treating diseases or disorders associated with the dysfunction or death of motor neurons and / or dopaminergic neurons, comprising administering a therapeutically effective amount of an antibody of the present disclosure.

[0059] The antibody may selectively bind to UNC5C over one or more other netrin receptors. The antibody may selectively bind to UNC5C over one or more (or all) of neogenin, DCC, and DSCAM, UNC5A, UNC5B, and UNC5. Selective binding may refer to preferential binding (e.g., a lower EC50) of one species (e.g., UNC5C) over another species (e.g., other netrin receptors), or no detectable binding to a comparative target (e.g., other netrin receptors). Selective binding may be verified by biolayer interferometry analysis as described herein. The antibody may bind to monomeric UNC5C and / or dimeric UNC5C and / or homodimeric UNC5C. The antibody may bind to monomeric UNC5C.

[0060] The antibody may comprise an scFv antibody molecule, a nanobody, an antibody constant region, or a whole antibody. The antibody may be a whole antibody. The antibody may be an IgG1 or a variant thereof, optionally the antibody is the IgG1 variant L234A / L235A (LALA).

[0061] The antibody may further comprise a light chain variable domain (VL) having the following CDRs: CDRL1 comprising the amino acid sequence of SEQ ID NO: 24, 31 or 32, CDRL2 comprising the amino acid sequence of SEQ ID NO: 34, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 40 or 47, or a set of CDRs comprising one, two, or three amino acid substitutions compared to the above sets of CDRs. The antibody of any embodiment may comprise a light chain variable domain (VL) having CDRs having at least 70%, 75%, 80%, 85%, or 90% sequence identity with the following CDRs: CDRL1 comprising the amino acid sequence of SEQ ID NO: 24, 31 or 32, CDRL2 comprising the amino acid sequence of SEQ ID NO: 34, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 40 or 47. The antibody may have CDRLs having the same length as those CDRLs. The antibody of any embodiment may comprise a light chain variable domain (VL) having the light chain CDRs of antibody ATL_5262, ATL_0006002, or ATL_0006003, or a set of CDRs that have one, two, or three amino acid substitutions, or that have at least 70%, 75%, 80%, 85%, or 90% sequence identity with these CDRs. The antibody of any embodiment may comprise a light chain variable domain (VL) having the light chain CDRs of antibody ATL_5262 or ATL_0006002, or a set of CDRs that have one, two, or three amino acid substitutions, or that have at least 70%, 75%, 80%, 85%, or 90% sequence identity with these CDRs. CDRL1, CDRL2, and CDRL3 of the VL domain may be within a germline framework. The antibody may further comprise a light chain variable domain (VL) having the following framework regions: LFWR1 comprising any of the amino acid sequences of SEQ ID NOs: 63 to 70, LFWR2 comprising any of the amino acid sequences of SEQ ID NOs: 71 to 76, LFWR3 comprising any of the amino acid sequences of SEQ ID NOs: 77 to 84, and LFWR4 comprising any of the amino acid sequences of SEQ ID NOs: 85 to 90, or a set of FWRs comprising 1 to 5 amino acid substitutions compared to the above sets of FWRs.The antibody may have a light chain variable domain (VL) having the following framework sequences: LFWR1 of SEQ ID NO: 63, LFWR2 of SEQ ID NO: 71, LFWR3 of SEQ ID NO: 77, and LFWR4 of SEQ ID NO: 85 or 87, or a set of FWRs containing 1 to 5 amino acid substitutions compared to the above sets of FWRs.

[0062] The antibody may have a light chain variable domain (VL) comprising SEQ ID NO: 9 (ATL_5262 VL), or a selected sequence having at least 95% sequence identity to a sequence selected from 10 to 19. The antibody may have a light chain variable domain (VL) having the following framework sequences: LFWR1 of SEQ ID NO: 63, LFWR2 of SEQ ID NO: 71, LFWR3 of SEQ ID NO: 77, and LFWR4 of SEQ ID NO: 85 or 87, or a set of FWRs comprising 1 to 5 amino acid substitutions compared to the above set of FWRs. The antibody may have a light chain variable domain (VL) comprising a sequence having at least 95% sequence identity to a sequence selected from SEQ ID NO: 9 (ATL_5262 VL), 16 (ATL_6002 VL), and SEQ ID NO: 17 (ATL_6003 VL). [Brief explanation of the drawings]

[0063] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention are discussed below with reference to the accompanying drawings. [Figure 1] 1 illustrates a schematic of a process for unbiased antibody discovery. [Figure 2]Figure 1 shows the results of an ELISA assay for binding of antibodies identified in FTD-resilient individuals to recombinant UNC5C. The pool of antibodies tested consisted of 18 antibodies identified from the FTD cohort, one antibody from the Huntington's cohort, and an anti-PDL1 antibody previously used as a positive control in the Retrogenix target deconvolution assay. Antibody ATL_5262 binds to UNC5C. The blue bar shows the raw absorbance signal for recombinant UNC5C, and the orange bar represents the absorbance signal for an unrelated protein, lysozyme. The figure shows absorbance at 450 nm. The anti-Fc tag bar shows the results for anti-Fc-HRP-treated wells, which serve as a control for ensuring that UNC5C was properly coated onto the assay plate. [Figure 3] Figure 1 shows the presence of UNC5C antibodies in subjects resilient to neurodegeneration in different neurodegenerative disease cohorts. A. UNC5C antibodies were found in resilient centenarians (subjects over 100 years old without symptoms of neurodegeneration), Alzheimer's disease (AD) patients (resilient Alzheimer's disease subjects showing slow cognitive progression), frontotemporal dementia (FTD) patients (three subjects resilient to FTD—carriers of high-risk mutations and older, but without progression to FTD), Parkinson's disease (PD) patients (prodromal REM sleep behavior disorder (RBD) but without a diagnosis of Parkinson's disease), and cognitively healthy controls (mainly individuals over 60 years old). Repertoires were searched for VH sequences containing the same V and J genes as ATL5262 and containing at most two amino acid mismatches across the junction region. Dots represent the number of matching sequences per repertoire, and colors indicate the isotype of the identified sequences. The majority of matching sequences were class-switched to IgG1, supporting similar function for these antibodies across individuals. Unclassified PD subject SU_0001278 was initially included as a healthy control in this cohort but was subsequently excluded due to suspected REM sleep behavior disorder, a prodromal marker of PD. Because they do not have a PD diagnosis, they are "potentially resilient." [Figure 4A] Figure 1 shows the results of a netrin-1 competition assay. ELISA plates were coated with rhUNC5C (recombinant human UNC5C) and preincubated with His-tagged recombinant netrin-1, followed by the addition of ATL_5262 or control antibody ATL_5338. A. Netrin-1 was detected using an anti-His (horseradish peroxidase) HRP antibody. B. The antibody (ATL_5262 or control) was detected by anti-F(ab)'2-HRP detection. The assay was set up in duplicate, whereby one replicate had netrin-1 binding detected with anti-His-HRP (A), and a second replicate was used to determine ATL_0005262 antibody binding using anti-Fab'2-HRP detection. B. The figure shows the absorbance at 450 nm. [Figure 4B] Figure 1 shows the results of a netrin-1 competition assay. ELISA plates were coated with rhUNC5C (recombinant human UNC5C) and preincubated with His-tagged recombinant netrin-1, followed by the addition of ATL_5262 or control antibody ATL_5338. A. Netrin-1 was detected using an anti-His (horseradish peroxidase) HRP antibody. B. The antibody (ATL_5262 or control) was detected by anti-F(ab)'2-HRP detection. The assay was set up in duplicate, whereby one replicate had netrin-1 binding detected with anti-His-HRP (A), and a second replicate was used to determine ATL_0005262 antibody binding using anti-Fab'2-HRP detection. B. The figure shows the absorbance at 450 nm. [Figure 5]Figure 1 shows the results of a concentration-dependent netrin-1 competition assay. ELISA plates were coated with rhUNC5C and preincubated with a 12-point dilution series of His-tagged recombinant netrin-1 (starting at 3 nM) before adding ATL_5262. Netrin-1 was detected using anti-His HRP detection and is shown in the figure as "Netrin-1 signal (+ATL_5262)." ATL_5262 was detected using anti-F(ab)'2-HRP detection and is shown in the figure as "ATL_0005262 signal (+Netrin-1)." Control wells contained netrin-1 only. Data are expressed as a percentage of the maximum absorbance signal. [Figure 6] Figure 1 shows the results of a netrin-1 competition assay. ELISA plates were coated with rhUNC5C and preincubated with ATL_5262 or the control antibody ATL_5338, after which His-tagged recombinant netrin-1 was added. Netrin-1 was detected using anti-His HRP detection and is shown in the figure as "Netrin signal (+ATL_0005262)" or "Netrin signal (+ATL_00005338)." ATL_5262 was detected using anti-F(ab)'2-HRP detection. The figure shows the absorbance at 450 nm. [Figure 7A] Figure 1 shows the binding of ATL_5262 to recombinant human (rh) UNC5C (A), recombinant mouse (rm) UNC5C (B), and lysozyme (C) using ELISA. The figure shows the absorbance at 450 nm. [Figure 7B] Figure 1 shows the binding of ATL_5262 to recombinant human (rh) UNC5C (A), recombinant mouse (rm) UNC5C (B), and lysozyme (C) using ELISA. The figure shows the absorbance at 450 nm. [Figure 7C]Figure 1 shows the binding of ATL_5262 to recombinant human (rh) UNC5C (A), recombinant mouse (rm) UNC5C (B), and lysozyme (C) using ELISA. The figure shows the absorbance at 450 nm. [Figure 8A] (A) shows the VH alignment of ATL_5262 and ATL_5262 homologs identified across a neurodegeneration cohort in resilient individuals. The figure shows IMGT numbering. (A) shows the sequences of the antibodies described herein (in particular, ATL_0005262 VH, SEQ ID NO: 1; ATL_0006187 VH, SEQ ID NO: 112; ATL_0006191 VH, SEQ ID NO: 113), highlighting variable positions in the heavy chain. [Figure 8B-1] (B-1 to B-4) show the heavy chain sequences of the antibody in (A) and ATL_6178 (VH of ATL_6178, SEQ ID NO: 93), aligned by IMGT position numbering. ATL_5262 and ATL_6178 have the same VL. Homologues were identified based on VH alone, so only the VH sequences are shown. [Figure 8B-2] (B-1 to B-4) show the heavy chain sequences of the antibody in (A) and ATL_6178 (VH of ATL_6178, SEQ ID NO: 93), aligned by IMGT position numbering. ATL_5262 and ATL_6178 have the same VL. Homologues were identified based on VH alone, so only the VH sequences are shown. [Figure 8B-3] (B-1 to B-4) show the heavy chain sequences of the antibody in (A) and ATL_6178 (VH of ATL_6178, SEQ ID NO: 93), aligned by IMGT position numbering. ATL_5262 and ATL_6178 have the same VL. Homologues were identified based on VH alone, so only the VH sequences are shown. [Figure 8B-4](B-1 to B-4) show the heavy chain sequences of the antibody in (A) and ATL_6178 (VH of ATL_6178, SEQ ID NO: 93), aligned by IMGT position numbering. ATL_5262 and ATL_6178 have the same VL. Homologues were identified based on VH alone, so only the VH sequences are shown. [Figure 9] The workflow for the 2-week and 4-week stability studies and the freeze-thaw (3xFT) stability study for ATL_5262 is shown. Quality control (QC) analyses were performed using size exclusion high-performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), capillary isoelectric focusing (cIEF), dynamic light scattering (DLS), and binding ELISA. [Figure 10] Results of a thermal shift assay for ATL_5262 at 1 mg / ml (left) and 5 mg / ml (right) are shown (ratio of unfolded to folded protein as a function of temperature). [Figure 11A] Electropherograms of charge variants of ATL_5262 tested by cIEF after 2 weeks (A) and 4 weeks (B) of storage at the indicated temperatures (-80°C, 4°C, and 40°C). C. is an enlarged version of A and B, showing the acidic species on the left side of the graph. [Figure 11B]Electropherograms of charge variants of ATL_5262 tested by cIEF after 2 weeks (A) and 4 weeks (B) of storage at the indicated temperatures (-80°C, 4°C, and 40°C). C. is an enlarged version of A and B, showing the acidic species on the left side of the graph. [Figure 11C] Electropherograms of charge variants of ATL_5262 tested by cIEF after 2 weeks (A) and 4 weeks (B) of storage at the indicated temperatures (-80°C, 4°C, and 40°C). C. is an enlarged version of A and B, showing the acidic species on the left side of the graph. [Figure 12A] SEC-HPLC chromatograms for ATL_5262 are shown after 2 weeks (FIG. 11A) or 4 weeks (FIG. 11B) at the indicated temperatures (−80° C., 4° C., and 40° C.). [Figure 12B] SEC-HPLC chromatograms for ATL_5262 are shown after 2 weeks (FIG. 11A) or 4 weeks (FIG. 11B) at the indicated temperatures (−80° C., 4° C., and 40° C.). [Figure 13A] SDS-PAGE results for ATL_5262 are shown after 2 weeks (A) or 4 weeks (B) at the indicated temperatures (-80°C, 4°C, and 40°C) or after 3x FT studies (B). FT = Freeze Thaw, NIST = NIST mAb control, R = Reduced, NR = Non-Reduced. [Figure 13B] SDS-PAGE results for ATL_5262 are shown after 2 weeks (A) or 4 weeks (B) at the indicated temperatures (-80°C, 4°C, and 40°C) or after 3x FT studies (B). FT = Freeze Thaw, NIST = NIST mAb control, R = Reduced, NR = Non-Reduced. [Figure 14A] CE-SDS results of ATL_5262 after 2 weeks (A) non-reduced and (C) reduced samples) or after 4 weeks (B) non-reduced and (D) reduced samples) at the indicated temperatures (-80, +4, and +40 °C) are shown. [Figure 14B]CE-SDS results of ATL_5262 after 2 weeks (A) non-reduced and (C) reduced samples) or after 4 weeks (B) non-reduced and (D) reduced samples) at the indicated temperatures (-80, +4, and +40 °C) are shown. [Figure 14C] CE-SDS results of ATL_5262 after 2 weeks (A) non-reduced and (C) reduced samples) or after 4 weeks (B) non-reduced and (D) reduced samples) at the indicated temperatures (-80, +4, and +40 °C) are shown. [Figure 14D] CE-SDS results of ATL_5262 after 2 weeks (A) non-reduced and (C) reduced samples) or after 4 weeks (B) non-reduced and (D) reduced samples) at the indicated temperatures (-80, +4, and +40 °C) are shown. [Figure 15A] Shown are the results of an ELISA testing the binding of ATL_5262 to rhUNC5C after 2 weeks (A) or 4 weeks (B) at the indicated temperatures (-80°C, 4°C, and 40°C) or after 3xFT studies (B). [Figure 15B] Shown are the results of an ELISA testing the binding of ATL_5262 to rhUNC5C after 2 weeks (A) or 4 weeks (B) at the indicated temperatures (-80°C, 4°C, and 40°C) or after 3xFT studies (B). [Figure 16] Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to rhUNC5C. ATL_5338 was included as a negative control. The figure shows the absorbance at 450 nm. [Figure 17A] Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to rhUNC5C (A), rmUNC5C (B), and lysozyme (C). ATL_5338 was included as a negative control. ATL_6002 and ATL_6003 (VL-pairing variants) were included for comparison. The figure shows the absorbance at 450 nm. [Figure 17B]Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to rhUNC5C (A), rmUNC5C (B), and lysozyme (C). ATL_5338 was included as a negative control. ATL_6002 and ATL_6003 (VL-pairing variants) were included for comparison. The figure shows the absorbance at 450 nm. [Figure 17C] Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to rhUNC5C (A), rmUNC5C (B), and lysozyme (C). ATL_5338 was included as a negative control. ATL_6002 and ATL_6003 (VL-pairing variants) were included for comparison. The figure shows the absorbance at 450 nm. [Figure 18A] Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to Fc-tagged rhUNC5C (A) or His-tagged rhUNC5C (B). ATL_5338 was included as a negative control. ATL_6002 and ATL_6003 (VL-pairing variants) were included for comparison. The figure shows the absorbance at 450 nm. [Figure 18B] Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to Fc-tagged rhUNC5C (A) or His-tagged rhUNC5C (B). ATL_5338 was included as a negative control. ATL_6002 and ATL_6003 (VL-pairing variants) were included for comparison. The figure shows the absorbance at 450 nm. [Figure 19A] Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to rhUNC5C (A), rmUNC5C (B), and lysozyme (C). ATL_5338 was included as a negative control. The figure shows the absorbance at 450 nm. [Figure 19B] Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to rhUNC5C (A), rmUNC5C (B), and lysozyme (C). ATL_5338 was included as a negative control. The figure shows the absorbance at 450 nm. [Figure 19C]Figure 1 shows the results of an ELISA testing the binding of different VH variants of ATL_5262 to rhUNC5C (A), rmUNC5C (B), and lysozyme (C). ATL_5338 was included as a negative control. The figure shows the absorbance at 450 nm. [Figure 20A] 1 shows the results of an in vivo mouse pharmacokinetic study assessing antigen levels in serum (A) and CSF (B) after administration of increasing doses of ATL_5262 or control antibody ATL_5338. [Figure 20B] 1 shows the results of an in vivo mouse pharmacokinetic study assessing antigen levels in serum (A) and CSF (B) after administration of increasing doses of ATL_5262 or control antibody ATL_5338. [Figure 21A-1] The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. Heavy chains aligned by IMGT position numbering. VH of ATL_0005262, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005: sequence number 1, VH of ATL_0006033: sequence number 2, VH of ATL_6034: sequence number 3, VH of ATL_0006035: sequence number 4, VH of ATL_0006036: sequence number 5, VH of ATL_0006037: sequence number 6, VH of ATL_0006038: sequence number 7, VH of ATL_0006039: sequence number 8, VH of ATL_0006177: sequence number 91, VH of ATL_0006178: sequence number 93. [Figure 21A-2]The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. Heavy chains aligned by IMGT position numbering. VH of ATL_0005262, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005: sequence number 1, VH of ATL_0006033: sequence number 2, VH of ATL_6034: sequence number 3, VH of ATL_0006035: sequence number 4, VH of ATL_0006036: sequence number 5, VH of ATL_0006037: sequence number 6, VH of ATL_0006038: sequence number 7, VH of ATL_0006039: sequence number 8, VH of ATL_0006177: sequence number 91, VH of ATL_0006178: sequence number 93. [Figure 21A-3] The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. Heavy chains aligned by IMGT position numbering. VH of ATL_0005262, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005: sequence number 1, VH of ATL_0006033: sequence number 2, VH of ATL_6034: sequence number 3, VH of ATL_0006035: sequence number 4, VH of ATL_0006036: sequence number 5, VH of ATL_0006037: sequence number 6, VH of ATL_0006038: sequence number 7, VH of ATL_0006039: sequence number 8, VH of ATL_0006177: sequence number 91, VH of ATL_0006178: sequence number 93. [Figure 21A-4]The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. Heavy chains aligned by IMGT position numbering. VH of ATL_0005262, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005: sequence number 1, VH of ATL_0006033: sequence number 2, VH of ATL_6034: sequence number 3, VH of ATL_0006035: sequence number 4, VH of ATL_0006036: sequence number 5, VH of ATL_0006037: sequence number 6, VH of ATL_0006038: sequence number 7, VH of ATL_0006039: sequence number 8, VH of ATL_0006177: sequence number 91, VH of ATL_0006178: sequence number 93. [Figure 21A-5] The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. Heavy chains aligned by IMGT position numbering. VH of ATL_0005262, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005: sequence number 1, VH of ATL_0006033: sequence number 2, VH of ATL_6034: sequence number 3, VH of ATL_0006035: sequence number 4, VH of ATL_0006036: sequence number 5, VH of ATL_0006037: sequence number 6, VH of ATL_0006038: sequence number 7, VH of ATL_0006039: sequence number 8, VH of ATL_0006177: sequence number 91, VH of ATL_0006178: sequence number 93. [Figure 21B-1]The sequences of the antibodies described herein are shown with variable positions in the heavy chain highlighted. B. Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0005996: SEQ ID NO: 10, VL of ATL_0005997: SEQ ID NO: 11, VL of ATL_0005998: SEQ ID NO: 12, VL of ATL_0005999: SEQ ID NO: 13, VL of ATL_0006000: SEQ ID NO: 14, VL of ATL_0006001: SEQ ID NO: 15, ATL_000 VL of 6002: sequence number 16, VL of ATL_0006003: sequence number 17, VL of ATL_0006004: sequence number 18, VL of ATL_0006005: sequence number 19, VL of ATL_0006033, 6034, 6035, 6036, 6037, 6038, 6039, 6177: sequence number 92, VL of ATL_0006178: sequence number 94. [Figure 21B-2] The sequences of the antibodies described herein are shown with variable positions in the heavy chain highlighted. B. Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0005996: SEQ ID NO: 10, VL of ATL_0005997: SEQ ID NO: 11, VL of ATL_0005998: SEQ ID NO: 12, VL of ATL_0005999: SEQ ID NO: 13, VL of ATL_0006000: SEQ ID NO: 14, VL of ATL_0006001: SEQ ID NO: 15, ATL_000 VL of 6002: sequence number 16, VL of ATL_0006003: sequence number 17, VL of ATL_0006004: sequence number 18, VL of ATL_0006005: sequence number 19, VL of ATL_0006033, 6034, 6035, 6036, 6037, 6038, 6039, 6177: sequence number 92, VL of ATL_0006178: sequence number 94. [Figure 21B-3]The sequences of the antibodies described herein are shown with variable positions in the heavy chain highlighted. B. Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0005996: SEQ ID NO: 10, VL of ATL_0005997: SEQ ID NO: 11, VL of ATL_0005998: SEQ ID NO: 12, VL of ATL_0005999: SEQ ID NO: 13, VL of ATL_0006000: SEQ ID NO: 14, VL of ATL_0006001: SEQ ID NO: 15, ATL_000 VL of 6002: sequence number 16, VL of ATL_0006003: sequence number 17, VL of ATL_0006004: sequence number 18, VL of ATL_0006005: sequence number 19, VL of ATL_0006033, 6034, 6035, 6036, 6037, 6038, 6039, 6177: sequence number 92, VL of ATL_0006178: sequence number 94. [Figure 21B-4] The sequences of the antibodies described herein are shown with variable positions in the heavy chain highlighted. B. Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0005996: SEQ ID NO: 10, VL of ATL_0005997: SEQ ID NO: 11, VL of ATL_0005998: SEQ ID NO: 12, VL of ATL_0005999: SEQ ID NO: 13, VL of ATL_0006000: SEQ ID NO: 14, VL of ATL_0006001: SEQ ID NO: 15, ATL_000 VL of 6002: sequence number 16, VL of ATL_0006003: sequence number 17, VL of ATL_0006004: sequence number 18, VL of ATL_0006005: sequence number 19, VL of ATL_0006033, 6034, 6035, 6036, 6037, 6038, 6039, 6177: sequence number 92, VL of ATL_0006178: sequence number 94. [Figure 21B-5]The sequences of the antibodies described herein are shown with variable positions in the heavy chain highlighted. B. Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0005996: SEQ ID NO: 10, VL of ATL_0005997: SEQ ID NO: 11, VL of ATL_0005998: SEQ ID NO: 12, VL of ATL_0005999: SEQ ID NO: 13, VL of ATL_0006000: SEQ ID NO: 14, VL of ATL_0006001: SEQ ID NO: 15, ATL_000 VL of 6002: sequence number 16, VL of ATL_0006003: sequence number 17, VL of ATL_0006004: sequence number 18, VL of ATL_0006005: sequence number 19, VL of ATL_0006033, 6034, 6035, 6036, 6037, 6038, 6039, 6177: sequence number 92, VL of ATL_0006178: sequence number 94. [Figure 22] 1 shows caspase 3 / 7 production in response to beta-amyloid 1-42 toxicity in SHSY5Y cells in the conditions indicated. [Figure 23] Western blot results showing protein expression of the indicated synaptic proteins (neurofilament-L, synaptophysin, PSD95 synapsin-1, synaptotagmin-1) after treatment of human iPS-derived dopaminergic neurons with ATL6178 are shown. [Figure 24A]Results of a netrin-1 depletion assay using human iPSC-derived spinal motor neurons pretreated with ATLX1282 and ATL-5262 are shown. (A) Graph showing imaging results and quantification of live / dead cell assays. Data are expressed as the average of live cells over total cell counts (calcein AM-positive and ethidium homodimer-positive combined). One-way ANOVA with Tukey's multiple comparison test was performed using four randomly selected wells. *P<0.05, **P<0.01, ***P<0.001. Error bars represent the mean + / - SEM. (B) Fluorescence microscopy images showing staining for DAPI and the synaptic protein GAP43. (C) Fluorescence microscopy images showing neurite outgrowth in microfluidic cultures containing BrainXell eGFP spinal motor neurons, and quantification of the average total number of axons (nerve fibers) (=total fiber counts). *P<0.05, **P<0.01 compared with isotype control and anti-netrin-1 groups, two-tailed unpaired t-test, n=3–4 independent units. [Figure 24B] Results of a netrin-1 depletion assay using human iPSC-derived spinal motor neurons pretreated with ATLX1282 and ATL-5262 are shown. (A) Graph showing imaging results and quantification of live / dead cell assays. Data are expressed as the average of live cells over total cell counts (calcein AM-positive and ethidium homodimer-positive combined). One-way ANOVA with Tukey's multiple comparison test was performed using four randomly selected wells. *P<0.05, **P<0.01, ***P<0.001. Error bars represent the mean + / - SEM. (B) Fluorescence microscopy images showing staining for DAPI and the synaptic protein GAP43. (C) Fluorescence microscopy images showing neurite outgrowth in microfluidic cultures containing BrainXell eGFP spinal motor neurons, and quantification of the average total number of axons (nerve fibers) (=total fiber counts). *P<0.05, **P<0.01 compared with isotype control and anti-netrin-1 groups, two-tailed unpaired t-test, n=3–4 independent units. [Figure 24C]Results of a netrin-1 depletion assay using human iPSC-derived spinal motor neurons pretreated with ATLX1282 and ATL-5262 are shown. (A) Graph showing imaging results and quantification of live / dead cell assays. Data are expressed as the average of live cells over total cell counts (calcein AM-positive and ethidium homodimer-positive combined). One-way ANOVA with Tukey's multiple comparison test was performed using four randomly selected wells. *P<0.05, **P<0.01, ***P<0.001. Error bars represent the mean + / - SEM. (B) Fluorescence microscopy images showing staining for DAPI and the synaptic protein GAP43. (C) Fluorescence microscopy images showing neurite outgrowth in microfluidic cultures containing BrainXell eGFP spinal motor neurons, and quantification of the average total number of axons (nerve fibers) (=total fiber counts). *P<0.05, **P<0.01 compared with isotype control and anti-netrin-1 groups, two-tailed unpaired t-test, n=3–4 independent units. [Figure 25A] Results of a multielectrode assay (MEA) using the ALS TDP43 iCell motor neuron cell line treated with ATLX-1282 are shown. (A) Graph showing the number of active electrodes in the indicated groups. (B) Graph showing the burst percentage in the indicated experimental groups. (C) Graph showing the mean resistance (left) and number of coated electrodes (right) as a measure of viability. (D) Graph showing the synchrony index (top) and mean firing rate (bottom). Three-way ANOVA (mixed-effects model) with post-hoc Tukey's for multiple comparisons. Error bars represent SEM. n=4-6 wells / treatment group. [Figure 25B]Results of a multielectrode assay (MEA) using the ALS TDP43 iCell motor neuron cell line treated with ATLX-1282 are shown. (A) Graph showing the number of active electrodes in the indicated groups. (B) Graph showing the burst percentage in the indicated experimental groups. (C) Graph showing the mean resistance (left) and number of coated electrodes (right) as a measure of viability. (D) Graph showing the synchrony index (top) and mean firing rate (bottom). Three-way ANOVA (mixed-effects model) with post-hoc Tukey's for multiple comparisons. Error bars represent SEM. n=4-6 wells / treatment group. [Figure 25C] Results of a multielectrode assay (MEA) using the ALS TDP43 iCell motor neuron cell line treated with ATLX-1282 are shown. (A) Graph showing the number of active electrodes in the indicated groups. (B) Graph showing the burst percentage in the indicated experimental groups. (C) Graph showing the mean resistance (left) and number of coated electrodes (right) as a measure of viability. (D) Graph showing the synchrony index (top) and mean firing rate (bottom). Three-way ANOVA (mixed-effects model) with post-hoc Tukey's for multiple comparisons. Error bars represent SEM. n=4-6 wells / treatment group. [Figure 25D] Results of a multielectrode assay (MEA) using the ALS TDP43 iCell motor neuron cell line treated with ATLX-1282 are shown. (A) Graph showing the number of active electrodes in the indicated groups. (B) Graph showing the burst percentage in the indicated experimental groups. (C) Graph showing the mean resistance (left) and number of coated electrodes (right) as a measure of viability. (D) Graph showing the synchrony index (top) and mean firing rate (bottom). Three-way ANOVA (mixed-effects model) with post-hoc Tukey's for multiple comparisons. Error bars represent SEM. n=4-6 wells / treatment group. [Figure 26]Figure 1 shows ATLX-1282-mediated rescue of AMPH-induced locomotion in netrin-1 haploinsufficient mice. The data show the results of a locomotion assay in netrin-1 haploinsufficient mice, demonstrating ATLX-1282-mediated rescue of amphetamine (AMPH)-induced locomotion. The graph shows the distance traveled by mice in the indicated groups 10 days after antibody or control (anti-fluorescein antibody, ATL5338 (LALA)) treatment. Saline was used as a control for AMPH treatment, and wild-type mice were used as a control for netrin-1 haploinsufficient mice. ****P<0.0001 One-way ANOVA with Sidak's multiple comparison test. [Figure 27] The percentage of contralateral contacts shown by rats 26 days after 6-OHDA or saline treatment is shown. Mice were administered isotype control (ATL5338) or ATLX1282 intraperitoneally once a week. ***P<0.001 One-way ANOVA with Sidak's multiple comparison test. [Figure 28] This figure shows the results of a biolayer interferometry assay to assess the binding of ATL_5262 and ATL_6178 to different netrin-1 receptors and related proteins. For each aligned antigen-antibody pair, the graph shows the reference-corrected sensor response (average 25-30 s signal) at the end of the measured association step of the BLI kinetic assay. The antigen was loaded as the ligand and exposed to the antibody as the analyte during association and to assay buffer during dissociation. [Figure 29A]This figure shows the results of a netrin-1 competition assay demonstrating that the binding of ATLX-1282 and ATL_5262 to mouse brain is blocked via netrin-1. (A) The graph shows the effect of netrin-1 in competing with ATL_5262 in tissues with high UNC5C expression ("high expression area" = mouse brain tissue) or control tissues with no or low UNC5C expression ("liver control" = mouse liver tissue). (B) Fluorescent and transmitted light tissue section images (20x) showing the binding of ATL_5262 to mouse brain or control liver tissue under various blocking conditions (no netrin block, netrin-1 block, clusterin block (control)). (C) The effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive mouse brain sections. (D) Effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive (i.e., untreated) mouse brain sections or liver control tissue ("Liver Control"). (E) Representative fluorescence and transmitted light tissue section images showing the effect of blocking ATLX-1282 binding netrin-4 and netrin-1 (resulting in reduced staining) in naive mouse brain sections. [Figure 29B]This figure shows the results of a netrin-1 competition assay demonstrating that the binding of ATLX-1282 and ATL_5262 to mouse brain is blocked via netrin-1. (A) The graph shows the effect of netrin-1 in competing with ATL_5262 in tissues with high UNC5C expression ("high expression area" = mouse brain tissue) or control tissues with no or low UNC5C expression ("liver control" = mouse liver tissue). (B) Fluorescent and transmitted light tissue section images (20x) showing the binding of ATL_5262 to mouse brain or control liver tissue under various blocking conditions (no netrin block, netrin-1 block, clusterin block (control)). (C) The effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive mouse brain sections. (D) Effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive (i.e., untreated) mouse brain sections or liver control tissue ("liver control"). (E) Representative fluorescence and transmitted light tissue section images showing the effect of blocking ATLX-1282 binding to netrin-4 and netrin-1 (resulting in reduced staining) in naive mouse brain sections. [Figure 29C]This figure shows the results of a netrin-1 competition assay demonstrating that the binding of ATLX-1282 and ATL_5262 to mouse brain is blocked via netrin-1. (A) The graph shows the effect of netrin-1 in competing with ATL_5262 in tissues with high UNC5C expression ("high expression area" = mouse brain tissue) or control tissues with no or low UNC5C expression ("liver control" = mouse liver tissue). (B) Fluorescent and transmitted light tissue section images (20x) showing the binding of ATL_5262 to mouse brain or control liver tissue under various blocking conditions (no netrin block, netrin-1 block, clusterin block (control)). (C) The effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive mouse brain sections. (D) Effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive (i.e., untreated) mouse brain sections or liver control tissue ("Liver Control"). (E) Representative fluorescence and transmitted light tissue section images showing the effect of blocking ATLX-1282 binding netrin-4 and netrin-1 (resulting in reduced staining) in naive mouse brain sections. [Figure 29D]This figure shows the results of a netrin-1 competition assay demonstrating that the binding of ATLX-1282 and ATL_5262 to mouse brain is blocked via netrin-1. (A) The graph shows the effect of netrin-1 in competing with ATL_5262 in tissues with high UNC5C expression ("high expression area" = mouse brain tissue) or control tissues with no or low UNC5C expression ("liver control" = mouse liver tissue). (B) Fluorescent and transmitted light tissue section images (20x) showing the binding of ATL_5262 to mouse brain or control liver tissue under various blocking conditions (no netrin block, netrin-1 block, clusterin block (control)). (C) The effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive mouse brain sections. (D) Effect of netrin-1 and netrin-4 blocking in competition with ATLX-1282 in naive (i.e., untreated) mouse brain sections or liver control tissue ("Liver Control"). (E) Representative fluorescence and transmitted light tissue section images showing the effect of blocking ATLX-1282 binding netrin-4 and netrin-1 (resulting in reduced staining) in naive mouse brain sections. [Figure 30A] Figure 1 shows the results of a flow cytometry assay demonstrating the binding of ATL5262 and ATLX-1282 to UNC5C and the UNC5C T835M variant (AD variant) overexpressed in HEK-293 cells. (A) Quantification of UNC5C binding as measured by % of IgG+ cells (antibody detected). [Figure 30B] (B) Flow cytometry assay results showing binding of ATL5262 and ATLX-1282 to UNC5C and the UNC5C T835M variant (AD variant) overexpressed in HEK-293 cells. (C) Quantification of UNC5C binding as measured by IgG mean fluorescence intensity (MFI). [Figure 31A] Figure 1 shows the X-ray crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain. (A) A schematic diagram of the complete Fab-UNC5C complex. [Figure 31B]Figure 1 shows the X-ray crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain. (B)-(F) show magnified views of the specific interaction between ATL_5262 and UNC5C shown in (A). [Figure 31C] Figure 1 shows the X-ray crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain. (B)-(F) show magnified views of the specific interaction between ATL_5262 and UNC5C shown in (A). [Figure 31D] Figure 1 shows the X-ray crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain. (B)-(F) show magnified views of the specific interaction between ATL_5262 and UNC5C shown in (A). [Figure 31E] Figure 1 shows the X-ray crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain. (B)-(F) show magnified views of the specific interaction between ATL_5262 and UNC5C shown in (A). [Figure 31F] Figure 1 shows the X-ray crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain. (B)-(F) show magnified views of the specific interaction between ATL_5262 and UNC5C shown in (A). [Figure 31G] (G) shows the X-ray crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain. (H) shows the protein alignment of human UNC5 family members B (SEQ ID NO: 260), A (SEQ ID NO: 261), and D (SEQ ID NO: 262) to the region of human UNC5C Ig-like domain 1 that interacts with ATL_5262 (SEQ ID NO: 259). [Figure 32A-1]The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. (A-1 to A-4) Heavy chains aligned by IMGT position numbering. VH of ATL_0005262: SEQ ID NO: 1, VH of ATL_0006178: SEQ ID NO: 93, VH of ATL_0006530: SEQ ID NO: 122, VH of ATL_0006531: SEQ ID NO: 123, VH of ATL_0006532: SEQ ID NO: 124, VH of ATL_0006533: SEQ ID NO: 125, VH of ATL_0006534: SEQ ID NO: 126, VH of ATL_0006535: SEQ ID NO: 127, VH of ATL_0006536: SEQ ID NO: 128, VH of ATL_0006537: SEQ ID NO: 129, VH of ATL_0006538: SEQ ID NO: 130, VH of ATL_0006539: SEQ ID NO: 131. [Figure 32A-2] The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. (A-1 to A-4) Heavy chains aligned by IMGT position numbering. VH of ATL_0005262: SEQ ID NO: 1, VH of ATL_0006178: SEQ ID NO: 93, VH of ATL_0006530: SEQ ID NO: 122, VH of ATL_0006531: SEQ ID NO: 123, VH of ATL_0006532: SEQ ID NO: 124, VH of ATL_0006533: SEQ ID NO: 125, VH of ATL_0006534: SEQ ID NO: 126, VH of ATL_0006535: SEQ ID NO: 127, VH of ATL_0006536: SEQ ID NO: 128, VH of ATL_0006537: SEQ ID NO: 129, VH of ATL_0006538: SEQ ID NO: 130, VH of ATL_0006539: SEQ ID NO: 131. [Figure 32A-3]The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. (A-1 to A-4) Heavy chains aligned by IMGT position numbering. VH of ATL_0005262: SEQ ID NO: 1, VH of ATL_0006178: SEQ ID NO: 93, VH of ATL_0006530: SEQ ID NO: 122, VH of ATL_0006531: SEQ ID NO: 123, VH of ATL_0006532: SEQ ID NO: 124, VH of ATL_0006533: SEQ ID NO: 125, VH of ATL_0006534: SEQ ID NO: 126, VH of ATL_0006535: SEQ ID NO: 127, VH of ATL_0006536: SEQ ID NO: 128, VH of ATL_0006537: SEQ ID NO: 129, VH of ATL_0006538: SEQ ID NO: 130, VH of ATL_0006539: SEQ ID NO: 131. [Figure 32A-4] The sequences of the antibodies described herein are shown with variable positions in the heavy chains highlighted. A. (A-1 to A-4) Heavy chains aligned by IMGT position numbering. VH of ATL_0005262: SEQ ID NO: 1, VH of ATL_0006178: SEQ ID NO: 93, VH of ATL_0006530: SEQ ID NO: 122, VH of ATL_0006531: SEQ ID NO: 123, VH of ATL_0006532: SEQ ID NO: 124, VH of ATL_0006533: SEQ ID NO: 125, VH of ATL_0006534: SEQ ID NO: 126, VH of ATL_0006535: SEQ ID NO: 127, VH of ATL_0006536: SEQ ID NO: 128, VH of ATL_0006537: SEQ ID NO: 129, VH of ATL_0006538: SEQ ID NO: 130, VH of ATL_0006539: SEQ ID NO: 131. [Figure 32B-1]The sequences of the antibodies described herein are shown with variable positions highlighted in the heavy chain. B. (B-1 to B-4) Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0006178: SEQ ID NO: 92, VL of ATL_0006530: SEQ ID NO: 132, VL of ATL_0006531: SEQ ID NO: 133, VL of ATL_0006532: SEQ ID NO: 134, VL of ATL_0006533: SEQ ID NO: 135, VL of ATL_0006534: SEQ ID NO: 136, VL of ATL_0006535: SEQ ID NO: 137, VL of ATL_0006536: SEQ ID NO: 138, VL of ATL_0006537: SEQ ID NO: 139, VL of ATL_0006538: SEQ ID NO: 140, VL of ATL_0006539: SEQ ID NO: 141. [Figure 32B-2] The sequences of the antibodies described herein are shown with variable positions highlighted in the heavy chain. B. (B-1 to B-4) Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0006178: SEQ ID NO: 92, VL of ATL_0006530: SEQ ID NO: 132, VL of ATL_0006531: SEQ ID NO: 133, VL of ATL_0006532: SEQ ID NO: 134, VL of ATL_0006533: SEQ ID NO: 135, VL of ATL_0006534: SEQ ID NO: 136, VL of ATL_0006535: SEQ ID NO: 137, VL of ATL_0006536: SEQ ID NO: 138, VL of ATL_0006537: SEQ ID NO: 139, VL of ATL_0006538: SEQ ID NO: 140, VL of ATL_0006539: SEQ ID NO: 141. [Figure 32B-3]The sequences of the antibodies described herein are shown with variable positions highlighted in the heavy chain. B. (B-1 to B-4) Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0006178: SEQ ID NO: 92, VL of ATL_0006530: SEQ ID NO: 132, VL of ATL_0006531: SEQ ID NO: 133, VL of ATL_0006532: SEQ ID NO: 134, VL of ATL_0006533: SEQ ID NO: 135, VL of ATL_0006534: SEQ ID NO: 136, VL of ATL_0006535: SEQ ID NO: 137, VL of ATL_0006536: SEQ ID NO: 138, VL of ATL_0006537: SEQ ID NO: 139, VL of ATL_0006538: SEQ ID NO: 140, VL of ATL_0006539: SEQ ID NO: 141. [Figure 32B-4] The sequences of the antibodies described herein are shown with variable positions highlighted in the heavy chain. B. (B-1 to B-4) Light chains aligned by IMGT position numbering. VL of ATL_0005262: SEQ ID NO: 9, VL of ATL_0006178: SEQ ID NO: 92, VL of ATL_0006530: SEQ ID NO: 132, VL of ATL_0006531: SEQ ID NO: 133, VL of ATL_0006532: SEQ ID NO: 134, VL of ATL_0006533: SEQ ID NO: 135, VL of ATL_0006534: SEQ ID NO: 136, VL of ATL_0006535: SEQ ID NO: 137, VL of ATL_0006536: SEQ ID NO: 138, VL of ATL_0006537: SEQ ID NO: 139, VL of ATL_0006538: SEQ ID NO: 140, VL of ATL_0006539: SEQ ID NO: 141. DETAILED DESCRIPTION OF THE INVENTION

[0064] Aspects and embodiments of the present invention are discussed below with reference to the accompanying drawings, and further aspects and embodiments will be apparent to those skilled in the art.

[0065] Disclosed herein are antibodies and fragments thereof that can specifically bind to UNC5C protein or fragments thereof. As used herein, an antibody that can "specifically bind" or "specifically bind" to a target is an antibody that can bind via the association of an epitope recognition site with an epitope in the target. This is different from non-specific binding, such as Fc-mediated binding, ionic and / or hydrophobic interactions. In other words, an antibody that specifically binds to a target does not generally recognize and bind to a specific protein structure therein, but rather to the protein itself.

[0066] The present disclosure refers to the antibodies described herein using references identified as "ATL_000xxxx," "ATL_xxxx," or "xxxx," where "xxxx" is a four-digit reference number unique to the antibody described herein. All of the above designations are used interchangeably to refer to the same antibody or portion thereof (e.g., the VH, VL, or portion thereof of the antibody). For example, antibody ATL_0005262 is referred to interchangeably herein as ATL_5262 and 5262.

[0067] Binding to UNC5C The UNC-5 netrin receptor C (UNC5C) belongs to the UNC5 family of plasma membrane netrin receptors, which are part of the immunoglobulin superfamily (Leonardo, E., et al. (1997)). UNC5 family members contain two extracellular Ig domains and two thrombospondin type 1 (TSP-1) domains. The intracellular C-terminus consists of a zonula occludens 5 (ZU5) domain, a DCC-binding domain, and a death domain involved in apoptosis regulation (Ackerman, S.L., Knowles, B.B., 1998). Four subtypes of UNC5, namely UNC5 A-D (referred to as UNC5 H1-H4 in other mammalian species), have been identified and are widely expressed in the central nervous system (CNS) (Rajasekharan, S., Kennedy, T.E. (2009)). Additional netrin receptors include Down's syndrome cell adhesion molecule (DSCAM), neogenin, deleted in colorectal cancer (DCC), and CD146 (also known as melanoma cell adhesion molecule (MCAM) or Mel-CAM). These receptors differ in the number of N-terminal Ig domains, the inclusion of a fibronectin type III domain, and an intracellular domain. For a review of plasma membrane netrin receptors, see Dun et al., 2017. The antibodies described herein can specifically bind UNC5C over other netrin receptors, such as neogenin, DCC, DSCAM, UNC5A, UNC5B, and UNC5D. Binding of the antibody to the candidate protein can be verified, for example, by biolayer interferometry analysis as described herein.

[0068] UNC5C is a dependence receptor responsible for regulating neuronal apoptosis, and whether UNC5C promotes or inhibits apoptosis depends on its binding to netrin-1 (Lambi et al. (2001); Poliak S, et al. 2015). The UNC5 receptor contains an intracellular death domain. This domain can interact with death-associated protein kinase (DAPK1) to induce apoptosis in the absence of ligand binding, while the receptor remains in a monomeric form. The T835M mutation in the death domain of UNC5C further increases apoptosis. This increased signal led to the involvement of death-associated protein kinase 1 / protein kinase D / apoptosis signal-regulating kinase 1 (ASK1) / JNK / NADPH oxidase / caspase in this signaling cascade (Hashimoto et al. 2016 Signal Transduction 291(23), 12282-12293). UNC5-induced cell death is augmented by caspase cleavage at its intracellular death domain, which can result in a feed-forward loop once apoptosis is initiated. All UNC5 receptors are cleaved in vitro by caspase-3 (Liambi et al., 2001). Cleavage of UNC5C by LGMN has also been proposed to increase UNC5C-mediated neuronal apoptosis associated with Alzheimer's disease (Chen et al., 2021). However, in the presence of netrin-1, UNC5C forms dimers and promotes neuronal survival, migration, and differentiation. Thus, it plays an anti-apoptotic role. Multimerization of the UNC5 receptor is sufficient to prevent activation of the apoptotic signaling pathway (Mille et al., 2009 Cell Death and Differentiation 16, 1344-1351), and netrin-1 directly inhibits apoptosis in neurons (Llambi et al., 2001). UNC5C has also been shown to form heterodimers with DCC or DSCAM ( Boyer and Gupton, 2018 ).The antibodies described herein can bind to monomeric and dimeric UNC5C. The antibodies described herein can bind to homodimeric UNC5C.

[0069] UNC5C is involved in Alzheimer's disease, and truncated UNC5C, which leads to enhanced neuronal apoptosis, has been found to be associated with accelerated AD pathology in mice (Chen et al., 2021). Furthermore, several SNPs in UNC5C are associated with the development of Alzheimer's disease (AD). For example, SNP T835M predisposes to AD and increases neuronal apoptosis (Li Q, et al., 2018). UNC5C is also involved in Parkinson's disease (PD), and truncation of UNC5C has been shown to cause neuronal loss in a mouse model of PD (Chen et al., 2022).

[0070] Netrin-1 is one of several netrins, all members of the laminin superfamily. Netrins are secreted proteins that direct axon outgrowth and cell migration during neurogenesis and regulate cell adhesion, cell morphological maturation, cell survival, and tumorigenesis (Rajasekharan, et al. (2009)). They are bifunctional proteins that act as attractants for some cell types and repellents for others. Receptors of the UNC-5 family mediate the repellent response to netrins. Netrin-1 consists of an N-terminal laminin-like domain (LN, also known as domain VI) followed by three epidermal growth factor (EGF) repeats (EGF1, EGF2, and EGF3, also known as domain V), and a C-terminal netrin-like domain (NTR) that shares homology with domains found in complement and other proteins (Dun et al. 2017).

[0071] In embodiments, the antibodies described herein specifically bind to UNC5C and mimic and / or compete with netrin-1 for binding to UNC5C. Competition with netrin-1 binding can be assessed by the ELISA assay described herein. Thus, the antibodies described herein can compete with netrin-1 for the same binding site on UNC5C as netrin-1. The antibodies described herein can bind to the same binding site on UNC5C as netrin-1. An antibody that mimics netrin-1 can refer to an antibody that interacts with UNC5C in a way that mimics the effect of netrin-1 on UNC5C structure and / or activity. An antibody that competes with netrin-1 for binding to UNC5C can refer to binding of the antibody to UNC5C that depends on the presence or absence of netrin-1 and / or binding of netrin-1 to UNC5C that depends on the presence or absence of the antibody. An antibody that competes with netrin-1 for binding to UNC5C can refer to the binding of the antibody to UNC5C being inhibited by netrin-1.For example, an antibody that competes with netrin-1 for binding to UNC5C can refer to the binding of the antibody to UNC5C being at least partially inhibited in the presence of netrin-1.This can be evaluated, for example, using an ELISA assay as described herein.For example, UNC5C can be pre-incubated with netrin-1 before incubation with the antibody, and the amount of bound antibody can be compared with a control condition that does not pre-incubate with netrin-1.A reduction in the amount of bound antibody can indicate that the antibody competes with netrin-1 for binding to UNC5C. In embodiments, the signal indicating the presence of antibody bound to UNC5C may be reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in the presence of netrin-1 (e.g., after pre-incubation with 200 μg / L netrin-1). In embodiments, the binding of the antibody to UNC5C may be reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in the presence of netrin-1 (e.g., after pre-incubation with 200 μg / L netrin-1).

[0072] The antibodies described herein may be such that the binding of the antibody to UNC5C is inhibited by netrin-1 in a concentration-dependent manner. This can be evaluated, for example, using the ELISA assay described herein or any other binding assay combined with incubation with multiple different concentrations of netrin-1 (e.g., a netrin-1 dilution series). A reduction in the binding of the antibody to UNC5C in the presence of netrin-1 (e.g., when the antibody is pre-incubated with netrin-1 followed by incubation with UNC5C) depends on the concentration of netrin-1 and can be considered to indicate netrin-1 concentration-dependent inhibition of the binding of the antibody to UNC5C. In an embodiment, a signal indicating the presence of the antibody bound to UNC5C is at least 10 -8 can be reduced by at least 50% in the presence of M netrin-1 (e.g., 10 -8 In embodiments, the binding of the antibody to UNC5C is at least 10 -8 can be reduced by at least 50% in the presence of M netrin-1 (e.g., 10 -8 In embodiments, the signal indicating the presence of antibody bound to UNC5C is at least 10 -7 can be reduced by at least 80% in the presence of M netrin-1 (e.g., 10 -7 In embodiments, the binding of the antibody to UNC5C is at least 10 -7 can be reduced by at least 80% in the presence of M netrin-1 (e.g., 10 -7 In embodiments, a signal indicating the presence of antibody bound to UNC5C is at least 5×10 -7 M or 1 x 10 -6 may be reduced by at least 90% or at least 95% in the presence of M netrin-1 (e.g., 5×10 -7 M or 1 x 10 -6 In embodiments, the binding of the antibody to UNC5C is at least 5×10 -7M or 1 x 10 -6 may be reduced by at least 90% or at least 95% in the presence of M netrin-1 (e.g., 5×10 -7 M or 1 x 10 -6 (e.g., after pre-incubation of M with netrin-1). The optional pre-incubation may be for a suitable period of time, such as, for example, about 30 minutes.

[0073] The antibodies described herein may have a binding site that at least partially overlaps with the netrin-1 binding site.The antibodies described herein may cause dimerization of UNC5C.

[0074] The human gene encoding UNC5C (Gene ID: 8633) is located on 4q22.3 and consists of 20 exons. Reference non-human UNC5C amino acid and coding sequences are available in public databases.

[0075] The sequence of human UNC5C is available under Uniprot identifier O95185. It contains 931 amino acids. Amino acids 62-159 form the Ig-like domain, amino acids 161-256 form the Ig-like C2-type domain, amino acids 260-314 and 316-368 form the TSP1 type 1 and TSP1 type 2 domains, amino acids 530-673 form the ZU5 domain, and amino acids 850-929 form the death domain. The protein is cleaved by caspase-3 at amino acids 415-416. The reference human UNC5C amino acid sequence is provided as SEQ ID NO:99.

[0076] The sequence of mouse UNC5C is available under Uniprot identifier O08747. It contains 931 amino acids. Amino acids 62-159 form the Ig-like domain (also referred to herein as the first immunoglobulin domain or N-terminal immunoglobulin domain), amino acids 161-256 form the Ig-like C2-type domain (also referred to herein as the second immunoglobulin domain), amino acids 260-314 and 316-368 form the TSP1 type 1 and TSP1 type 2 domains, amino acids 530-673 form the ZU5 domain, and amino acids 850-929 form the death domain. This protein is cleaved by caspase-3 at amino acids 415-416. A reference mouse UNC5C amino acid sequence is provided as SEQ ID NO: 100. However, as used herein, the term "UNC5C" encompasses truncations, derivatives, and variants of the UNC5C sequences provided herein and may refer to any protein having at least 80%, at least 90%, or at least 95% sequence identity. The antibodies described herein can specifically bind to a peptide or protein having or comprising the amino acid sequence of SEQ ID NO: 99 (including a full-length UNC5C protein comprising said sequence or a fragment of said protein comprising said sequence), or a fragment thereof, such as a fragment comprising or consisting of amino acids 41-380 of human or mouse (murine) UNC5C (e.g., the amino acid sequence of SEQ ID NO: 101 or 102), or a corresponding homologous sequence. The antibodies described herein can specifically bind to a peptide or protein comprising the extracellular domain of UNC5C or a portion thereof, such as a portion comprising amino acids 41-380 of human or mouse UNC5C, or a corresponding homologous sequence. The antibodies described herein can specifically bind to human UNC5C and the human mutant UNC5C T835M. The antibodies described herein can bind to mutant forms of human UNC5C that contain one or more mutations that are not present in the extracellular domain of the protein. The antibodies can bind to epitopes located in the extracellular domain of UNC5C. In particular, the antibodies can bind to the N-terminal immunoglobulin domain (also referred to as the "first immunoglobulin domain") of UNC5C.The antibody may bind to one or more residues of UNC5C involved in the binding of netrin-1 to UNC5C. Thus, the antibody may bind to one or more residues of UNC5C located in the first immunoglobulin domain of UNC5C.

[0077] The antibodies described herein bind to UNC5C (e.g., murine and / or human) with high affinity. High affinity binding can be assessed by measuring the EC50 value, the concentration at which the antibody results in half-maximal binding, for example, via ELISA as described herein. For example, antibody affinity can be assessed by binding to plate-bound recombinant human UNC5C as described herein. High affinity, as referred to herein, can be at most 1E-06M (i.e., 1000nM), at most 2E-06M, at most 3E-06M, at most 4E-06, at most 5E-06M, at most 6E-06M, at most 7E-06, at most 8E-06M, at most 9E-06M, at most 1E-07M (i.e., 100nM), at most 5E-07M, at most 1 ... E-08M, up to 2E-08M, up to 3E-08M, up to 4E-08M, up to 5E-08M, up to 6E-08M, up to 7E-08M, up to 8E-08M, up to 9E-08M, up to 1E-09M, up to 2E-09M, up to 3E-09M, up to 4E-09M, up to 5E-09M, up to 6E-09M, up to 7E-09 M, up to 8E-09M, up to 9E-09M, up to 1E-10M, up to 2E-10M, up to 3E-10M, up to 4E-10M, up to 5E-10M, up to 6E-10M, up to 7E-10M, up to 8E-10M, up to 9E-10M, up to 1E-11M, up to 2E-11M, up to 3E-11M, up to 4E-11M, up By "antibody binding" is meant an EC50 value of up to 5E-11M, up to 6E-11M, up to 7E-11M, up to 8E-11M, up to 9E-11M, up to 1E-12M, up to 2E-12M, up to 3E-12M, up to 4E-12M, 5E-12M, up to 6E-12M, up to 7E-12M, up to 8E-12M, up to 9E-12M or 1E-13M. In embodiments, the antibodies described herein bind to UNC5C with an EC50 of up to 100 nM as assessed by ELISA (e.g., as described herein).

[0078] In some embodiments, the antibody binds to human UNC5C with an EC50 of at most 1E-08M, at most 1.1E-08M, at most 2.24E-08, at most 2.40E-08, at most 9.81E-08M, at most 4.52E-09, at most 5.5E-09M, at most 9.62E-09M, at most 5.50E-10, at most 7.10E-10, at most 1.00E-11, at most 1.30E-11, at most 3.90E-11, at most 2.80E-12, or at most 9.20E-12.

[0079] In some embodiments, the antibody binds to human UNC5C with an EC50 of about 1.1E-08M, about 2.24E-08, about 2.40E-08, about 9.81E-08M, about 4.52E-09, about 5.5E-09M, about 8.00E-09, about 9.62E-09M, about 5.50E-10, about 7.10E-10, about 1.00E-11, about 1.30E-11, about 3.90E-11, about 2.80E-12, or about 9.20E-12.

[0080] In some embodiments, the EC50 value is 1E-07M to 1E-13M, 5E-07M to 1E-13M, 1E-08M to 1E-13M, 5E-08M to 1E-13M, 1E-09M to 1E-13M, 5E-09M to 1E-13M, 1E-10M to 1E-13M, 5E-10M to 1E-13M, 1E-11M to 1E-13M, 5E-11M to 1E-13M, 1E-12M to 1E-13M, 5E-12M~1E-13M, 1E-07M~1E-12M, 5E-07M~1E-12M, 1E-08M~1E-12M, 5E-08M~1E-12M, 1E-09M ~1E-12M, 5E-09M~1E-12M, 1E-10M~1E-12M, 5E-08M~1E-12M, 1E-11M~1E-12M, 5E-11M~1E-12M, 1E-07 M~1E-11M, 5E-07M~1E-11M, 1E-08M~1E-11M, 5E-08M~1E-11M, 1E-09M~1E-11M, 5E-0 9M~1E-11M, 1E-10M~1E-11M, 5E-10M~1E-1M, 1E-07M~1E-10M, 5E-07M~1E-10M, 1E-0 8M~1E-10M, 5E-08M~1E-10M, 1E-09M~1E-10M, 5E-09M~1E-10M, 1E-07M~1E-09M, 5E-07M~1E-09M, 1E-08M~1E-09M, 5E-08M~1E-09M, 1E-07M~1E-08M, 5E-07M~1E-08M.

[0081] In some embodiments, the antibodies described herein can specifically bind to an UNC5C protein or a protein fragment comprising or consisting of an UNC5C variant amino acid sequence. In some embodiments, the UNC5C variant protein or fragment comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 99. In some embodiments, the antibodies disclosed herein can specifically bind to an UNC5C amino acid sequence or an UNC5C fragment comprising at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the UNC5C variant sequence.

[0082] The UNC5C may be human UNC5C or mouse UNC5C. Preferably, the UNC5C may be human UNC5C. UNC5C may refer to human UNC5C unless the context indicates otherwise. In other embodiments, for example, when the individual to be treated is a non-human mammal, the UNC5C may be non-human UNC5C.

[0083] Structural properties The antibodies of the present disclosure can specifically target a single epitope on the UNC5C protein. As used herein, the term epitope, also known as antigenic determinant, refers to any protein determinant that can specifically bind to an immunoglobulin or a fragment thereof. Epitope determinants or antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0084] The antibodies described herein may bind to a discontinuous epitope of UNC5C. The antibodies described herein may bind to an epitope comprising one or more or all of the following residues of UNC5C provided as SEQ ID NO: 99: Thr89, Gln90, Gln102, Lys103, Val107, Asp108, Glu109, Arg110, Val111, Ile118, Arg120, or the corresponding residues in a homologous sequence. In some embodiments, for example, when the antibody is a heavy chain-only antibody, the antibody binds to residues Thr89, Gln90, Gln102, Lys103, Val107, Glu109, Ile118, and Arg120.

[0085] In embodiments, one or more (or all) of VH residues 55, 63, 64, 65, 66, 74, 108, 110 (according to the IMGT numbering) interact with one or more residues in the epitope. In embodiments, one or more (or all) of VH residues 55 (Tyr), 63 (Gly), 64 (T, H, or S, more particularly Thr), 65 (T, or S, more particularly Thr), 66 (Asn), 74 (Ser), 108 (Arg), 110 (Met) (according to the IMGT numbering) interact with one or more residues in the epitope. All of these residues are conserved across the VHs of antibodies 5262, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005, 6033, 6034, 6035, 6036, 6037, 6038, 6039, 6177, and 6178. All of these residues, except for 64 (Thr) and / or 65 (Thr), are conserved across the VHs of antibodies 6187 (H at position 64 and T at position 65) and 6191 (S at position 64 and S at position 65), indicating that substitutions at these positions are possible while maintaining binding to the epitope. Thus, in embodiments, one or more (or all) of VH residues 55 (Tyr), 63 (Gly), 64 (Thr or a conservative substitution, e.g., H or S), 65 (Thr or a conservative substitution, e.g., S), 66 (Asn), 74 (Ser), 108 (Arg), 110 (Met) according to the IMGT numbering interact with one or more residues in the epitope. In embodiments, one or more (or all) of VL residues 38, 107, 108, 109, and 114 according to the IMGT numbering interact with one or more residues in the epitope. In embodiments, one or more (or all) of VL residues 38 (Y, D, L, or T, e.g., in certain embodiments, Tyr), 107 (S, Y, F, or W, e.g., in certain embodiments, Ser), 108 (Y, D, T, or G, e.g., in certain embodiments, Tyr), 109 (S, D, K, or absent, e.g., in certain embodiments, Ser), and 114 (T, N, S, R, G, or A, e.g., in certain embodiments, Thr) according to the IMGT numbering interact with one or more residues in the epitope.Position 38 is Tyr in antibodies 5262, 6178, 6530, 6531, 6533, 6534, 6537, 6539, D in antibody 6335, L in antibody 6336, and T in antibody 6538. Position 107 is S in antibodies 5262 and 6178 (same VL), Y in antibodies 6530, 6532, 6533, 6534, 6535, and 6536, F in 6531, and W in antibodies 6537, 6538, and 6539. Position 108 is Y in antibodies 5262 and 6178, D in antibodies 6530, 6532, 6533, 6534, 6535, 6537, and 6538, T in antibodies 6531 and 6536, and G in antibody 6539. Position 109 is S in antibodies 5262, 6178, 6530, 6533, 6534, 6535, 6536, and 6537, D in antibodies 6531 and 6538, K in antibody 6532, and absent in antibody 6539. Position 114 is T in antibodies 5262, 6178, and 6536, N in antibodies 6530, 6533, and 6538, S in antibodies 6531 and 6537, R in antibody 6532, G in antibody 6534, and A in antibody 6535. Thus, in embodiments, the VH of an antibody described herein has a Tyr at residue 55, a Gly at residue 63, a T, H, or S, more particularly Thr, at residue 64, a T or S, more particularly Thr, at residue 65, an Asn at residue 66, a Ser at residue 74, an Arg at residue 108, and a Met at residue 110, where all positions are according to IMGT numbering. Further, in embodiments, the VL of an antibody described herein has a Y, D, L, or T, e.g., in certain embodiments, a Tyr, at residue 38, a S, Y, F, or W, e.g., in certain embodiments, a Ser, at residue 107, a Y, D, T, or G, e.g., in certain embodiments, a Tyr, at residue 108, a S, D, K, or absent (deleted), e.g., in certain embodiments, a Ser, at residue 109, and a T, N, S, R, G, or A, e.g., in certain embodiments, a Thr, at residue 114, where all positions are according to IMGT numbering. Any of the amino acids at any of these positions can be combined with each other, and all resulting combinations are expressly contemplated.

[0086] In some cases, (a) VH residue 64 (e.g., Thr) of an antibody described herein interacts with Thr 89 of the discontinuous epitope, and / or (b) VH residue 63 (e.g., Gly) of an antibody described herein interacts with Gln 90 of the discontinuous epitope, and / or (c) VH residue 65 (e.g., Thr) of an antibody described herein interacts with Gln 102 of the discontinuous epitope, and / or (d) VH residue 74 (e.g., Ser) of an antibody described herein interacts with Lys 103 of the discontinuous epitope, and / or (e) VH residue 66 (e.g., Asn) of an antibody described herein interacts with Val 1 of the discontinuous epitope. 107, and / or (f) VH residues 55 (e.g., Tyr), 66 (e.g., Asn), 108 (e.g., Arg), and / or 110 (e.g., Met) of an antibody described herein interact with Glu109 of the discontinuous epitope, and / or (g) VH residues 108 (e.g., Arg), and / or 110 (e.g., Met) of an antibody described herein interact with Ile118 of the discontinuous epitope, and / or (h) VH residues 55 (e.g., Tyr), 64 (e.g., Thr), and / or 65 (e.g., Thr) of an antibody described herein interact with Arg120 of the discontinuous epitope. In some cases, (i) VL residue 114 (e.g., Thr) of an antibody described herein interacts with Val107 and / or Asp108, and / or Glu109 of the discontinuous epitope, and / or (j) VL residue 109 (e.g., Ser) of an antibody described herein interacts with Glu109 and / or Arg110 of the discontinuous epitope, and / or (k) VL residue 108 (e.g., Tyr) of an antibody described herein interacts with Arg110 and / or Val111 of the discontinuous epitope, and the UNC5C residues are at a particular position in SEQ ID NO: 99 or the corresponding position in a homologous sequence.Those skilled in the art are familiar with methods for mapping epitopes, including, but not limited to, X-ray cocrystallography as described herein, cryo-electron microscopy as described, for example, in Renaud et al. (2018), and mutagenesis strategies such as alanine scanning mutagenesis as described, for example, in Cunningham et al. (1989).

[0087] An "antigen-binding domain" describes the portion of a molecule that binds to all or part of a target antigen. Antibodies generally have six complementarity-determining regions (CDRs): three in the VH region: HCDR1, HCDR2, and HCDR3, and three in the VL region: LCDR1, LCDR2, and LCDR3. The six CDRs together define the paratope of the antigen-binding domain, which is the portion of the antigen-binding domain that binds to the target antigen. The paratope of the antibodies described herein may include the following VH residues according to the IMGT numbering system: 55 (e.g., Tyr), 63 (e.g., Gly), 64 (e.g., Thr, His, or Ser), 65 (e.g., Thr or Ser), 66 (e.g., Asn), 74 (e.g., Ser), 108 (e.g., Arg), and 110 (e.g., Met). The paratope of the antibodies described herein may comprise the following VL residues according to the IMGT numbering: 38 (e.g., Tyr, D, L, or T), 107 (e.g., Ser, Y, F, or W), 108 (e.g., Tyr, D, T, or G), 109 (e.g., Ser, D, K, or -(deletion)), 114 (e.g., Thr, N, S, R, G, or A). Thus, an antibody according to any embodiment of the disclosure may comprise a VH having the following residues according to the IMGT numbering: Tyr at position 55, Gly at position 63, Thr at position 64, Thr at position 65, Asn at position 66, Ser at position 74, Arg at position 108, and Met at position 110. Additionally, an antibody according to any embodiment of the present disclosure may comprise a VL having the following residues according to the IMGT numbering: Tyr at position 38, Ser at position 107, Tyr at position 108, Ser at position 109, and Thr at position 114. The present disclosure primarily relates to antibody molecules, whole antibodies (e.g., IgGs such as IgG1) or antibody fragments (e.g., single-chain variable fragments (scFvs), antibody fragments (Fabs) or bivalent antibody fragments (F(ab')s), single-domain antibodies (sdAbs). Antibody antigen-binding regions (also referred to as "antigen-binding portions") are provided, as are antibody heavy chain variable (VH) and light chain variable (VL) domains. Within the VH and VL domains, complementarity-determining regions (CDRs) are provided, which may be provided within different framework regions (FRs) to form the VH or VL domain, as the case may be.The antigen-binding site may consist of an antibody VH domain and / or VL domain. Thus, an antibody according to the present disclosure may be an scFv antibody molecule, a nanobody, or a whole antibody. The antibody may comprise an antibody constant region. The antibody constant region may be a human constant region, such as a human IgG1 constant region. The antibody may be a whole antibody. The antibody may be an IgG, such as an IgG1 or a variant thereof. The antibody may be the IgG1 variant L234A / L235A (LALA).

[0088] Antibodies according to the present disclosure may be provided in isolated form. The term "antibody" includes fragments or derivatives thereof, or synthetic antibodies or synthetic antibody fragments.

[0089] The antigen-binding portion can be a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., scFV). Suitable antibodies against a selected antigen can be prepared by known techniques, such as those disclosed in "Monoclonal Antibodies: A manual of techniques," by H. Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications," by J. G. R. Hurrell (CRC Press, 1982). Chimeric antibodies are discussed by Neuberger et al. (1988, 8th International Biotechnology Symposium Part 2, 25 792-5 799).

[0090] The antibody or fragment thereof may be a monoclonal antibody (mAb), which is a homogeneous population of antibodies that specifically target a single epitope on an antigen.

[0091] Antibody fragments, such as Fab and F(ab')2 fragments, can also be provided, as can genetically engineered antibodies and antibody fragments. The VH and VL domains of antibodies are involved in antigen recognition, a fact first recognized by early protease digestion experiments. Further confirmation was found by the "humanization" of rodent antibodies. Variable domains of rodent origin can be fused to constant domains of human origin so that the resulting antibody retains the antigen specificity of the rodent parent antibody (Morrison et al. (1984) Proc. Natl. Acad. Sd. USA 81, 6851-6855).

[0092] Antigen specificity is conferred by the variable domains, and is independent of the constant domains, as is known from experiments involving bacterial expression of antibody fragments, all of which contain one or more variable domains. These molecules include Fab-like molecules (Better et al. (1988) Science 240, 1041), Fv molecules (Skerra et al. (1988) Science 240, 1038), single-chain Fv (ScFv) molecules in which the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al. (1988) Science 20 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sd. USA 85, 5879), and single-domain antibodies (dAbs) containing isolated V domains (Ward et al. (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349, 293-299.

[0093] The term "ScFv molecule" refers to a molecule in which the VH and VL partner domains are covalently linked, for example, by a flexible oligopeptide. Fab, Fv, ScFv and sdAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the fragments.

[0094] Whole antibodies and F(ab')2 fragments are "bivalent." The term "bivalent" means that the antibodies and F(ab')2 fragments have two antigen-binding sites. In contrast, Fab, Fv, ScFv, and dAb fragments are monovalent and have only one antigen-binding site.

[0095] Antibodies according to the present disclosure may be detectably labeled, or at least detectable. For example, antibodies may be labeled with radioactive atoms, colored molecules, fluorescent molecules, or any other molecule that can be easily detected. Suitable detectable molecules include fluorescent proteins, luciferase, enzyme substrates, and radioactive labels. The binding moiety (antibody or fragment thereof) may be directly labeled with a detectable label or may be indirectly labeled. For example, the binding moiety may be an unlabeled antibody that can be detected by another antibody that is itself labeled. Alternatively, the second antibody may have biotin attached to it, and the binding of labeled streptavidin to the biotin is used to indirectly label the first antibody.

[0096] A "fragment" of an antibody may contain any number of residues of a "parent" antibody while retaining target binding ability. A fragment may lack effector function, e.g., may be completely unable to bind Fc receptors or may exhibit reduced binding to Fc receptors compared to the parent. Fragments are typically smaller than the parent antibody. A fragment may contain 50%, 60%, 70%, 80%, 90%, 95%, or more of the contiguous or non-contiguous amino acids of the parent antibody. A fragment may contain 50, 100, 150, 200, 250, 300, or more contiguous or non-contiguous amino acids of the parent antibody. A fragment may contain deletions in or of the Fc region. A fragment may retain the CDRs and / or variable domains of the parent antibody unaltered. In some embodiments, the fragment is a Fab fragment or a F(ab')2 fragment.

[0097] CDR sequences are described herein using the IMGT numbering (Lefranc, M.-P., Immunology Today, 18, 509 (1997)).

[0098] An antibody according to this disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_5262, ATL_6036, ATL_6039, ATL_6177, or ATL_6178. An antibody according to this disclosure may have the following heavy chain CDRs: CDRH1: SEQ ID NO: 20, CDRH2: SEQ ID NO: 21 or SEQ ID NO: 22, and CDRH3: SEQ ID NO: 23.

[0099] The present disclosure further provides an antibody comprising a VH having CDRs of the heavy chain variable domain (VH) of antibody ATL_6187 or ATL_6191: CDRH1 comprising the amino acid sequence of SEQ ID NO: 115 or SEQ ID NO: 116, CDRH2 comprising the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 118, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having CDRs of the heavy chain variable domain (VH) of antibody ATL_6187, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 115, CDRH2 comprising the amino acid sequence of SEQ ID NO: 117, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 119. Furthermore, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6191, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 116, CDRH2 comprising the amino acid sequence of SEQ ID NO: 118, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 120. In an antibody according to the present disclosure, at least one of the VH CDR1-3 sequences may be altered. For example, a variant may have a VH CDR with up to three amino acid mutations across the above VH CDR.

[0100] The present disclosure further provides antibodies comprising a heavy chain variable domain (VH) having the CDRs of the VH of antibodies ATL_6530, ATL_6531, ATL_6532, ATL_6533, ATL_6534, ATL_6535, ATL_6536, ATL_6537, ATL_6538, and ATL_6539, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NOs: 142 to 149, CDRH2 comprising the amino acid sequence of SEQ ID NOs: 150 to 158, and CDRH3 comprising the amino acid sequence of SEQ ID NOs: 159 to 168. In particular, antibodies according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6530, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 142, CDRH2 comprising the amino acid sequence of SEQ ID NO: 150, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 159. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6531, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 143, CDRH2 comprising the amino acid sequence of SEQ ID NO: 151, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 160. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6532, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 144, CDRH2 comprising the amino acid sequence of SEQ ID NO: 152, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 161. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6533, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 145, CDRH2 comprising the amino acid sequence of SEQ ID NO: 153, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 162. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6534, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 144, CDRH2 comprising the amino acid sequence of SEQ ID NO: 152, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 163.In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6535, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 143, CDRH2 comprising the amino acid sequence of SEQ ID NO: 154, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 164. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6536, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 146, CDRH2 comprising the amino acid sequence of SEQ ID NO: 155, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 165. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6537, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 147, CDRH2 comprising the amino acid sequence of SEQ ID NO: 156, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 166.

[0101] In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6538, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 148, CDRH2 comprising the amino acid sequence of SEQ ID NO: 157, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 167. In particular, an antibody according to the present disclosure may have a heavy chain variable domain (VH) having the CDRs of the heavy chain variable domain (VH) of antibody ATL_6539, i.e., CDRH1 comprising the amino acid sequence of SEQ ID NO: 149, CDRH2 comprising the amino acid sequence of SEQ ID NO: 158, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 168. In an antibody according to the present disclosure, at least one of the VH CDR1-3 sequences may be altered. For example, a variant may have a VH CDR with up to three amino acid mutations across the above VH CDRs.

[0102] In antibodies according to the present disclosure, at least one of the VH CDR1-3 sequences may be altered. That is, the set of CDRs described above may contain one, two, or three mutations. As used herein, "mutation" refers to an amino acid substitution, insertion, or deletion. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. A variant may have one or two amino acid mutations, such as substitutions, compared to the set of VH CDR1-3 described above. In embodiments, an antibody according to the present disclosure comprises a CDR having a sequence with one or two mutations, such as substitutions, compared to the VH CDR sequence of any antibody described herein. For example, an antibody according to the present disclosure may comprise a VH CDR having the sequence of any antibody described above, except that one or two of the CDRHs contain substitutions, and the total number of substitutions across the CDRHs does not exceed two. In embodiments, a variant may have one, two, or three, preferably at most one or two, substitutions in each of one or more of the VH CDR1-3 described above. The CDRH1 region of any antibody or fragment described herein may have a length of 8 amino acids. The CDRH2 region of any antibody or fragment described herein may have a length of 7 amino acids. The CDRH3 region of any antibody or fragment described herein may have a length of 13 amino acids. In embodiments, variants may have VH CDRs that have at least 70%, at least 80%, or at least 90% sequence identity with any set of VH CDRs described herein. For example, a variant combining CDRHs 1-3 that is 28 amino acids in length may have up to 8, up to 5, or up to 2 amino acid substitutions compared to the set of VH CDRs described herein. In embodiments, variants may have one or two substitutions, including or consisting of a substitution at position 58 of CDRH2. The substitution may be N58Y.

[0103] An antibody according to the present disclosure may have a VH having the following CDRH: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20, CDRH2 comprising the amino acid sequence of SEQ ID NO: 21, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 23. These are the VH CDRs of ATL_5262 and all of its variants except ATL_6178 (i.e., ATL_6033, 6034, 6035, 6036, 6037, 6038, 6039, 6177).

[0104] An antibody according to the present disclosure may have a VH having the following CDRH: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20, CDRH2 comprising the amino acid sequence of SEQ ID NO: 22, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 23. These are the VH CDRs of ATL_6178.

[0105] An antibody according to the present disclosure may have a VH having the following CDRHs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 144, CDRH2 comprising the amino acid sequence of SEQ ID NO: 152, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 161. These are the VH CDRs or ATL_6532. These are the CDRs of ATL_6533. An antibody according to the present disclosure may have a light chain variable domain (VL) having the CDRs of the light chain variable domain (VL) of antibody ATL_5262 (identical to those of ATL_6178), ATL_6002, or ATL_6003, or ATL6187, or more specifically ATL_5262, or ATL_6002. An antibody according to the present disclosure may have the following light chain CDRs: CDRL1: SEQ ID NO: 24, 31 or 32, CDRL2: SEQ ID NO: 34, and CDRL3: may have SEQ ID NO: 40 or 47 or 121.

[0106] In antibodies according to the present disclosure, at least one of the VL CDR1-3 sequences may be altered. Variants may have one, two, or three amino acid mutations, such as substitutions, compared to the set of VL CDR1-3 described above. In embodiments, antibodies according to the present disclosure comprise CDRs having sequences with one to three mutations, such as substitutions, compared to the VL CDR sequences of any antibody described herein. For example, an antibody according to the present disclosure may comprise a VL CDR having the sequence of any antibody described above, except that one to three of the CDRLs contain substitutions, and the total number of substitutions across the CDRLs does not exceed three. In embodiments, variants may have one, two, or three, preferably at most one or two, substitutions in each of one or more of the VL CDR1-3 described above. The CDRL1 region of any antibody or fragment described herein may be 6 to 12 amino acids in length, preferably 6 amino acids in length. The CDRL2 region of any antibody or fragment described herein may be 3 amino acids in length. The CDRL3 region of any antibody or fragment described herein can have a length of 9 or 10 amino acids, preferably 9. In embodiments, variants can have VL CDRs that have at least 70%, at least 80%, or at least 90% sequence identity with any set of VL CDRs described herein. For example, a variant having a CDRL 1-3 combination that is 18 amino acids in length (such as a variant of CDRLs ATL_0005262 or ATL0006003) can have up to 5, up to 3, or up to 1 amino acid substitution compared to the set of VL CDRs described herein.

[0107] An antibody according to the disclosure may have a VL having the following CDRLs: CDRL1: SEQ ID NO: 24, CDRL2: SEQ ID NO: 34, CDRL3: SEQ ID NO: 40. These are the VL CDRs of antibodies ATL_5262, 6033, 6034, 6035, 6036, 6037, 6038, 6039, 6177, 6178, 6187, and 6191.

[0108] The antibody according to the present disclosure comprises: (a) Framework sequences of ATL_5262 (and 6002, 6003, 6187, 6191): LFWR1 of SEQ ID NO: 63, LFWR2 of SEQ ID NO: 71, LFWR3 of SEQ ID NO: 77, and LFWR4 of SEQ ID NO: 85; (b) Framework sequences of ATL_5996: LFWR1 of SEQ ID NO: 64, LFWR2 of SEQ ID NO: 71, LFWR3 of SEQ ID NO: 78, and LFWR4 of SEQ ID NO: 86; (c) Framework sequences of ATL_5997: LFWR1 of SEQ ID NO: 65, LFWR2 of SEQ ID NO: 72, LFWR3 of SEQ ID NO: 79, and LFWR4 of SEQ ID NO: 87; (d) Framework sequences of ATL_5998: LFWR1 of SEQ ID NO: 66, LFWR2 of SEQ ID NO: 73, LFWR3 of SEQ ID NO: 80, and LFWR4 of SEQ ID NO: 87; (e) Framework sequences of ATL_5999: LFWR1 of SEQ ID NO: 67, LFWR2 of SEQ ID NO: 73, LFWR3 of SEQ ID NO: 81, and LFWR4 of SEQ ID NO: 87; (f) Framework sequences of ATL_6000: LFWR1 of SEQ ID NO: 68, LFWR2 of SEQ ID NO: 74, LFWR3 of SEQ ID NO: 82, and LFWR4 of SEQ ID NO: 88; (g) Framework sequences of ATL_6001: LFWR1 of SEQ ID NO: 63, LFWR2 of SEQ ID NO: 75, LFWR3 of SEQ ID NO: 77, and LFWR4 of SEQ ID NO: 89; (h) Framework sequences of ATL_6004: LFWR1 of SEQ ID NO: 69, LFWR2 of SEQ ID NO: 72, LFWR3 of SEQ ID NO: 83, and LFWR4 of SEQ ID NO: 85; (i) Framework sequences of ATL_6005: LFWR1 of SEQ ID NO: 70, LFWR2 of SEQ ID NO: 76, LFWR3 of SEQ ID NO: 84, and LFWR4 of SEQ ID NO: 90; (j) or a VL comprising a framework sequence that contains up to 5 or up to 10 mutations, eg, substitutions, compared to the above sequences.

[0109] An antibody according to the present disclosure may have a VL comprising the framework sequence of any of ATL_6530, ATL_6531, ATL_6532, ATL_6533, ATL_6534, ATL_6535, ATL_6536, ATL_6537, ATL_6538, ATL_6539.

[0110] The VH CDR1-3 and optionally the VL CDR1-3 of any of the above antibodies may also be particularly useful in conjunction with several different framework regions. Thus, light and / or heavy chains having CDR1-3 as described above may have alternative framework regions. Suitable framework regions are known in the art and are described, for example, in M. Lefranc & G. Le Franc (2001) "The Immunoglobulin Facts Book," Academic Press.

[0111] Antibodies of the disclosure may have VH domains CDRH1, CDRH2, and CDRH3 in a germline framework, in some embodiments, antibodies of the disclosure may have a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3 in a germline framework, provided that position 67 is Q according to standard IMGT numbering.

[0112] An antibody of the disclosure may have a heavy chain variable domain (VH) having framework sequences FWRH1, FWRH2, FWRH3, and FWRH4 of any of the ATL_5262 variants (i.e., antibodies ATL_0005996, ATL_0005997, ATL_0005998, ATL_0005999, ATL_0006000, ATL_0006001, ATL_0006002, ATL_0006003, ATL_0006004, ATL_0006005, ATL_0006036, ATL_0006039, ATL_0006177, ATL_0006178).

[0113] Antibodies according to the present disclosure may have a VH having the following framework sequences: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 51, HFWR3 of SEQ ID NO: 55, and HFWR4 of SEQ ID NO: 62, or framework sequences containing 1 to 5 mutations, e.g., substitutions, compared to these framework sequences.

[0114] Mutations in the FWRH sequence may include one or more or all of: (i) HFWR2: positions 40, 49; (ii) HFWR3: positions 80, 82, 86. In embodiments, the mutations in the framework sequences are substitutions and are selected from the following positions according to standard IMGT numbering: (i) HFWR2: position 40: T40S, position 49: R49G; (ii) HFWR3: position 80: I80V, position 82: T82K, position 86: H86Q (according to standard IMGT numbering).

[0115] The mutation in the FWRH sequence may not involve a substitution at position 67 in the standard IMGT numbering.

[0116] Antibodies according to the present disclosure may have a VH having the following framework sequences: HFWR1 of SEQ ID NO:50, HFWR2 of SEQ ID NOs:51-54, HFWR3 of SEQ ID NOs:55-61, and HFWR4 of SEQ ID NO:62.

[0117] Antibodies according to the present disclosure may have the following framework sequence: HFWR1:QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 50) of ATL_6187 or ATL_6191, HFWR2:WSWIRQPPGKGLEWIGY (SEQ ID NO: 52) or WSWIRQTPGKGLEWIGY (SEQ ID NO: 195) of ATL_6187 or ATL_6191, HFWR3 of ATL_6187 or ATL_6191: NQNPSLKSRVTMSVDSSKSQLSLKLTSVTAADTAVYYC (SEQ ID NO: 196) or NYNPSLKSRVTISVDTFKNQFSLKLTSVTAADTAVYYC (SEQ ID NO: 197), HFWR4:WGQGILVIVSS (SEQ ID NO: 198) or WGQGTLVTVSS (SEQ ID NO: 62) of ATL_6187 or ATL_6191, or The heavy chain variable domain (VH) may comprise framework sequences that contain 1 to 10 or 1 to 5 mutations, for example substitutions, compared with these framework sequences.

[0118] Mutations in FWRH may include one or more or all of the following: - in HFWR1: at position 25, optionally the mutation is the substitution V25Q; - in HFWR2: at position 43, optionally the mutation is the substitution I42V; at position 49, optionally the mutation is the substitution R49A; - in HFWR3: at position 68, optionally, the mutation is the substitution N68S; at position 78, optionally, the mutation is the substitution I78M; at position 81, optionally, the mutation is the substitution D81Y; at position 83, optionally, the mutation is the substitution S83F; at position 84, optionally, the mutation is the substitution K84T; at position 87, optionally, the mutation is the substitution F87L; - in HFWR4: at position 122, optionally the mutation is the substitution I122K; at position 125, optionally the mutation is the substitution T125I.

[0119] An antibody according to the present disclosure may have a heavy chain variable domain (VH) comprising the framework sequence of any of antibodies ATL_6530, 6531, 6532, 6533, 6534, 6535, 656, 6537, 6538, or 6539, or a framework sequence containing 1 to 5 mutations, e.g., substitutions, relative to these framework sequences. (a) HFWR1 of QVQLLETGGGLVQPGGSLRLSCAAS (SEQ ID NO: 199), HFWR2 of MNWVRQAPGKGLEWVSS (SEQ ID NO: 200), HFWR3 of YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 201), HFWR4 of WGQGTLVTVSS (SEQ ID NO: 62) (framework sequence of ATL_6530), (b) HFWR1 of EVQLVESGGVVVQPGGSLRLSCAAS (SEQ ID NO: 202), HFWR2 of MHWVRQAPGKGLEWVSL (SEQ ID NO: 203), HFWR3 of YYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYC (SEQ ID NO: 204), HFWR4 of WGQGTLVTVSS (SEQ ID NO: 62) (framework sequence of ATL_6531) (c) HFWR1 of QVTLKESGAEVKKPGASVKVSCKAS (SEQ ID NO: 205), HFWR2 of MHWVRQAPGQGLEWMGR (SEQ ID NO: 206), HFWR3 of NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC (SEQ ID NO: 207), HFWR4 of WGQGTMVTVSS (SEQ ID NO: 208) (framework sequence of ATL_6532) (d) HFWR1 of QVQLQQSGGGLVKPGGSLRLSCAAS (SEQ ID NO: 209), HFWR2 of MSWIRQAPGKGLEWVSY (SEQ ID NO: 210), HFWR3 of NYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC (SEQ ID NO: 211), HFWR4 of WGQGTMVTVSS (SEQ ID NO: 208) (framework sequence of ATL_6533) (e) HFWR1 of QVQLVESGAEVKKPGASVKVSCKAS (SEQ ID NO: 212), HFWR2 of MHWVRQAPGQGLEWMGR (SEQ ID NO: 206), HFWR3 of NYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC (SEQ ID NO: 207), HFWR4 of WGQGTMVTVSS (SEQ ID NO: 208) (framework sequence of ATL_6534) (f) HFWR1 of EVQLLETGGGLVQPGRSLRLSCAAS (SEQ ID NO: 13), HFWR2 of MHWVRQAPGKGLEWVSG (SEQ ID NO: 14), HFWR3 of GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC (SEQ ID NO: 15), HFWR4 of WGQGTMVTVSS (SEQ ID NO: 208) (framework sequence of ATL_6535) (g) HFWR1 of EVQLLESAGGVVQLGRSLRLSCAAS (SEQ ID NO: 216), HFWR2 of MHWVRQAPGKGLEWVAI (SEQ ID NO: 217), HFWR3 of YYAGSVGGRFTISRDNSKNTLYLQMDSLRSDDTAVYYC (SEQ ID NO: 218), HFWR4 of WGHGTMVTVSS (SEQ ID NO: 219) (framework sequence of ATL_6536) (h) HFWR1 of QVQLVQSGAEVKKPGESLKISCKGS (SEQ ID NO: 220), HFWR2 of IGWVRQMPGKGLEWMGI (SEQ ID NO: 221), HFWR3 of RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYC (SEQ ID NO: 222), HFWR4 of WGQGTTVTVSS (SEQ ID NO: 223) (framework sequence of ATL_6537) (i) HFWR1 of QVQLQESGPGLVKPSGTLSLTCAVS (SEQ ID NO: 224), HFWR2 of WSWVRQPPGKGLEWIGE (SEQ ID NO: 225), HFWR3 of NYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYC (SEQ ID NO: 226), HFWR4 of WGQGTLVTVSS (SEQ ID NO: 62) (framework sequence of ATL_6538) (j) HFWR1 of QVQLVESGGGLVQPGGSLRLSCSAS (SEQ ID NO: 227), HFWR2 of MSWVRQAPGKGLEWVST (SEQ ID NO: 228), HFWR3 of YYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYC (SEQ ID NO: 229), HFWR4 of WGQGTMVTVSS (SEQ ID NO: 208) (framework sequence of ATL_6539) (k) or a VH having a framework sequence containing 1 to 5 mutations, for example substitutions, compared to these framework sequences.

[0120] Mutations in FWRH can include mutations at one or more or all of the following positions: - In HFWR1: 1, optionally, the mutation is the substitution Q1E; 1, optionally, the mutation is the substitution Q3T; 6, optionally, the mutation is the substitution E6Q; 7, optionally, the mutation is the substitution S7T; 12, optionally, the mutation is the substitution L12V; 13, optionally, the mutation is the substitution V13K; 15, optionally, the mutation is the substitution P15L; 16, optionally, the mutation is the substitution S16G; 19, optionally, the mutation is the substitution L19V; 21, optionally, the mutation is the substitution L21V; 22, optionally, the mutation is the substitution S22T; - in HFWR2: 48, optionally wherein the mutation is the substitution K48Q; - in HFWR3: 74, optionally, the mutation is the substitution S74G; 75, optionally, the mutation is the substitution R75Q; 76, optionally, the mutation is the substitution V76F; 78, optionally, the mutation is the substitution I78M; 79, optionally, the mutation is the substitution S79T; 83, optionally, the mutation is the substitution S83A; 84, optionally, the mutation is the substitution K84I; 85, optionally, the mutation is the substitution N85S; 89, optionally, the mutation is the substitution L89M; 93, optionally, the mutation is the substitution S93R; 96, optionally, the mutation is the substitution A96S; - In HFWR4:120, optionally the mutation is the substitution Q120H.

[0121] As used herein, the antibodies may have a VH (and optionally a VL) region comprising an amino acid sequence that has a high percentage of sequence identity to the VH and / or VL amino acid sequences set forth above.

[0122] For example, antibodies according to the present disclosure include antibodies that bind to UNC5C and have a VH region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH region amino acid sequence of any antibody described herein, such as ATL_5262, ATL_6036, ATL_6039, ATL_6177, ATL_6178, ATL_6187, ATL_6191, ATL_6530-6539 (SEQ ID NOs: 1, 5, 8, 91, 93, 112, 113, 122-131). It should be noted that any sequence obtained by applying one or more conservative amino acid substitutions to the sequences described herein is also encompassed, provided that the overall sequence identity is within the range provided and the resulting antibody still binds to UNC5C and competes with / mimics netrin-1 binding to UNC5C.

[0123] Antibodies of the disclosure may have VL domains CDRL1, CDRL2, and CDRL3 within a germline framework. Antibodies of the disclosure may have a light chain variable domain (VL) with framework sequences LFWR1, LFWR2, LFWR3, and LFWR4 of any of the ATL_5262 variants, including ATL_5262, ATL_6036, ATL_6039, ATL_6177, or ATL_6178.

[0124] Thus, an antibody of the present disclosure may have a light chain variable domain (VL) having the following framework sequences: LFWR1 of SEQ ID NO: 63, LFWR2 of SEQ ID NO: 71, LFWR3 of SEQ ID NO: 77, and LFWR4 of SEQ ID NO: 85 or 87, or a set of FWRs containing 1 to 5 amino acid substitutions compared to the set of FWRs above.

[0125] Alternatively, or in addition, an antibody of the present disclosure may have a VL region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL region amino acid sequence of any antibody described herein, such as ATL_5262, ATL_5998, ATL_6001, ATL_6002, ATL_6003, ATL_6177, ATL_6178, ATL_6187, ATL_6191, ATL6530-6539 (SEQ ID NOs: 9, 12, 15, 16, 17, 92, 94, 114, 132-141). For example, an antibody of the present disclosure may have a VL region comprising an amino acid sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL region amino acid sequence of antibody ATL_5262 or ATL_6002 (SEQ ID NOs: 9, 16). For example, an antibody of the present disclosure may have a VL region comprising an amino acid sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL region amino acid sequence of antibody ATL_5262 or ATL_6178 (SEQ ID NOs: 9, 94).

[0126] The antibodies of the present disclosure can have L-type or K-type VL regions.

[0127] The antibodies described herein may comprise a heavy chain variable domain (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1, and a light chain variable domain (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9.

[0128] The antibodies described herein may comprise a heavy chain variable domain (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93, and a light chain variable domain (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94.

[0129] The antibodies described herein include antibodies having a heavy chain variable domain (VH) comprising the sequence of the VH of any antibody described herein and a light chain variable domain (VL) comprising the sequence of the VH of any antibody described herein. In particular, the antibodies described herein comprise (i) a heavy chain variable domain (VH) comprising the sequence of VH of ATL_5262, ATL_6177, ATL_6178, ATL_6033, ATL_6034, ATL6035, ATL_6036, ATL_6037, ATL_6038, ATL_6039, ATL_5998, ATL_6001, ATL_6002, ATL_6003, ATL_6187, ATL_6191, ATL_6530, ATL_6531, ATL_6532, ATL_6533, ATL_6534, ATL_6535, ATL_6536, ATL_6537, ATL_6538, ATL_6539 H), and (ii) antibodies having a light chain variable domain (VL) comprising the sequence of the VL of ATL_5262, ATL_6177, ATL_6178, ATL_6033, ATL_6034, ATL6035, ATL_6036, ATL_6037, ATL_6038, ATL_6039, ATL_5998, ATL_6001, ATL_6002, ATL_6003, ATL_6187, ATL_6191, ATL_6530, ATL_6531, ATL_6532, ATL_6533, ATL_6534, ATL_6535, ATL_6536, ATL_6537, ATL_6538, or ATL_6539. In particular, (i) a heavy chain variable domain (VH) comprising the sequence of the VH of ATL_5262, ATL_6177, ATL_6178, ATL_6033, ATL_6034, ATL6035, ATL_6036, ATL_6037, ATL_6038, ATL_6039, ATL_5998, ATL_6001, ATL_6002, ATL_6003, ATL_6187, ATL_6191, and (ii) Also described are antibodies having a light chain variable domain (VL) comprising the sequence of the VL of ATL_5262, ATL_6177, ATL_6178, ATL_6033, ATL_6034, ATL6035, ATL_6036, ATL_6037, ATL_6038, ATL_6039, ATL_5998, ATL_6001, ATL_6002, ATL_6003, ATL_6187, ATL_6191.Also described are antibodies having (i) a heavy chain variable domain (VH) comprising the sequence of the VH of ATL_6530, ATL_6531, ATL_6532, ATL_6533, ATL_6534, ATL_6535, ATL_6536, ATL_6537, ATL_6538, ATL_6539, and (ii) a light chain variable domain (VL) comprising the sequence of the VL of ATL_6530, ATL_6531, ATL_6532, ATL_6533, ATL_6534, ATL_6535, ATL_6536, ATL_6537, ATL_6538, ATL_6539.

[0130] The antibodies of the present disclosure are derived from human antibodies and may therefore be referred to as human antibodies. The antibodies of the present disclosure were obtained by pairing a human VH domain with a VL domain using a transformer-based model. Thus, the antibodies of the present disclosure may differ from naturally occurring antibodies at least by their VH-VL pairing. The antibodies of the present disclosure may comprise human VH and / or VL sequences. The antibodies of the present disclosure may be formulated as human IgG1 antibodies or variants thereof. The antibodies of the present disclosure may have human framework sequences and / or human Fc domains.

[0131] The overall identity percentage of the variable region or full-length heavy / light chain sequences can be combined with a particular CDR sequence from the same antibody.

[0132] The percentage (%) of sequence identity is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the comparison sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence identity, without considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of each sequence. If the aligned sequences are of different lengths, the sequence identity of the shorter comparison sequence can be determined over the entire length of the longer given sequence, or if the comparison sequence is longer than the given sequence, the sequence identity of the comparison sequence can be determined over the entire length of the shorter given sequence. Sequence identity can be defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters can be used, with a gap creation penalty of 12 and a gap extension penalty of 4. Although the use of GAP may be preferred, other algorithms may also be used, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215:405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), SSEARCH (Smith and Waterman (1981) J. Mol. Biol. 147:195-197;), HMMER3 (Johnson LS et al BMC Bioinformatics. 2010 Aug 18;11():431) or, generally, the TBLASTN program of Altschul et al. (1990) supra, using default parameters (e.g., Pearson Curr Prot Bioinformatics (2013) Chapter 3 Univ. 3.1). doi:10.1002 / 0471250953.bi0301s42). In particular, the psi-Blast algorithm may be used (Altschul et al. Nucl. Acids Res. (1997) 25 3389-3402).Sequence identity and similarity can also be determined using Genomequest™ software (Gene-IT, Worcester MA USA). Sequence comparison is preferably performed over the entire length of the relevant sequences being compared.

[0133] The antibodies of the present disclosure may contain one or more substitutions within the framework of the VH and / or VL regions. As used herein, a "substitution" refers to the replacement of one amino acid with another amino acid at a particular position compared to the same position in a baseline molecule. In some embodiments, the baseline molecule is an antibody exemplified herein, e.g., ATL_5262, ATL_6177, ATL_6178, ATL_6002, or ATL_6003 (VH sequences of SEQ ID NOs: 1, 91, 93; VL sequences of SEQ ID NOs: 9, 16, 17).

[0134] In some embodiments, an antibody or fragment thereof according to the present disclosure can cross the blood-brain barrier. In a preferred embodiment, an antibody of the present disclosure is a bispecific antibody. For example, an antibody according to the present disclosure may have one scFv chain that binds to a receptor in the brain, such as the transferrin receptor (Yu et al., Sci Transl Med. 2014 Nov 5;6(261):261ral54), and one scFv chain that binds to UNC5C described herein. In some embodiments, an antibody according to the present disclosure comprises an antibody or fragment thereof that binds to UNC5C described herein (e.g., an antibody, scFv, sdAb, etc.) and an additional binding moiety that binds to another target. The other target may be a receptor in the brain, such as the transferrin receptor. The additional binding moiety may be an antibody, scFv, nanobody, or aptamer. The two binding moieties of such a bispecific molecule may form a fusion protein.

[0135] Also described herein are single domain antibodies (sdAbs), also known as nanobodies, which comprise the heavy chain CDR and / or VH sequences of any of the antibodies described herein. Accordingly, also described herein are antibodies or fusion molecules comprising a nanobody that binds to UNC5C as described herein and a nanobody that binds to a receptor in the brain. Also described herein are antibodies that are fusion molecules comprising an scFV chain or nanobody that binds to UNC5C as described herein and an aptamer that binds to a receptor in the brain.

[0136] Isolated nucleic acids encoding the antibodies, antigen-binding fragments, or polypeptides described herein are provided. Also provided are vectors containing the nucleic acids described herein and host cells containing the vectors. For example, the host cells can be eukaryotic or mammalian cells, such as Chinese Hamster Ovary (CHO) cells, or prokaryotic cells (e.g., E. coli). In some embodiments, the vector is a viral vector, such as a bacteriophage.

[0137] Further provided is a method for producing an antibody or antibody fragment described herein, the method comprising culturing a host cell described herein under conditions suitable for expression of a vector encoding the antibody or antibody fragment, and isolating and / or purifying the antibody or antibody fragment. The method further comprises formulating the antibody or antibody fragment into a composition comprising at least one additional component.

[0138] Therapeutic Uses and In Vitro / In Vivo Effects The antibody described herein can reduce UNC5C-mediated apoptosis.For example, the antibody described herein can reduce UNC5C-mediated apoptosis in neurons, for example, induced pluripotent stem cell (iPSC)-derived neurons, and / or neuroblastoma cells, such as SH-SY5Y cells.Those skilled in the art will be familiar with methods for measuring apoptosis, such as by measuring caspase 3 / 7 production as described herein, or by measuring cell viability by staining cells with live / dead cell markers, such as DAPI (4',6-diamidino-2-phenylindole).

[0139] As described above, the antibody of the present disclosure has been found to mimic / compete the binding of netrin-1 to UNC5C. Netrin-1 levels correlate with the severity of dementia, and netrin-1 is reduced in mild cognitive impairment and AD patients (Ju T, et al., 2022).

[0140] Netrin-1 has been shown to protect against β-amyloid-induced neurotoxicity through an NF-κB / Nrf2-dependent mechanism (Zamani et al., 2020). The antibodies described herein can reduce β-amyloid-induced neurotoxicity. This can be assessed by measuring apoptosis, for example, caspase 3 / 7 production, in neurons treated with β-amyloid as described herein.

[0141] Netrin-1 has been shown to be involved in synaptic plasticity, i.e., the ability of neurons to change connections between neuronal networks in response to stimuli, in adults. UNC5C is a netrin-1 receptor that plays a role in axon growth and guidance. This is a mechanism that has been most studied in development and may be important for preventing neuronal degeneration. Therefore, the antibodies and fragments thereof described herein may be effective and / or used to promote synaptic health by promoting synaptic plasticity, which can be assessed, for example, by quantifying the effect of the antibody on the number of processes per cell, the length of neurite outgrowth, and branching. For example, the methods described herein and in Spiijkers et al., 2021, can be used.

[0142] The effect of promoting synaptic health can also be evaluated by quantifying the expression of synaptic proteins, for example, using Western blot. The synaptic proteins can be selected from the group consisting of PSD95, synaptophysin, synapsin-1, synaptotagmin-1, neurofilament-L, and / or GAP-43, and increased expression of any or all of these indicates improved synaptic health. Measuring neuronal synchrony and firing rate using electrophysiological assessment methods familiar to those skilled in the art, such as measuring extracellular field potentials using electrodes, is another method of evaluating synaptic health, with increased synchrony indicating enhanced synaptic health (e.g., increased synaptic connectivity).

[0143] The antibodies described herein may also improve neuronal function, such as neuronal activity and / or burst potential, which may be determined by measuring neuronal extracellular field potentials, for example, by using a microelectrode array as described herein.

[0144] Antibodies according to the present disclosure may improve neuronal survival and / or synaptic function. For example, antibodies of the present disclosure may reduce amyloid toxicity. Antibodies of the present disclosure may reduce cell death of amyloid-treated neurons in vitro. Cell death may be measured by measuring caspase 3 / 7 signaling in response to beta-amyloid-induced cytotoxicity. Antibodies of the present disclosure may increase the expression of one or more synaptic proteins, such as GAP-43. Antibodies of the present disclosure may at least partially rescue neuronal cell death and axonal damage caused by netrin-1 depletion. Antibodies of the present disclosure may improve neuronal connectivity and / or firing rate and / or synchrony in motor neurons. Antibodies of the present disclosure may improve neuronal connectivity and / or firing rate and / or synchrony in motor neurons of subjects with ALS. Antibodies of the present disclosure may improve the function and / or survival of dopaminergic neurons. For example, antibodies of the present disclosure may at least partially rescue the effects of UNC5C or netrin-1 haploinsufficiency on dopaminergic neuron innervation to the medial prefrontal cortex and / or dopamine content in the medial prefrontal cortex. This can be assessed by determining the increased response to amphetamine in netrin-1 haploinsufficient mice treated with antibodies of the present disclosure. Antibodies of the present disclosure may reduce neurodegeneration of dopaminergic neurons. This can be assessed by measuring neurodegeneration upon exposure to a neurotoxin such as 6-hydroxydopamine (6-OHDA), as evidenced by reduced contralateral limb use. Thus, also described herein are methods for improving the survival and / or function (including synaptic function) of motor neurons and / or dopaminergic neurons in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody of the present disclosure. Similarly, also described herein are methods for treating diseases or disorders associated with the dysfunction or death of motor neurons and / or dopaminergic neurons, comprising administering a therapeutically effective amount of an antibody of the present disclosure.

[0145] The described antibodies and fragments thereof may find use in therapy.

[0146] The subject to be treated or diagnosed may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be male or female. The subject may be a patient. Therapeutic use may be in humans or animals (veterinary use).

[0147] Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by several routes, including, but not limited to, parenteral, intravenous, intraarterial, intramuscular, oral, and nasal. Medicaments and compositions may be formulated for injection.

[0148] Pharmaceutical compositions can be prepared using pharmaceutically acceptable "carriers" composed of materials considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe," e.g., physiologically tolerated, and typically do not produce allergic or similar adverse reactions, such as stomach upset, when administered to humans. In some embodiments, the term refers to molecular entities and compositions approved by a U.S. federal or state regulatory agency as a GRAS listed under Sections 5, 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, subject to premarket review and approval by the FDA or similar listing, the U.S. Pharmacopeia, or another generally recognized pharmacopeia, for use in animals, more specifically, humans. The term "carrier" refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and methods of formulating pharmaceutical compositions using such carriers.

[0149] The pharmaceutical compositions provided herein may contain one or more excipients, such as solvents, solubility enhancers, suspending agents, buffers, isotonicity agents, antioxidants, or antimicrobial preservatives. When used, the excipients of the composition do not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient, i.e., the anti-CFH antibody used in the composition. Thus, a skilled artisan will understand that a composition is provided in which there is no incompatibility between any of the components of the dosage form. The excipient may be selected from the group consisting of a buffer, a solubilizer, an isotonicity agent, a chelating agent, an antioxidant, an antimicrobial agent, and a preservative.

[0150] Administration is preferably in a "therapeutically effective amount," which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescribing treatment, e.g., determining dosage, is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20 th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0151] Conditions treatable according to the present disclosure include any in which UNC5C plays a role, including neurodegenerative disorders, particularly those characterized by increased neuronal apoptosis, impaired synaptic health, and / or altered synaptic plasticity (e.g., reduced or abnormal synaptic plasticity). Antibodies of the present disclosure were initially identified through analysis of resilient frontotemporal dementia (FTD) patients. These antibodies were subsequently found in other resilient and control individuals with Alzheimer's disease (AD), Parkinson's disease (PD), and in resilient centenarians, demonstrating relevance to other neurodegenerative diseases beyond FTD. Accordingly, antibodies of the present disclosure for use as pharmaceuticals are also described herein. Antibodies of the present disclosure for use in the treatment or prevention of neurodegenerative diseases are also described herein. Antibodies of the present disclosure for use in the manufacture of pharmaceuticals, such as pharmaceuticals for the treatment or prevention of neurodegenerative diseases or disorders, are also described herein. Also described herein is a method of treating a subject diagnosed with or at risk of having a neurodegenerative disorder, comprising administering to the subject a therapeutically effective amount of an antibody described herein. The neurodegenerative disease or disorder may include one or more of the following: FTD, AD, HD, Parkinson's disease (PD), human immunodeficiency virus (HIV)-induced encephalitis, chronic traumatic encephalopathy (CTE), vascular dementia, prion disease, Lewy body disease, spinal muscular atrophy (SMA); motor neuron disease (MND), such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinocerebellar ataxia (SCA) types 1, 2, 6, 7, and 17, Machado-Joseph disease (MJD / SCA3), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy type 1 (SMAX1 / SBMA), Anderson-Fabry (X-linked Fabry disease), and DNAJB6 myopathy. In embodiments, the neurodegenerative disease is selected from FTD, AD, HD, MND, such as ALS and PD. In embodiments, the neurodegenerative disease is an MND, such as ALS. In embodiments, the neurodegenerative disease is FTD, AD, HD, or PD.

[0152] The compounds of the present disclosure may be used in therapy with an additional therapeutic agent. As used herein, an "additional therapeutic agent" is an additional compound, protein, vector, antibody, cell, or entity that has a therapeutic effect. The antibody may be co-administered with the additional therapeutic agent. The antibody may be co-formulated with the additional therapeutic agent. The antibody may be administered sequentially, before or after the additional therapeutic agent.

[0153] The antibodies described herein can be used as biomarkers that indicate a subject is likely to respond to therapy using the antibodies or antibody fragments described herein. A method for determining whether a subject is likely to respond to treatment with the antibodies or fragments thereof described herein, comprising obtaining BCR sequence data from the subject and using the sequence data to determine whether the subject's BCR repertoire includes one or more antibodies that can bind to UNC5C (e.g., an antibody described herein, e.g., an antibody having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to any particular antibody or antibody fragment described herein), wherein a subject whose BCR repertoire does not include one or more antibodies that can bind to UNC5C is likely to respond to treatment with the antibodies or antibody fragments thereof described herein.

[0154] Thus, also described herein is a method for treating a subject diagnosed as having or likely to have a disease in which UNC5C plays a role (e.g., a neurodegenerative disorder), the method comprising obtaining BCR sequence data from the subject, using the sequence data to determine whether the subject's BCR repertoire contains one or more antibodies that are likely to bind to UNC5C, and administering a therapeutically effective amount of an antibody or antibody fragment thereof described herein to a subject whose BCR repertoire does not contain one or more antibodies that are likely to bind to UNC5C.

[0155] Some methods of the present disclosure include a sample containing cells. The sample may be a culture of cells grown in vitro. For example, the culture may include a suspension of cells or cells cultured in a culture plate or dish.

[0156] Methods according to the present disclosure may be performed or products may exist in vitro, ex vivo, or in vivo. The term "in vitro" is intended to encompass experiments using materials, biological substances, cells and / or tissues in laboratory conditions or culture, while the term "in vivo" is intended to encompass experiments and procedures using intact multicellular organisms. "Ex vivo" refers to something that exists or occurs outside of an organism, e.g., outside the human or animal body, and may be on tissues (e.g., whole organs) or cells removed from an organism.

[0157] According to some aspects of the present disclosure, there is provided a kit of parts comprising an antibody according to the invention, hi some embodiments, the kit comprises an antibody according to the invention and one or more of: a reagent for use in immunochemistry, an antibody immobilized on a solid support, a means for labeling the antibody, a means for linking the antibody to a cytotoxic moiety, and an additional therapeutic agent.

[0158] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, and expressed in their specific form or as means for performing a disclosed function or as methods or processes for obtaining a disclosed result, may be utilized, as appropriate, separately or in any combination of such features, to realize the invention in diverse forms thereof.

[0159] While the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes can be made to the described embodiments without departing from the spirit and scope of the invention.

[0160] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of improving the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0161] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0162] Throughout this specification, including the claims which follow, unless the context indicates otherwise, the words "comprise" and "include", and variations such as "comprises" and "comprising", are understood to mean the inclusion of a stated integer, step, group of integers, or group of steps, but not the exclusion of any other integer, step, group of integers, or group of steps.

[0163] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in connection with numerical values ​​is optional and may mean, for example, + / - 10%.

[0164] All references cited herein are incorporated by reference in their entirety. For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0165] array Exemplary sequences of the antibodies described herein are provided in Figures 21, 8B, 32 (aligned by IMGT position numbering) and in the table below.

[0166] [Table 1-1]

[0167] [Table 1-2]

[0168] [Table 1-3]

[0169] [Table 1-4]

[0170] [Table 1-5]

[0171] [Table 1-6]

[0172] [Table 1-7]

[0173] [Table 1-8]

[0174] [Table 1-9]

[0175] [Table 1-10]

[0176] [Table 1-11]

[0177] [Table 1-12]

[0178] [Table 1-13]

[0179] [Table 1-14]

[0180] [Table 1-15] [Example]

[0181] These examples demonstrate the identification of potential therapeutic antibodies from a cohort of subjects believed to have or be at risk for developing frontotemporal dementia or Parkinson's disease (PD) (Example 1), and their in vitro and in vivo characterization (Examples 2-5, 7-19) and optimization (Example 6). Additional antibodies exhibiting properties similar to those of Examples 1 and 6 (Example 20) were subsequently identified.

[0182] Materials and Methods Human and mouse UNC5C ELISA UNC5C recombinant human antigen (Fc-tagged R&D Systems, no. 1005-UN; or in-house production from HEK293F cells with an N-terminal His or FLAG tag (SEQ ID NO: 98) - in-house human antigen was used to confirm that the results were not driven by the Fc tag) or mouse antigen (in-house production from HEK393F cells with an N-terminal His or FLAG tag (SEQ ID NO: 97 - the expressed mouse UNC5C extracellular domain contains AA 41-380 of the mouse UNC5C sequence); rat antigen (SEQ ID NO: 103); or cynomolgus monkey antigen (SEQ ID NO: 104) were directly absorbed onto the ELISA plates at 3 μg / ml (50 μl per well) and incubated overnight at 4°C. The plates were washed with PBS. The plates were blocked with 200 μl / well of blocking solution (1% BSA w / v in PBS) for 1 hour at room temperature. After this time, the blocking solution was removed, and the antibody to be evaluated was diluted in a dilution series (1 μM to 0.05 nM) in blocking solution (1% BSA w / v in PBS) and applied to the plate. The plate was incubated for 1 hour at room temperature. The plate was washed with PBS / 0.1% Tween. Anti-human IgG HRP (Jackson ImmunoResearch, no. 109-035-097) was added to the plate and incubated for 1 hour at room temperature to detect antibody binding. The plate was washed with PBS / 0.1% Tween and TMB solution (Life Technology, no. 002023). The plate was incubated for 5 minutes at room temperature, after which stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm on a Molecular Devices FilterMaxF5 plate reader. Analysis was performed using a nonlinear curve-fitting algorithm on GraphPad Prism, and EC50 values ​​were calculated.

[0183] Targeted Deconvolution Using Retrogenix Proteomics The pool of 20 antibodies used in this screen included 18 antibodies identified from the FTD sample cohort analysis, one antibody from the Huntington's cohort, and one positive control anti-PDL1 antibody. Antibodies were mixed in equal proportions (concentrations) to create the pool. The antibody pool was screened at 4 μg / ml, and therefore, each individual antibody within the pool was screened at 0.2 μg / ml. The antibody pool was screened for binding to fixed HEK293 cells / slides expressing the "Retrogenix library," which contains replicates of 6,019 human plasma membrane proteins, secreted and cell surface-anchored human secreted proteins, and 397 human heterodimers. Screening was performed in duplicate. Antibody binding was detected using an AlexaFluor 647-labeled secondary antibody and scanned slides. UNC5C was identified as a weak binder in one replicate and a very weak binder in the second replicate.

[0184] Following this result, each antigen from the pool was individually tested for binding to rhUNC5C protein by ELISA.

[0185] Netrin-1 competition assay UNC5C recombinant human antigen (R&D Systems, no. 1005-UN) or mouse antigen (produced in-house) was directly absorbed onto the ELISA plate at 3 μg / ml (50 μl per well) and incubated overnight at 4°C. The plate was washed with PBS. The plate was blocked with 200 μl / well of blocking solution (1% BSA w / v in PBS) for 1 h at room temperature. Following this, recombinant human netrin-1 with a C-terminal His tag (R&D Systems, no. 6419-N1) or vehicle was added to the plate for a 30-min preincubation step at room temperature. The netrin-1 concentration was 200 μg / ml for the single-point competition assay, with a dilution range of 200 μg / ml to 0.003 μg / ml. The assay was set up in duplicate wells to detect both ATL_0005262 antibody binding and netrin-1 binding. ATL_0005262 (4 μg / ml) was applied to wells containing netrin-1 and incubated for 1 hour at room temperature. The plate was then washed with PBS / 0.1% Tween. Anti-human IgG HRP (Jackson ImmunoResearch, no. 109-035-097) was added to the plate and incubated for 1 hour at room temperature to detect antibody binding to UNC5C. Anti-His-HRP (Invitrogen no. MA1-21315-HRP) was added to the wells to detect netrin-1 binding to UNC5C. The plate was washed with PBS / 0.1% Tween and TMB solution (LifeTechnology, no. 002023). The plate was incubated for 5 minutes at room temperature, after which stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm on a Molecular Devices FilterMaxF5 plate reader.

[0186] Netrin-1 was directly absorbed onto ELISA plates at 3 μg / ml (50 μl per well) using ATL_0005252, a UNC5C recombinant human antigen (R&D Systems, no. 1005-UN) and incubated overnight at 4°C. The plates were washed with PBS. The plates were blocked with 200 μl / well of blocking solution (1% BSA in PBS) for 1 hour at room temperature. After this, ATL_0005262 or the negative control antibody ATL_0005338 was added to the plates in a 12-point, 3-fold dilution curve starting at 666 nM. The plates were incubated for 30 minutes at room temperature. Netrin-1 was then added to the wells at a final concentration of 75 nM and incubated for 1 hour at room temperature. The assay was set up in duplicate wells to detect both ATL_0005262 antibody binding and netrin-1 binding. The plates were then washed with PBS / 0.1% Tween. Anti-human IgG HRP (Jackson ImmunoResearch, no. 109-035-097) was added to the plate and incubated for 1 hour at room temperature to detect antibody binding to UNC5C. Anti-His-HRP (Invitrogen no. MA1-21315-HRP) was added to the wells to detect netrin-1 binding to UNC5C. The plate was washed with PBS / 0.1% Tween and TMB solution (LifeTechnology, no. 002023). After incubating the plate at room temperature for 5 minutes, stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm on a Molecular Devices FilterMaxF5 plate reader.

[0187] Biolayer Interferometry Assay The binding interactions of ATL_5262 or ATL_6178 with different netrin-1 receptors were assessed by bio-layer interferometry (BLI) using an Octet Red 96e instrument. Purified recombinant antigens were purchased from commercial suppliers or prepared and purified in-house, as detailed in Table 2. Recombinant human antigens, as detailed in the table below, were captured via their his-tags on Sartorius Octet HIS1K biosensors in assay buffer (Cytiva HBS-EP + kinetic buffer). The loaded sensors were equilibrated in assay buffer and then immersed in antibody analytes (ATLX-1282, ATL5262 hIgG1-LALA), and the response (association) was measured. The sensors were then immersed in binding buffer, and the response (dissociation) was measured.

[0188] Biosensors were hydrated and conditioned before use by soaking in assay buffer and regeneration buffer (10 mM glycine pH 1.5, Cytiva) and equilibrated in assay buffer before loading.

[0189] The binding signal of a control antibody soaked in the same sample was subtracted from the raw data. Reference data were collected from loaded sensors soaked in a concentration-matched control antibody analyte.

[0190] [Table 2]

[0191] Capillary isoelectric focusing (cIEF) Charge variant analysis was performed by preparing a master mix to dilute antibody samples and running them on a cIEF cartridge on a Maurice instrument (Protein Simple). The master mix contained a final concentration of 0.35% methylcellulose (Protein Simple, 101876), 4% Pharmalyte (Protein Simple, 17-0456-01) at pH 3-10, 10 mM arginine (Protein Simple, 042-691), and 0.01% pI markers 4.05 and 9.99 (Protein Simple, 046-029 and 046-034). Samples were diluted to 0.15-0.25 mg / ml in the master mix and run at 1500 volts for 1 minute, followed by 3000 volts for 4.5 minutes. A system suitability standard (Protein Simple, 046-044) was also run at the beginning of the run. Stability data generated at 2 and 4 weeks were overlaid and charge species profiles were compared.

[0192] Capillary isoelectric focusing (cIEF) of ATL_6187 was performed on a Maurice instrument (ProteinSimple). 20 μg of sample was added to a master mix containing carrier amphoteric, pI markers pI 7 and 10, methylcellulose, and urea, and loaded onto a cIEF cartridge (ProteinSimple). Separation was performed over a pH range of pI 7.0 to 10.0, with detection at 280 nm.

[0193] Size Exclusion Chromatography (SEC-HPLC) Antibody samples were diluted to 1.2 mg / ml in 20 mM histidine acetate, 150 mM NaCl, pH 5.5, and run on a Zorbax GF-250 SEC-HPLC column (Agilent) on a Vanquish Flex (Thermo Scientific). Samples were separated by size in a mobile phase of 20 mM sodium phosphate, 300 mM sodium sulfate, and 100 mM arginine at a flow rate of 0.75 ml / min at +25°C for 25 minutes per sample. Chromatograms were integrated using Chromeleon software (Thermo Scientific).

[0194] SDS-PAGE Two micrograms of antibody samples were prepared by dilution in water and 2x Laemmli Sample Buffer (Bio-Rad). Samples were analyzed under both reduced and non-reduced conditions; 50 mM DTT (Sigma-Aldrich) was added to reduced samples. All samples were heated to 95°C for 5 minutes and loaded into wells of a Bolt 4-12% Bis-Tris Plus Protein Gel (Thermo Scientific). Five microliters of molecular weight markers (Precision Plus Protein Dual Color Standards (Bio-Rad, 1610737)) were also loaded onto each gel and run in Bolt MES SDS Running Buffer (Thermo Scientific). Samples were run at a constant voltage of 200 volts for 23 minutes, followed by staining with InstantBlue Coomassie stain for a minimum of 1 hour. Gels were washed with water, destained, and scanned using a GelDoc Go Imaging system (Bio-Rad) for imaging.

[0195] Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) CE-SDS was performed on a Maurice instrument separating samples on a CE-SDS Plus Cartridge (Protein Simple). Antibody samples were prepared by mixing 5 μL of 12.6 mg / mL sample with 55 μL of 1× sample buffer (Protein Simple) for a test solution concentration of 1 mg / mL. For reduced samples, 2.5 μL of 2-mercaptoethanol (Sigma-Aldrich ref: M6250) was added to the mixture; for non-reduced samples, 2.5 μL of iodoacetamide (Sigma-Aldrich) was added. The mixture was then thoroughly vortexed and heated at 70°C for 10 minutes, followed by heating on ice for 5 minutes. The samples were then briefly vortexed, spun down, and 50 μL of the sample mixture was transferred to a 96-well plate, centrifuged at 1000 × g for 10 minutes, and then placed in the Maurice instrument. For reducing conditions, samples were run at 4600 volts for 25 seconds for injection followed by 5750 volts for 25 minutes for separation. For non-reducing conditions, samples were run at 4600 volts for 25 seconds for injection followed by 5750 volts for 35 minutes for separation, and data were analyzed using Chromeleon software (Thermo Scientific).

[0196] Stability conditions 100 μl vials of 12.6 mg / ml mAb (ATL_5262) ​​or 50 mg / ml (ATL_6178) were stored at 40°C for 2 or 4 weeks in a static incubator, 4°C refrigerator, or -80°C freezer. Triplicate vials were prepared for each condition. For freeze-thaw conditions, 12.6 mg / ml mAb samples were freeze-thawed from -80°C storage to room temperature (RT) for three cycles within an 8-hour period.

[0197] A 50 mg / ml sample (ATL_6178) for freeze-thaw evaluation was temperature cycled between -70°C and room temperature, with the sample being allowed to thaw completely each time for either 3 or 5 cycles. All stressed samples were compared to a control sample frozen at -70°C.

[0198] Samples requiring agitation were placed in a shaking incubator at 25°C and 300 rpm.

[0199] Samples requiring low pH conditions were adjusted to pH 3.5 using hydrochloric acid, placed at 25°C for 6 or 24 hours, and then frozen at -70°C until required for analysis.

[0200] Melting conditions Prior to solubility evaluation, purified ATL_6178 was concentrated to 50 mg / mL, 100 mg / mL, or up to 288.9 mg / mL by ultrafiltration centrifugation. The 288.9 mg / mL sample was diluted to 200 mg / mL for analysis. Samples were then analyzed before or after incubation at 25°C for 7 days.

[0201] Thermal shift assay Protein thermal shift measurements were performed using Uncle (Unchained Labs). Antibodies were diluted to 1 mg / ml or 5 mg / ml in 20 mM histidine acetate, 150 mM NaCl, pH 5.5 buffer and run through a temperature gradient from 25 to 95°C increasing at a rate of 0.5°C / min. Samples were run in triplicate, and 8.8 μl was loaded into three different wells of the uni (Unchained Labs). Laser settings were set to achieve an initial fluorescence in the 300-350 nm range of 10,000-50,000 counts. Melting temperatures (Tm1 / Tm2) and aggregation temperatures (Tagg / Tonset) were analyzed using Uncle Analysis software v6 (Unchained Labs). Tm measurements were calculated using the 350 / 330 nm ratio, while Tonset and Tag were obtained from SLS readings at 266 nm.

[0202] UNC5C PK To determine the PK of the UNC5C antibody, in vivo PK experiments were performed with ATL_0005262. Male C57BL / 6J mice aged 6-8 weeks were treated IP with ATL_0005262 at 1, 10, and 60 mg / kg, and serum was collected at 1, 4, 8, 24, 72, and 144 hours post-dose. Brain and cerebrospinal fluid (CSF) were collected at 24, 72, and 144 hours post-dose. ATL_5338 was administered at 10 mg / kg as a control antibody. Antibody levels were assessed by ELISA.

[0203] Dopaminergic neuron analysis Experiments were performed on dopaminergic neurons (Fujifilm, iCell DopaNeurons 01279) cultured in vitro for 7 days according to the manufacturer's protocol using iCell Neural Base Medium 1, iCell Neural Supplement B, and iCell Nervous System Supplement provided by the manufacturer. On day 5 of culture, neurons were exposed to treatment with ATLX-1282 (100 nM), ATL5252 (100 nM), isotype control (100 nM), and recombinant netrin-1 (1 μg / mL) as a positive control (obtained from R&D systems, catalog number 6419N1025CF).

[0204] On day 7, cells were lysed and proteins were extracted using RIPA cell lysis buffer consisting of 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 1% SDS, supplemented with protease and phosphatase inhibitors (Sigma). The effects of various treatments were assessed by quantitative Western blot analysis performed by RayBiotech, Inc. (Peachtree Corners, GA, USA) using their Auto Western Blot Service. Extracted protein samples at a concentration of 0.1 mg / ml were loaded onto an automated capillary electrophoresis instrument. The following antibodies were used for protein detection: UNC5C (ThermoFisher, Catalog No. PA5-67780), Neurofilament L (Cell Signaling, Catalog No. 2837), Synaptophysin (Cell Signaling, Catalog No. 36406), PSD95 (Cell Signaling, Catalog No. 3450), Synapsin-1 (Cell Signaling, Catalog No. 5297), and Synaptotagmin-1 (Cell Signaling, Catalog No. 14558). Secondary antibodies from the RayBiotech, Inc. auto Western Blot Service catalog were used. As a loading control, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody provided by RayBiotech from its service library was used.

[0205] Netrin-1 depletion assay To evaluate the effects of netrin-1 deficiency on motor neurons, experiments were performed on human iPSC-derived spinal motor neurons (BX-0100, Brain Xell, USA) cultured in vitro for 7 days. On day 5 of culture, these neurons were pretreated with ATLX-1282 (100 nM) or ATL5262 (100 nM) for 1 hour. After this pretreatment, the neurons were exposed to further treatments (detailed below). 40 hours later, on day 7, the neurons were harvested for a series of assays to assess their cellular response and health. These assays included: (a) live / dead assay, (b) GAP-43 expression analysis, and (c) neurite outgrowth assessment (using XonaChips compartmentalized microfluidic devices from Xona Microfluidics, USA).

[0206] For cell viability and GAP-43 expression assays, cells were exposed to (1) the isotype control antibody ATL5338 alone, (2) ATL5338 and anti-netrin-1 antibody 2F5 (10 μg / ml) (AdipoGEN, NC2134483), (3) ATL5262 (100 nM) and anti-netrin-1 antibody 2F5 (10 μg / ml), or (4) ATLX-1282 (100 nM) and anti-netrin-1 antibody 2F5 (10 μg / ml) for pretreatment. Cell viability was assessed using a live / dead staining kit from Thermo Fisher Scientific. Motor neurons were incubated with 1 μM calcein AM and 2 μM ethidium homodimer-1 (EthD1) in PBS for 30 min. After washing with PBS, cells were imaged using an electronic fluorescence microscope (Echo Revolve). Analysis included four frames per chip at ×10 magnification, with particle counting using Fiji / ImageJ. GAP-43 immunostaining was performed after fixation with 4% paraformaldehyde and permeabilization with 1% saponin in PBS. Blocking was performed in 10% donkey serum in PBS containing 0.1% saponin for 30 minutes. The primary antibody (GAP-43, ThermoFisher, PIPA579299) was diluted in 10% donkey serum in PBS containing 1% saponin and incubated overnight at 4°C. After three PBS washes, cells were incubated with a 1:300 dilution of fluorescently labeled secondary antibody (Abcam, ab175470) at room temperature for 1 hour, followed by DAPI nuclear staining.

[0207] For neurite outgrowth assessment, after pretreatment, cells were exposed to (1) the isotype control antibody ATL5338 alone, (2) ATL5338 and the anti-netrin-1 antibody 2F5 (10 μg / ml) (AdipoGEN, NC2134483), or (3) ATLX1282 (100 nM) and the anti-netrin-1 antibody 2F5 (10 μg / ml). Microfluidic cultures contained BrainXell eGFP spinal motor neurons (BX-0101) cultured on XonaChip SF150X4 "X4" culture units, with each chip containing 40-60k neurons per unit. After 48 h of incubation, the soma and axon compartments were imaged using a 20x objective, capturing both differential interference contrast (DIC) and 488 fluorescence channels. Visualization was performed after three PBS washes and images were acquired using an Echo Revolve. Quantitative analysis of the axonal compartment included measurement of total neurite length and total neurite number.

[0208] MEA-based assay iCell Motor Neuron Isogenic Control (Cat. No. C1048), iCell Motor Neuron ALS TDP43 Q331K (Cat. No. R1144), and iCell Astrocytes (Cat. No. R1092) were all supplied by FUJIFILM Cellular Dynamics, Inc. and maintained according to the manufacturer's protocol. Culture surfaces were prepared by first treating them with 0.1% polyethyleneimine solution (Sigma-Aldrich), followed by overnight incubation. The treated surfaces were then thoroughly washed three times with sterile distilled water and air-dried for 1 hour. The dried surfaces were then coated with 5 μg / ml laminin (Sigma-Aldrich), followed by an additional overnight incubation. After this incubation, the laminin solution was aspirated, and the cells were cultured in the provided manufacturer's medium at 1 cm. 2Cells were immediately plated at a density of 55,000 cells per 1000 neurons. Specifically, the cell cultures contained a 15% ratio of astrocytes to neurons (equivalent to 1.5K astrocytes per 10K neurons). Cultured motor neurons received a 50% medium change every 2-3 days and were analyzed between days 15 and 20 of culture for all experiments.

[0209] To assess population-level function in motor neuron cultures, we used a 48-well multielectrode array (MEA) plate and the Axion Biosystems Maestro MEA system. During data acquisition, standard recording settings for spontaneous neuronal spikes were applied using Axis software (version 2.5). Cells were maintained at a constant temperature of 37°C and 5% CO2 throughout the 2-minute recording period. Standard settings included a 130x gain, recording at 1–25,000 Hz, and a 2 kHz low-pass digital filter for noise reduction. Spike detection consisted of 5 times the standard deviation of the noise, and network burst detection was recorded when at least 25% of the electrodes in a given well exhibited synchronous activity. The results reported herein were obtained by averaging all electrodes in each well and then averaging data from six wells. Experimental treatments consisted of ATL-5338 (isotype control) and ATLX-1282, both administered at a specific final concentration of 100 nM. These treatments began on day 15 of the experiment and continued for 142 hours (dose 1: time point 0, dose 2: 120 hours after the first dose). To evaluate the effects of these treatments on cellular behavior, daily recordings were performed from day 15 to day 20, with one recording per day. Recorded data were processed using Axion Biosystems software for spike detection and waveform analysis. Statistical analysis was performed using appropriate statistical tests to compare the effects of different compounds on neuronal network activity. Results were considered statistically significant at p<0.05.

[0210] Caspase assay Nuclight Red (Sartorius Catalog No. 4475) SH-SY5Y cells (DSMZ) were seeded at a density of 5000 cells / well in 96-well plates (Corning Catalog No. 3606) in DMEM / F12 (Gibco Catalog No. A4192001) containing 10% FBS and allowed to adhere overnight (37°C, 5% CO2). The medium was replaced with a 24-hour pretreatment in complete medium containing Incycyte® Caspase 3 / 7 Green dye (Sartorius Catalog No. 4440). Pretreatment was performed with 80 nM ATL5262, 80 nM ATLX-1282, 80 nM isotype control (ATL-5338), 10 nM netrin-1, 10 nM clusterin (a control protein with a size similar to netrin-1 (100 kD)), or a negative control lacking pretreatment (anti-fluorescein ATL_5338, same IgG1 isotype). Subsequently, β-amyloid peptide 1-42 (rPeptide, catalog no. A-1170, Abcam, catalog no. AB120301) diluted in complete medium containing caspase 3 / 7 green dye (final concentration 20 μM) was added. Plates were then read using the Red / Green optical module for 72 h using an Incucyte live imaging system (Sartorious). Analysis was performed by normalizing green counts to cell number (red counts) after removal of any outliers (Grubbs and ROUT - no outliers were detected). Areas under the curve on Graphpad Prism were generated with a one-way anova using Sidak's multiple comparison test to determine significance between treatments.

[0211] In vivo netrin-1 haploinsufficiency studies The mice used were 12-week-old male netrin-1 haploinsufficient B6.129(Cg)-Ntn1tm1.2Tek / J mice (stock number 028070) and non-transgenic littermates from Jackson Laboratories. Netrin-1 haploinsufficient mice were treated with 60 mg / kg ATLX-1282 via IP twice weekly. Amphetamine was administered via IP at 4 mg / kg starting on day 8 after ATLX-1282. It was administered every other day for 10 days (5 doses). Prior to amphetamine treatment, mice were brought to the experimental room for at least 1 hour of habituation. Locomotor activity, rearing frequency, and stereotypic counts were recorded in 5-minute bins for a 30-minute baseline, after which mice were given either amphetamine (4 mg / kg) or saline and returned to the OF chamber for another 60-minute session. Saline was used as an amphetamine control, and ATL5338 was used as an isotype control for ATLX-1282.

[0212] In vivo 6-OHDA studies The rats used were female Sprague-Dawley rats (Charles River, approximately 290 g at the time of surgery). On day 1 of the study, all animals received either 6-OHDA or vehicle (saline) administered unilaterally at three sites in the right striatum using stereotaxic techniques. ATLX-1282 and the isotype control (ATL5338) were administered IP at a dose volume of 5 ml / kg once weekly (QW) for 4 weeks. On day 26 of the study, rats underwent behavioral evaluation via the cylinder test (assessment of spontaneous forelimb use). The number of times each paw touched the side of the cylinder during each individual rear was determined from post-hoc analysis of the video by an observer blinded to the given treatment, and the results are plotted as % contralateral contact.

[0213] Netrin-1 competition assay To determine whether netrin-1 could displace phage-derived antibodies, UNC5C recombinant human antigen (R&D Systems, no. 1005-UN) was directly absorbed onto ELISA plates and incubated overnight at 4 °C. The plates were washed with PBS. The plates were blocked with 1% BSA w / v in PBS blocking solution at room temperature for 1 h. After this, phage-derived antibodies (ATL6530-ATL6539) or the negative control antibody ATL_0005338 were added to the plates at a dilution curve ranging from 1 μM to 0.1 pM (final concentrations, netrin-1 was added once). The plates were incubated at room temperature for 30 min. Netrin-1 (R&D Systems, no. 6419-N1) was then added to the wells at a final concentration of 75 nM and incubated at room temperature for 1 h. The assay was set up in duplicate wells to detect both antibody-bound UNC5C in the presence and absence of netrin-1, as well as netrin-1-bound UNC5C in the presence of antibody. The plate was then washed with PBS / 0.1% Tween. Anti-human IgG HRP (Jackson ImmunoResearch, no. 109-035-097) was added to the plate and incubated at room temperature for 1 hour to detect antibody binding to UNC5C. Anti-His-HRP (Invitrogen no. MA1-21315-HRP) was added to the wells to detect netrin-1 binding to UNC5C. The plate was washed with PBS / 0.1% Tween and TMB solution (LifeTechnology, no. 002023). The plate was incubated at room temperature for 5 minutes, after which stop solution (0.5 M sulfuric acid) was added. Absorbance was read at 450 nm on a Molecular Devices FilterMaxF5 plate reader. Analysis was performed using a nonlinear fit curve fitting algorithm on GraphPad Prism and EC50s were calculated.

[0214] immunohistochemistry Naive C57BL / 6 mouse brains were cryopreserved by quick freezing in OCT, sectioned onto glass slides using a cryostat, and stored at -20°C until use. Slides were thawed and air-dried, then fixed in neutral-buffered formalin (10%) for 10 minutes and washed in PBS-Triton X (0.5%) for 30 minutes. A hydrophobic barrier was drawn around each tissue section and blocked for 1 hour in the appropriate blocking solution (1% BSA, 10% FBS, 5 mM EDTA, 0.3 M glycine in PBS-Tx 0.5%) supplemented with either clusterin (R&D Systems catalog no. 2937-HS-050), netrin-4 (R&D Systems catalog no. 1254-N4-025 / CF), or netrin-1 (R&D Systems catalog no. 6419-N1-025 / CF) [10 nM]. The blocking solution was removed, and primary antibodies were added in the same blocking buffer (ATLX-1282 / ATL_5262 [10 μg / ml]) overnight at 4°C. Slides were washed three times with PBS-Tx (0.5%) for 5 minutes each, followed by the addition of secondary antibodies anti-human IgG DyLight® 594 (Abcam catalog no. ab97005) [2.5 μg / ml] and DAPI [0.1 μg / ml] (Thermo Scientific catalog no. 62248) in their respective buffers for 90 minutes at room temperature, washed, and coverslipped using VectaShield antifade mounting medium (Vector Labs catalog no. H-1200-10). Images of the slides were acquired using Molecular Devices PICO, and analysis was performed using FIJI. Images were thresholded, measured, and a GraphPad t-test was performed for significance.

[0215] Flow cytometry Nucleotide sequences encoding canonical UNC5C (SEQ ID NO: 99) and the mutant UNC5C T835M variant (SEQ ID NO: 110) were cloned into the lentiviral transfer vector pCDH-EF1α-MCS-(PGK-GFP-T2A-Puro) (System Biosciences, no. CD813A-1) and confirmed by Sanger sequencing. Lentiviral particles were produced in the 293T cell line (ATCC, no. CRL-3216) using TransIT-VirusGEN Transfection Reagent (Mirus, no. MIR 6700) and the pPACKH1 HIV Lentivector Packaging Kit (System Biosciences, no. LV500A-1). Stable cell lines were generated by transducing the HEK-293 cell line (ECACC, number CRL-1573) with lentiviral particles and then selecting successfully transduced cells with 1 μg / ml puromycin (Gibco™, number A1113803). Overexpressing cell lines were monitored for GFP expression and cultured in the presence of the selection antibiotic.

[0216] For surface antigen detection, HEK-293 cells and cell lines expressing UNC5C, mutant UNC5C T835M, and empty vector (EV) control were harvested using non-enzymatic dissociation buffer (Corning, catalog no. 25-056-Cl). Cells were then labeled with dead cell labeling reagent eF780 (Invitrogen, catalog no. 65-0865), washed with FACS buffer (DPBS, Gibco; catalog no. 14190-169, 2% FBS, LSG; catalog no. S-001A-USDA), and incubated in triplicate with 100 μg / ml (0.69 nM) of antibody at 4°C for 1 hour. Cells were then washed twice with FACS buffer and incubated with secondary detection anti-IgG APC antibody (clone IS11-3B2.2.3, Miltenyi; catalog no. 130-119-772) at 4°C for 30 minutes. The cells were then washed twice, resuspended in FACS buffer, and analyzed using a NovoCyte Penteon flow cytometer (Agilent). Compensation was performed automatically using NovoExpress software (Agilent) and samples labeled with individual fluorochromes. The detection parameters for each fluorochrome are listed in Table 3 below. Data analysis was performed using FlowJo software (BD).

[0217] [Table 3]

[0218] APC positive cells (IgG + The percentage of APC (IgG) and IgG-APC median fluorescence intensity (MFI) were measured in the population gated on single / viable / cells. + ) was used as a negative control for gating, and background signal was subtracted from the results.

[0219] Retrogenix analysis This was performed by Charles River (UK) using their proprietary Retrogenix Cell Microarray Technology (see www.criver.com / products-services / discovery-services / screening-and-profiling-assays / retrogenix-cell-microarray-technology).

[0220] X-ray crystallography Recombinant human UNC5C was produced in ExpiHEK293 by transient transfection of constructs corresponding to Ig domains 1 and 2 and purified via a C-terminal hexahistidine tag (SEQ ID NO: 111). The Fab fragment of ATL_5262 was produced by transient transfection of ExpiCHO and purified via a C-terminal hexahistidine tag (SEQ ID NO: 111). HThe complex was purified via the 1 domain. Both components were purified by SEC, mixed together to form a complex, and screened for crystallization using a commercial crystallization screen. X-ray diffraction data were collected from crystals grown in 22% PEG Smear Broad, 0.1 M Bicine pH 9.3 (among many other formulations with similar composition and pH). Data were automatically processed with Xia2 / Dials and subjected to molecular replacement in the program Phaser, using the truncated "Netrin receptor UNC5C" monomer (UniProt ID D6RE16 AlphaFold model) and Fab heterodimer (PDB ID 6CNR) as search models. The original data from the automated processing were reindexed to P6522 (space group for protein crystals), and the model was taken through several rounds of interaction model rebuilding with the program Coot and refinement with Refmac, and finally manual and automated solvent building with ARP / wARP to generate the final model. Data collection and refinement statistics are shown in Table 4. This model contains one molecule each of the Fab heavy and light chains and one molecule of UNC5C in the asymmetric unit. The asymmetric unit is a region in the crystal lattice containing molecules that are repeated throughout the crystal. The finding of only one copy each of the Fab and antigen, combined with an analysis of how the repeating units interact with each other, indicates that other biologically relevant interactions, such as target dimerization, are not present.

[0221] [Table 4]

[0222] phage display A phage library of scFv molecules displayed on M13 phage was generated by cloning the heavy chain variable region (VH) repertoires of subjects SU_0000656 and SU_0000697 into a sublibrary of a phagemid vector carrying light chain variable region (VL) sequences from a healthy donor. A phage display library of 1.3e8 clones in size was generated.

[0223] The following reagents were used: - Hyperphage (1.5e12 cfu / ml) from Progen (German catalogue number PRHYPE K07deltapIII) (used in phage display and phage ELISA), - Helper phage (1e11 cfu / ml) from Invitrogen (USA) catalog number 18311-019 (used in phage display and phage ELISA), In-house produced UNC5C_HUMAN antigen (SEQ ID NO: 194) (in-house number UNC5C_HUMAN_008 (Alchemab) res41-380 Batch number 004) (used in phage display and phage ELISA) UNC5C_human (used in phage display) from R&D Systems (USA) catalog number 1005-UN-050

[0224] Phage ELISA for phage display-derived scFv Phages were prepared by culturing each phagemid-carrying TG1 clone from a glycerol stock in 100 μl of 2TYAG (2TY medium supplemented with 100 μg / ml ampicillin and 2% glucose) at 37°C with aeration to an optical density (OD) of 0.6, followed by rescue with helper or hyperphage (Invitrogen, Catalog No. 18311-019 and Progen, Catalog No. PRHYPE, K07deltapIII, respectively) added at an MOI of 10 for 1 hour, and then replacing the medium with 2TYAK (2TY medium supplemented with 100 μg / ml ampicillin and 50 mg / ml kanamycin). The cultures were then incubated overnight at 25°C with good aeration, and the next day, phages were separated from the bacteria by centrifugation at 3200 rpm for 10 minutes. The phage-containing supernatant was transferred to a new plate and blocked with 3% milk w / v in PBS.

[0225] UNC5C recombinant human antigen (in-house, UNC5C_HUMAN_008 (Alchemab) res41-380 Batch No. 004) or negative control lysozyme (MP Biomedicals No. 195303) was directly absorbed onto the ELISA plate at 3 μg / ml (50 μl per well) and incubated overnight at 4°C. Each antigen-coated plate was washed with PBS and blocked with 200 μl / well of blocking solution (3% milk w / v in PBS) at room temperature for 1 hour. After this, the blocking solution was removed, and the blocked phage sample to be evaluated was applied to the plate. The plate was incubated at room temperature for 1 hour. Each plate was washed with PBS / 0.1% Tween and incubated with anti-M13 HRP (Sino Biological, No. 11973-MM05T-H) at room temperature for 1 hour to detect phage binding. The plate was then washed with PBS / 0.1% Tween and TMB solution (Life Technology, No. 002023). Plates were incubated at room temperature for 5 minutes before adding stop solution (0.5 M sulfuric acid). Absorbance was read at 450 nm on a Molecular Devices FilterMaxF5 plate reader.

[0226] Screening for IgG binding to UNC5C To determine the binding of IgG antibodies to UNC5C, UNC5C_Fc (R&D Systems #1005-UN), or in-house produced from HEK293F cells with an N-terminal His tag, or negative control lysozyme (MP Biomedicals #195303), the antibodies were directly absorbed onto ELISA plates at 1 μg / ml and incubated overnight at 4°C. The plates were washed with PBS. The plates were blocked with 1% BSA w / v in PBS blocking solution for 1 hour at room temperature. After this, the blocking solution was removed, and the antibody to be evaluated was diluted in a dilution series (2 μM to 0.4 pM) in blocking solution (1% BSA w / v in PBS) and applied to the plate. The plate was incubated for 1 hour at room temperature. The plate was washed with PBS / 0.1% Tween. Anti-human IgG HRP (Jackson ImmunoResearch, #109-035-097) was added to the plate and incubated for 1 hour at room temperature to detect antibody binding. Plates were washed with PBS / 0.1% Tween and TMB solution (Life Technology, no. 002023). Plates were incubated at room temperature for 5 minutes before the addition of stop solution (0.5 M sulfuric acid). Absorbance was read at 450 nm on a Molecular Devices FilterMaxF5 plate reader. Analysis was performed on GraphPad Prism using a nonlinear fit curve fitting algorithm, and EC50s were calculated.

[0227] Example 1 - Convergence Analysis Convergent sequence clusters derived from the antibody repertoire of resilient individuals can be used to identify disease-specific antibody sequences.The present inventors attempted to identify candidate protective antibodies from FTD-resilient individuals from a cohort of frontotemporal dementia (FTD) patients (Genetic FTD Initiative (GENFI), www.genfi.org / ).FTD encompasses several types of dementia that affect the frontal and temporal lobes of the brain, and the main genetic risk factors are GRN and C9orf72 mutations.

[0228] Resilience to FTD was defined as individuals who were within the top 33% of cumulative ages of onset for frontotemporal dementia and who remained disease-free despite the presence of GRN or C9orf72 mutations. From a total cohort of over 1,000 individuals, we selected the 22 most resilient individuals. Sequencing of the antibody repertoires of these resilient patients revealed convergent heavy chain variable (VH) sequences among the three highly resilient individuals (see Figure 1 for the workflow used to identify convergent VH sequences from the FTD dataset). The probability that all three individuals would produce this antibody by chance is extremely low. This particular sequence is highly selected and therefore likely favors the individual.

[0229] The VH sequences were then paired with the VL sequences using a Transformer-based model comprising an encoder-decoder model trained on a corpus of paired VH-VL sequences. Further details on how such models can be trained and used are provided in WO2022 / 223451. The trained model takes a VH sequence as input and produces a single complementary VL sequence as output.

[0230] The resulting antibody, ATL_5262 (ATL_0005262), was included in a proteomics array for target deconvolution, and the netrin receptor UNC5C was identified as its target (data not shown). Binding of ATL_5262 to UNC5C was confirmed by ELISA (Figure 2).

[0231] Interestingly, UNC5C antibodies were subsequently identified across repertoires derived from subjects resilient to neurodegeneration (Figure 3). In particular, the heavy chain sequence of ATL_5262 was used as a probe to search for similar sequences in unpublished repertoire databases derived from neurodegeneration. Highly homologous sequences were found in resilient Alzheimer's disease subjects (individuals showing slow cognitive progression), Parkinson's disease resilient subjects (prodromal REM sleep behavior disorder (RBD) but without a Parkinson's disease diagnosis), three FTD resilient subjects (carriers of high-risk mutations and elderly, but without progression to FTD), and resilient centenarians without neurodegenerative symptoms (Figure 3). The sequences were also found in cognitively healthy controls, primarily individuals over 60 years of age. Notably, there is an absence of UNC5C antibodies in the so-called "progressors" from these cohorts, with the exception of SU_0001278, who has a diagnosis of Parkinson's disease and mild cognitive impairment. PD subject SU_0001278 was initially included in the cohort as a healthy control and was subsequently reclassified due to suspected REM sleep behavior disorder. Because they did not have a PD diagnosis, they are "potentially resilient." All identified sequences, referred to herein as "ATL_5262 homologs," share the same V and J genes as the ATL5262 VH, with at most two AA mismatches across the junction region. Figure 8 shows the VH sequence alignment of representative ATL_5262 homolog sequences ATL_6187 and ATL_6191 (SEQ ID NOs: 112-113). Representative VH sequences of ATL_5262 homologs were then paired with the ATL_5262 VL (SEQ ID NO: 9) and produced as IgG1 Fc NULL antibodies. The VHs of these antibodies shared at least 80% sequence identity with the VH sequence of ATL_5262 (specifically, 6187: 84.87%, 6191: 89.92% - for reference, the 5262 derivative antibody 6178 shared 94.96% VH sequence identity with the VH of ATL_5262, all determined using Clustal). -7The antibodies had an EC50 for binding to a target antigen of less than 100 kJ / mL (determined by ELISA). These antibodies had zero or one mutation in VH CDR3 compared to ATL_5262 (maximum one mutation at position 117 in the IMGT numbering, Y117S) and up to three mutations in each of VH CDR2 and CDR1 compared to ATL_5262. Both ATL_6178 and ATL_6191 had mutated variable regions and IGHG1 constant regions, indicating that class switch recombination and affinity maturation via somatic hypermutation had occurred, and thus most likely a germinal center-induced immune response to the antigen was present (see Table 5). These highly mutated class switch sequences are likely derived from memory B cells, which are typically more specific for the antigen than naive B cells.

[0232] The fact that UNC5C antibodies were found in resilient individuals in these cohorts highlights the relevance of UNC5C as a therapeutic target in a variety of neurodegenerative conditions.

[0233] The identified homologues were then tested by ELISA for binding to UNC5C and the results are shown in Table 14.

[0234] [Table 5]

[0235] Example 2 - Competition with Netrin-1 To determine whether ATL_5262 competes for the same binding site as the UNC5C ligand, netrin-1, a competition assay was performed.

[0236] Briefly, ELISA plates were coated with 45 nM UNC5C and preincubated with 3 μM netrin-1 for 30 minutes, followed by the addition of 27 nM ATL_5262 or a negative control anti-fluorescein antibody labeled "ATL_5338." Figure 4A shows that netrin-1 was detected in wells containing only UNC5C, UNC5C and ATL5262, and UNC5C and ATL_5338, indicating that netrin-1 inhibits the binding of ATL_5262 to UNC5C. Figure 4B shows that ATL_5262 was detected only in wells not preincubated with netrin-1, confirming that ATL_5262 binds to UNC5C and that this binding is inhibited by netrin-1. Figure 4B further shows that the control antibody ATL_5338 does not bind to UNC5C.

[0237] To determine whether netrin-1 inhibits the binding of ATL_5262 to UNC5C in a concentration-dependent manner, a 12-point dilution series of netrin-1 starting from 3 μM was performed, and the binding of ATL_5262 to UNC5C was assessed using ELISA (Figure 5). Figure 5 shows that netrin-1 inhibits the binding of ATL_5262 to UNC5C in a concentration-dependent manner.

[0238] To test whether netrin-1 can compete with ATL_5262 already bound to UNC5C, ELISA plates were coated with 45 nM UNC5C and preincubated with a 12-point dilution series of ATL_5262 starting at 666 nM for 30 min before adding 75 nM netrin-1 (Figure 6). Figure 6 shows that netrin-1 can outcompete ATL_5262 binding, as indicated by the right-shifted "ATL_0005262 + netrin-1" curve compared to "ATL_0005262 alone." That is, only at high concentrations of ATL_5262, ATL_5262 can partially outcompete netrin-1. Similarly, only at the highest concentration of ATL_5262 was a slight decrease in netrin signal detected. As expected, no binding signal was observed for wells incubated with UNC5C and the control antibody ATL_5338.

[0239] Taken together, these data show that netrin-1 inhibits the binding of ATL_5262 to UNC5C in a concentration-dependent manner, indicating that ATL_5262 competes for the same binding site on UNC5C as netrin-1. Thus, ATL_5262 may act as a netrin-1 mimetic. To our knowledge, the antibody described herein is the first antibody that binds to UNC5C and competes with netrin-1 binding.

[0240] Example 3 - Mouse cross-reactivity The ability of ATL_5262 to bind to both mouse and human recombinant UNC5C was tested using ELISA (Figure 7).

[0241] Human and mouse UNC5C share 97% homology, and ATL_5262 can bind to both rhUNC5C (Figure 7A) and rmUNC5C (Figure 7B). The control antibody ATL_5338 shows negligible binding to rhUNC5C (Figure 7A) and rmUNC5C (Figure 7B). ATL_5262 does not bind to the unrelated protein lysozyme (Figure 7C), indicating that the antibody does not exhibit nonspecific binding.

[0242] In conclusion, ATL_5262 binds to both human and mouse recombinant UNC5C.

[0243] Example 4 - Binding to other netrin receptors and related proteins We then sought to investigate the selectivity of ATL_5262 for UNC5C. To identify other potential antibody binding targets, we performed a BLAST search of human protein sequences with greater than 50% sequence identity using the reference UNC5C sequence (accession O95185) in the UniProtKB database (www.uniprot.org / ). This returned the related proteins UNC5A, UNC5B, and UNC5D. Furthermore, to address possible structural or functional mimicry of netrin, we considered the human netrin receptors neogenin, DCC, and DSCAM as potential binding partners.

[0244] The binding of ATL_5262 and ATL_6178 (variants of ATL_5262, see Example 6) to other netrin receptor family members and related proteins was tested using biolayer interferometry analysis (Figure 28). Figure 28 shows that ATL_5262 and ATL6178 bind to UNC5C in this assay, but do not bind to any other netrin receptors or related proteins (UNC5A, B, D, DCC, and neogenin-1 ectodomain). At concentrations up to 100 nM, no evidence of binding to non-UNC5C antigens was observed by either antibody, demonstrating the selectivity of these ATL_5262 and ATL_6178 for UNC5C.

[0245] To further confirm the selectivity of ATL_6178, it was analyzed in binding assays against fixed HEK293 cells expressing 6,105 individual full-length human plasma membrane proteins and cell surface-tethered secreted human proteins, as well as an additional 400 human heterodimers, followed by a series of confirmatory screens using the Retrogenix platform with operators blinded to the antibody target. Other netrin-1 receptors in the screening panel, including UNC5A, UNC5B, and UNC5D, NEO1, DCC, and DSCAM, did not show binding to ATL_6178. UNC5C was the only confirmed target of ATL_6178.

[0246] These data demonstrate the specificity of ATL_5262 and ATL_6178 for UNC5C and show that they do not bind to other netrin receptors or related proteins.

[0247] Example 5 - Stability Studies To ensure that ATL_5262 was stable and therefore suitable for further optimization, a stability study shown in Figure 9 was performed.

[0248] ATL_5262 was tested in the thermal shift assay at both 1 mg / ml and 5 mg / ml. The results are shown in Table 6 and Figure 10. This indicates that Tm1 and Tm2 were within the normal range for a monoclonal antibody (mAb), that Tagg occurred before Tonset, and that aggregation was induced by stronger unfolding than by protein-protein or protein-buffer interactions. m1 is T agg , which indicates that proteins start to aggregate at a ratio of unfolded to folded protein of less than 50%.

[0249] [Table 6]

[0250] Capillary isoelectric focusing (cIEF) was used to analyze the charge heterogeneity of ATL_5262 after 2 weeks (Figure 11A) or 4 weeks (Figure 11B) of storage at -80°C, 4°C, and 40°C. An increase in acidic charge variants was observed at 40°C (49.27% ​​at 2 weeks and 53.39% at 4 weeks) (Figure 11C, Table 4), while minimal changes in the charge species present were observed in all other conditions (Table 7).

[0251] [Table 7]

[0252] Protein aggregation during antibody storage must be kept to a minimum as it can cause immunogenic reactions. Size exclusion chromatography (SEC-HPLC) was used to assess antibody purity and aggregation in a 2 / 4 week stability study.

[0253] FIG. 12 shows that no soluble aggregates were formed after incubation at −80° C., 4° C., or 40° C. for 2 weeks (FIG. 12A) or 4 weeks (FIG. 12B).

[0254] Table 8 further supports the excellent stability of ATL_5262, showing low levels of high molecular weight species (HMWS) and low molecular weight species (LMWS), indicating low tendency of the antibody to aggregate and degrade up to 40°C, which is further supported by the high percentage of monomer in all of the samples (>95%).

[0255] [Table 8]

[0256] SDS-PAGE was used to analyze the purity of ATL_5262 after storage at -80°C, 4°C, or 40°C for 2 weeks (Figure 13A) or 4 weeks (Figure 13B), or after three FT cycles (Figure 13B). Reduced IgG antibodies typically yield a glycosylated heavy chain of approximately 50 kDa and a light chain of approximately 25 kDa on SDS-PAGE (as indicated by "R" in Figure 13), and a 150 kDa band for nonreduced antibodies (as indicated by "NR" in Figures 13A and B). After temperature and freeze-thaw stability time courses, minimal changes to the molecular weight separation of the samples are detected for both reduced and nonreduced samples. NIST mAb was run as a molecular weight control for human IgG1 mAb.

[0257] In addition to SDS-PAGE, CE-SDS was used to analyze the molecular weight and purity of species along the temperature time course stability samples. Non-reduced samples showed a 3-5% decrease in the major species at both 2 weeks (Figure 14A) and 4 weeks (Figure 14B) at 40°C, but not at other storage temperatures (Table 9), while under reduced conditions, minimal changes were detected throughout the temperature time course stability study at both 2 weeks (Figure 14C) and 4 weeks (Figure 14D) (Table 10).

[0258] [Table 9]

[0259] [Table 10]

[0260] Finally, the binding of ATL_5262 to rhUNC5C was tested using ELISA after 2 weeks (Figure 15A) or 4 weeks (Figure 15B) at -80°C, 4°C, or 40°C, or after 3 x FT cycles (Figure 15B). ATL_5262 showed consistent binding to UNC5C under all conditions, reflected in consistent EC50 values ​​(Table 11), indicating that the binding capacity of ATL_5262 is not affected by temperature changes or freeze-thaw conditions.

[0261] [Table 11]

[0262] In conclusion, ATL_5262 is stable at -80°C and 4°C through three freeze-thaw cycles at -80°C for up to 4 weeks as tested, and although some biophysical changes were observed at 40°C, ELISA indicates that binding to UNC5C is unaffected by these changes. Thus, ATL_5262 is stable and suitable for further development.

[0263] Example 6 - Antibody variants To identify a lead antibody with optimal development properties, variants of the VH (SEQ ID NO: 1) and VL sequences (SEQ ID NO: 9) of ATL_5262 were generated and their ability to bind to UNC5C was tested using direct ELISA for binding to human UNC5C and mouse UNC5C.

[0264] To generate the VH variants, the ATL_5262 sequence was compared to the VH germline sequence and found to have five amino acid substitutions compared to the germline sequence. Reverting a sequence to germline typically reduces immunogenicity and improves antibody expression and stability. Therefore, one VH variant tested was a germline-reversed VH framework sequence, ATL_6033 (SEQ ID NO: 2). To identify whether any specific reversion to germline would be detrimental to antibody expression or binding activity, partial germline-reversed VH framework sequences were generated with four of the five germline mutations—i.e., all germline reversions minus one position that remained identical to the VH of ATL_5262 (ATL_6034 (SEQ ID NO: 3), ATL_6035 (SEQ ID NO: 4), ATL_6036 (SEQ ID NO: 5), ATL_6037 (SEQ ID NO: 6), ATL_6038 (SEQ ID NO: 7)). Additionally, the VH sequence of ATL_5262 (using IMGT numbering) (ATL_6039) (SEQ ID NO: 8) with the burden-relieving mutation T82K was also generated (see Table 12). All antibodies were expressed using the ATL_5262 VL chain (SEQ ID NO: 9).

[0265] All antibodies tested were IgG1 variant L234A / L235A (LALA), an IgG1 variant containing L234A / L235A substitutions that reduce binding to the IgG Fc receptors FcγRI, FcγRII, and FcγRIII and complement component C1q, reducing Fc-mediated toxicity (Lund J, et al. 1991).

[0266] [Table 12]

[0267] Reversing the entire ATL_5262 VH sequence to germline increases the EC50 and therefore reduces potency by more than 10-fold compared to ATL_5262 (Table 12 and Figure 16). All variants (IMGT) with the germline reversion Q67Y (ATL_6033, 6034, 6035, 6037, and 6038) exhibited reduced binding activity in ELISA (Figure 16), reflected by increased EC50 values. Therefore, to maintain the degree of UNC5C binding observed for ATL_5262, it is desirable to maintain a glutamine (Q) at IMGT67. We further observed that by mutating IMGT82 of the VH to lysine (K) to reduce the risk of aspartic acid (T) isomerization, the binding of the resulting antibody (ATL_6039) to UNC5C was enhanced approximately three-fold compared to ATL_5262 (Figure 16). Thus, these data indicate that IMGT residues 67 and 82 are important for UNC5C binding. These mutations were then combined in the VHs of ATL_6177 (SEQ ID NO: 91) and ATL_6178 (SEQ ID NO: 93), both of which mutated IMGT position 82 (T82K) and retained Q67. In addition, ATL_6178 (SEQ ID NO: 93) also removes additional potential sequence bias by mutating the asparagine (N) of IMGT at position 58, which is part of a predicted asparagine deamidation site in CDR2 (NS motif), to tyrosine (Y) (N58Y). By combining these beneficial mutations, ATL_6177 and ATL_6178 are expected to have improved stability and binding capacity compared to other ATL_5262 variants.

[0268] The VH variants tested show similar binding profiles for human (Figure 17A) and mouse UNC5C (Figure 17B), but show minimal binding to lysozyme at the highest concentration of antibody used (Figure 17C).

[0269] UNC5C exists in a monomeric form in the absence of its ligand, netrin-1, and in a dimeric form upon binding to netrin-1. To test whether ATL_5262 binds to both monomeric and dimeric UNC5C, ELISA was performed using Fc-tagged dimeric rhUNC5C and His-tagged monomeric rhUNC5C. Similar binding profiles were observed for dimeric Fc-tagged rhUNC5C (Figure 18A) and monomeric His-tagged rhUNC5C (Figure 18B), reflected by similar EC50 values ​​(Table 13), indicating that ATL_5262 can bind to both monomeric and dimeric UNC5C.

[0270] [Table 13]

[0271] Antibody variants of ATL_5262 with different light chains were also generated and tested.

[0272] Additional candidate VLs were obtained by a two-step process. First, 600 different VLs were computationally synthesized for the VH of ATL_5262 using a modified version of the model described in WO2022 / 223451, where the encoder and decoder models were replaced with a pre-trained AntiBERTa model as described in Leem et al., 2022. As a second step, the 600 different VH-VL pairs were prioritized using a separate AntiBERTa model described in GB2215218.5, filed October 14, 2022. Briefly, the model from GB2215218.5 takes a VH-VL pair (natural or synthetic) as input and calculates the probability that the two chains will form a pairing.

[0273] Ten VLs were selected for synthesis based on predicted probability from the model (selecting candidate VLs with a high predicted matching probability with the VH of ATL_5262), low sequence confidence content, and sequence diversity. The VL variants tested included the VLs of ATL_5996 (SEQ ID NO: 10), ATL_5997 (SEQ ID NO: 11), ATL_5998 (SEQ ID NO: 12), ATL_5999 (SEQ ID NO: 13), ATL_6000 (SEQ ID NO: 14), ATL_6001 (SEQ ID NO: 15), ATL_6002 (SEQ ID NO: 16), ATL_6003 (SEQ ID NO: 17), ATL_6004 (SEQ ID NO: 18), and ATL_6005 (SEQ ID NO: 19). All of the above VL variant antibodies contain the VH of ATL_5262.

[0274] Different light chain variants were screened for binding to rhUNC5C and rmUNC5C by ELISA and compared to ATL_5262. The results show that ATL_6002 (same VH but different VL as ATL_0005262) ​​binds to rhUNC5C in a manner very similar to ATL_0005252 (Figure 19A). Similar results were observed for binding to rmUNC5C (Figure 19B). Variant ATL_6003 showed reduced binding (approximately 10-fold) to both rhUNC5C and rmUNC5C.

[0275] This data demonstrates that other VL pairings, for example using the VL of ATL_6002, are able to maintain the functionality of the VH described herein. The data further demonstrate that the first VH-VL pairing obtained (resulting in ATL_5262) ​​was already very good.

[0276] To measure the binding ability of the antibodies to the UNC5C protein, the antibodies were evaluated for binding to human UNC5C in a direct ELISA format. Furthermore, a selection of these antibodies was also screened for binding to mouse, cynomolgus monkey, and rat UNC5C in a direct ELISA format. Lysozyme was run as a control antigen and showed no discernible binding to any of the antibodies evaluated at concentrations below 1 μM. The anti-fluorescein antibody ATL-5338 was used as a negative isotype control and showed no binding to any of the UNC5C antigens evaluated up to antibody concentrations <1 μM. In addition, some of the antibodies were evaluated in additional assays to evaluate their ability to compete with netrin-1 for binding to UNC5C. The results of these tests are shown in Table 14.

[0277] [Table 14]

[0278] ATL_0005262, ATL_0005998, ATL_0006001, ATL_0006002, ATL_0006003, ATL_0006033, ATL_0006034, ATL_0006035, ATL_0006036, ATL_0006037, ATL_0006038, ATL_0006039, ATL_0006177, ATL_0006178, ATL_0006187, ATL_0006191 all showed binding to human UNC5C with varying potency (see Table 14). For ATL_0006001 and ATL_6039, signals at least three times the level of lysosomal regulation were observed, but because full dose-response curves could not be obtained, an EC50 cannot be reported. Therefore, in both cases, the data indicate that the antibodies bind to the target. In the case of ATL_6039, an EC50 could not be obtained because the antibody is so potent that a lower concentration range would have been required to test it. This data indicates that the antibody binds reliably, but a potency number cannot be reported.

[0279] ATL_0005262, ATL_0005998, ATL_0006001, ATL_0006002, ATL_0006003, ATL_0006178, ATL_0006191 demonstrated binding to mouse UNC5C. ATL_0005262, ATL_0006178, and ATL_0006191 demonstrated binding to cynomolgus monkey UNC5C. ATL_0005262, ATL_0006178, and ATL_0006191 were tested and demonstrated binding to rat UNC5C. In addition, netrin-1 competed with ATL_0005262, ATL_0006002, ATL_0006036, ATL_0006039, ATL_0006177 and ATL_0006178 for binding to human UNC5C.

[0280] These data demonstrate that at least ATL_0006178 exhibits binding to human, mouse, rat, and cynomolgus UNC5C and can compete with netrin-1 for binding to UNC5C.

[0281] Example 7 - PK Analysis In vivo pharmacokinetic studies were performed in mice to assess the circulating levels of ATL_5262 and its ability to penetrate the CNS compared to the control antibody ATL_5338. Antibody levels were assessed by ELISA in serum samples collected at 1, 4, 8, 24, 72, and 144 hours after intraperitoneal administration and in CSF samples collected at 4, 24, and 144 hours. ATL_5262 exhibits linear serum (Figure 20A) and CSF PK (Figure 20B) with a clearance profile comparable to that of the non-binding IgG1 control, ATL_5338. Importantly, ATL_5262 demonstrates evidence of CNS penetration with serum:CSF ratios of approximately 0.1-0.2%.

[0282] Example 8 - UNC5C Apoptosis Amyloid treatment of SHSY5Y cells, a neuroblastoma cell line, has been demonstrated to induce cell death. Netrin-1 has been reported to reduce this. To determine whether ATL6178 (also referred to herein as ATLX-1282) and ATL5262 can reproduce this effect, rescue experiments were performed in which antibodies were added to amyloid-treated SHSY5Y cells, followed by measurement of caspase-3 / 7 production as a measure of amyloid toxicity. Figure 22 shows the results of this assay. We demonstrated that ATLX1282 (ATL_6178), ATL5262, and netrin-1 can all reduce caspase-3 / 7 signaling in response to beta-amyloid 1-42-induced cytotoxicity.

[0283] The antibodies described herein are expected to enhance synaptic health (e.g., plasticity) by acting as netrin-1 mimetics, as predicted by their binding profiles. This can be tested using in vitro assays that directly assess measures of synaptic health, including, but not limited to, parameters such as the number of processes per cell, neurite outgrowth length, and branching (e.g., using methods provided in Spijkers et al., Sci Rep, 2021).

[0284] Taken together, this data indicates that the UNC5C antibodies described herein are likely to exhibit therapeutic efficacy in the treatment and / or prevention of neurodegenerative diseases such as FTD and AD, and provides antibodies that can achieve this by binding to the same binding site as netrin-1.

[0285] Example 12 - Synaptic plasticity in human iPSC dopaminergic neurons To examine the effects of ATLX-1282 and ATL5262 on synaptic proteins, a series of experiments were performed on human iPSC-derived dopaminergic neurons. As shown in Figure 23, treatment of dopaminergic neurons with ATL6178 resulted in a significant increase in the expression of synaptic proteins (specifically, PSD95, synaptophysin, synapsin-1, synaptotagmin-1, and neurofilament-L), similar to the effect observed with netrin-1.

[0286] These findings demonstrate that ATL6178, as expected from their binding profile, enhances synaptic health by acting as a netrin-1 mimetic using in vitro assays that directly assess measures of synaptic health, which may include parameters such as, but are not limited to, the number of processes per cell, neurite outgrowth length, and branching (e.g., using methods provided in Spijkers et al., 2021).

[0287] Example 13 - Netrin-1 depletion assay The effects of netrin-1 depletion in human iPSC-derived motor neurons were investigated using an anti-netrin-1 antibody to induce netrin-1 depletion. This was found to result in reduced levels of the axon growth cone protein GAP-43 (Figure 24A), increased neuronal cell death (Figure 24B), and axonal damage (Figure 24C). The figures show that ATLX-1282 and ATL-5262 effectively counteract these effects (Figures 24A-C).

[0288] These findings indicate that ATLX-1282 and ATL5262 enhance synaptic health by acting as netrin-1 mimetics.

[0289] Example 14 - Effect of anti-UNC5C antibodies on neuronal function TDP-43 is a highly conserved ribonucleoprotein that accumulates as ubiquitinated inclusions in both ALS and FTD patients. iPSC-derived motor neurons harboring the ALS-associated TDP43 Q331K mutation have been reported to exhibit synaptic dysfunction as assessed by multi-electrode arrays (Smith, Alec ST et al. 2021). Co-culture of such neurons with iCell astrocytes provides an elegant co-culture model, enabling a more comprehensive understanding of neural network behavior and its response to various compounds. To investigate this, iPSC-derived motor neuron and astrocyte co-cultures were treated with ATLX-1282 or an isotype control (ATL-5338) for up to 142 hours, and activity was assessed by multi-electrode assay (MEA), which measures the extracellular field potentials produced by neurons. ALS TDP43 Q331K iCell motor neurons exhibit significant deficits in overall activity, viability, and burst kinetics when compared to their isogenic control counterparts. These ALS TDP43 Q331K iCell motor neurons also show discernible changes in connectivity and mean firing rate compared to isogenic iCell motor neurons.

[0290] ATLX-1282 attenuated the time-dependent loss of activity seen in TDP43 Q331K motor neurons and promoted enhanced burst activity (Figures 25A-B). Viability remained relatively stable over time within each cell line (Figure 25C), suggesting that any changes in activity were a putative change and promotion of neuronal health rather than simply a result of cell death. Notably, ATLX-1282 also plays a crucial role in enhancing synaptic connectivity, thus promoting enhanced synchronization between ALS TDP43 Q331K motor neurons, as indicated by the increased synchrony index and higher firing rate of ATLX-1282-treated TDP43 cells compared to isotype controls (Figure 25D). These findings represent compelling demonstration of the efficacy of ATLX-1282 treatment within motor neurons.

[0291] Example 15 - In vivo mouse studies Haploinsufficiency of UNC5C or netrin-1 results in altered innervation of the medial prefrontal cortex by dopaminergic neurons and disrupted dopamine content in the medial prefrontal cortex (mPFC). This manifests as a reduced response to the stimulant amphetamine (Pokinko et al., 2015). To determine whether ATLX-1282 can act as a ligand mimetic for netrin-1, netrin-1 haploinsufficient mice were treated with ATLX-1282 and their response to amphetamine was assessed. To determine the optimal window in which any altered response to amphetamine could be observed, amphetamine was administered on various days, starting on day 8 after the first ATXL-1282 treatment. As reported in the literature, a reduced response to amphetamine was observed in netrin-1 haploinsufficient mice compared to non-transgenic (wild-type) littermates. This was statistically significant by day 10 of the study (second amphetamine treatment). Therefore, the ability of ATLX-1282 to rescue the netrin-1 haploinsufficient phenotype was tested at this time point.

[0292] On day 10, ATLX-1282 treatment resulted in a statistically significant increased response to amphetamine in netrin-1 haploinsufficient mice compared to isotype controls (see Figure 26). These data suggest that ATLX-1282 can effectively engage UNC5C in its native CNS environment and reduce the deficits induced by netrin-1 haploinsufficiency, and that ATLX-1282 can act as a ligand mimetic of netrin-1.

[0293] Example 16: In vivo rat studies 6-hydroxydopamine (6-OHDA) is a neurotoxin that has been demonstrated to cause degeneration of dopaminergic neurons in the midbrain. Netrin-1 administered intracerebroventricularly (ICV) has been shown to have neuroprotective effects against 6-OHDA-mediated degeneration (Jasmin et al., 2021).

[0294] ATLX-1282 and the isotype control ATL-5338 were tested in an efficacy study in 6-OHDA-treated rats to determine whether they could reproduce this effect (whether ATLX-1282 has a neuroprotective effect similar to netrin-1 in this model). This was assessed using the contralateral limb cylinder test to determine the extent of dopaminergic cell loss in treated rats. Rats treated with 6-OHDA showed a mean decrease in contralateral limb use compared to saline-treated controls, indicating successful 6-OHDA treatment (i.e., demonstrating 6-OHDA-mediated neurodegeneration). The mean contralateral contact of rats treated with ATLX1282 and 6-OHDA tended to improve compared to isotype control and 6-OHDA-treated rats (see Figure 27), indicating that ATLX-1282 may have a neuroprotective effect in PD similar to netrin-1.

[0295] Taken together, these data demonstrate that the UNC5C antibodies described herein exhibit therapeutic efficacy in the treatment and / or prevention of neurodegenerative diseases such as PD, FTD and AD, and provide antibodies that can achieve this by binding to the same binding site as netrin-1.

[0296] Example 17 - Solubility and safety of ATL_6178 (ATLX_1282) The manufacturability of ATL_6178 (also referred to herein as ATXL_1282) was tested after exposure to different stress conditions. Such conditions included freeze-thaw cycles, agitation, thermal stability, and low pH. Stressed samples were evaluated for changes in appearance, purity, aggregation, size, and charge variants using MFI, SEC-HPLC, CE-SDS, cIEF, and DLS. Samples were also concentrated to high densities (50 mg / mL, 100 mg / mL, and 200 mg / mL) and analyzed at time 0 and after 7 days of incubation at room temperature. Solubility samples were analyzed for appearance, viscosity, and purity using SEC-HPLC.

[0297] ATL_6178 was expressed from CHO-K1 stable pool transfections and after 14 days of fed-batch culture, the culture was harvested and the monoclonal antibody purified using protein A capture and a second cation exchange chromatography step, followed by centrifugation using a 30 kDa cutoff and buffer exchange into formulation buffer 20 mM histidine HCl, 8% sucrose, 0.04% Tween 80, pH 6.0, followed by sterile filtration.

[0298] The solubility of ATLX-1282 was tested at 50 mg / mL, 100 mg / mL, and 200 mg / mL. Samples were evaluated at time 0 and after 7 days at 25°C. The results are shown in Tables 15 and 16. Visual evaluation indicated that the protein was free of particles and unchanged in appearance (Table 15), while SEC-HPLC analysis demonstrated substantial changes in the monomeric form present after concentration and incubation at 25°C for 7 days, with only slight changes in the low molecular weight species (LMWS) and high molecular weight species (HMWS) (Table 16). This indicates that the molecule is stable at high concentrations. The viscosity reached a maximum of 24 cP at 200 mg / mL, indicating that the molecule is manufacturable.

[0299] [Table 15]

[0300] [Table 16]

[0301] To further evaluate the stability of ATL_6178, the antibody was subjected to a panel of different stress conditions, including temperature, freeze-thaw cycles, agitation, and low pH. Visual analysis of the samples before and after the stress conditions showed that the samples were slightly yellow, slightly milky, and free of macroscopic particles, with no change in appearance after the conditions evaluated, except for heat stress at 40°C for 4 weeks, which showed low levels of macroscopic particles. Table 17 shows the results of these stress studies.

[0302] [Table 17]

[0303] Microflow imaging (MFI) analysis shows an increase in sub-visible particles >2-25 μm detected for the 40°C stress condition. Particle size and distribution were analyzed using dynamic light scattering (DLS). Z-average showed no significant change in any of the samples compared to the unstressed ones stored at -70°C. Polydispersity index (PDI) indicates that the samples were monodisperse (Table 18).

[0304] [Table 18]

[0305] SEC-HPLC analysis shows no change after 3 or 5 freeze-thaw cycles or 1 or 3 days of agitation. A slight decrease in the main peak % monomer was detected after 4 weeks of incubation at 40°C, with an increase of 0.7% and 0.5% of HMWS and LMWS present, respectively. A low pH hold at 25°C, pH 3.4, resulted in a 1.3% increase in HMWS (Table 19).

[0306] [Table 19]

[0307] Charge heterogeneity of ATL_1678 was analyzed using cIEF. A decrease in the major charge variant was observed at 40°C (-22.6 pp at 2 weeks and 34.3 pp at 4 weeks) along with an increase in both acidic and basic species, while minimal changes in the charge species present were observed at all other conditions (Table 20).

[0308] [Table 20]

[0309] Molecular weight species and purity were analyzed after exposure of samples to stress conditions using CE-SDS. Samples incubated at 40°C for 4 weeks showed a decrease in purity of 3.5 pp for the non-reduced sample and 1 pp for the reduced sample. Minimal changes were observed for samples experiencing other stress conditions (Table 21).

[0310] [Table 21]

[0311] The above data indicate that ATL_6178 is highly stable under various stress conditions.

[0312] Example 18 - Binding to cellular UNC5C To assess whether ATL_6178 (also referred to herein as ATLX-1282) and ATL5262 can bind to UNC5C in the native central nervous system (CNS) context, antibody binding to frozen mouse brain sections was assessed.

[0313] ATLX-1282 (ATL_6178) and ATL_5262 showed similar binding patterns, which were blocked by 10 nM netrin-1 but not by similarly sized proteins such as clusterin and netrin-4 (which do not bind to UNC5C), as shown in Figures 29A-29D. This is consistent with the in vitro data above showing the ability of ATLX-1282 and ATL_5262 to selectively bind to UNC5C.

[0314] To confirm antibody binding to UNC5C expressed on the cell surface, the antibodies were evaluated in a flow cytometry assay. The standard human UNC5C isoform (O95185) and the AD-linked UNC5C T835M variant (VAR_081368) were constitutively overexpressed in HEK-293 cell lines using a lentiviral system. The antibodies were then tested for binding using flow cytometry. An empty vector (EV)-transduced cell line and an antibody specific for an unrelated target expressed in the relevant IgG format (control ATL) were used as negative controls.

[0315] ATL5262 and ATLX-1282 showed increased binding to HEK-293 cells overexpressing UNC5C and UNC5C T835M compared to cells transfected with the empty vector control (Figures 30A and 30B).

[0316] These data demonstrate that ATL5262 and ATLX-1282 can bind to UNC5C and the AD-linked UNC5C T835M variant (VAR_081368) when in their native cell membrane environment. This data indicates that the antibodies bind to Alzheimer's disease-associated forms of UNC5C. The antibodies described herein have been shown to bind to the extracellular domain of UNC5C, and this mutation affects the intracellular domain. Therefore, all of the described antibodies are expected to bind to AD-associated forms of UNC5C.

[0317] Example 19 - Determination of the crystal structure of the ATL_5262 Fab fragment bound to the human UNC5C Ig domain To better understand the binding of ATL_5262 to UNC5C, an X-ray crystal structure was obtained for a complex containing the Fab fragment of ATL_5262 bound to the immunoglobulin (Ig) domain of human UNC5C. A schematic of the complete Fab-UNC5C complex is shown in Figure 31A, and expanded views of the specific interactions between the proteins are shown in Figures 31B-31E. A summary of the interchain contacts is provided below in Table 22, and details of specific amino acid interactions are provided in Table 23.

[0318] [Table 22]

[0319] [Table 23]

[0320] Figure 30F shows a protein alignment of human UNC5 family members B, A, and D to the region of human UNC5C Ig-like domain 1 that interacts with ATL_5262. Identical sequences are highlighted in red. Amino acids in UNC5C that are involved in the interaction with ATL_5262 and summarized in Table 23 are indicated by arrows. These data indicate that the specificity of ATL_5262 for UNC5C over other UNC5 family members may be driven by amino acids that differ between UNC5C and other UNC5 proteins.

[0321] Example 20 - Further antibodies Phage display selection was performed to discover additional functional and resilient patient-derived antibodies against UNC5C. A phage display library was generated from the VH repertoire of the patient carrying the original antibody ATL_0005262 and the VL repertoire from a healthy donor. Phage display selection was performed by panning on two UNC5C antigens. A subset of enriched antibodies was screened by phage ELISA against the UNC5C antigen, and 10 antibodies were selected for IgG conversion.

[0322] Ten phage-derived IgGs (ATL_6530 to ATL_6539) were screened for binding to UNC5C by ELISA, and the EC50 values ​​estimated from the UNC5C binding curves are listed in Table 24 below. All antibodies showed high affinity binding to UNC5C. ATL_6178 was also included for reference. Figure 32 shows the alignment of the VH and VL sequences of these antibodies.

[0323] [Table 24]

[0324] Next, these antibodies were screened to determine whether they could compete with recombinant netrin-1 for binding to UNC5C. UNC5C was coated on an ELISA plate, and titrations of each antibody were individually preincubated with UNC5C protein, followed by the addition of a set concentration of netrin-1. In each case, the binding of each antibody to netrin-1 was determined using a secondary detection antibody specific for each reagent. If the presence of netrin-1 was able to shift the antibody binding curve to the right (i.e., a higher concentration of antibody was required to see a similar level of signal for binding to UNC5C), this was observed as competition in the assay. Furthermore, if the presence of the antibody was able to reduce the signal for netrin-1 binding detected in the assay, this was also observed as competition (see Table 25). All antibodies showed competition with netrin-1.

[0325] [Table 25]

[0326] [Table 26]

[0327] Thus, this example identifies additional antibodies that bind to UNC5C with high affinity, can compete with netrin-1 for UNC5C binding, and are therefore expected to have properties similar to ATL_5262 and ATLX-1282, including reducing UNC5C-mediated apoptosis, reducing beta-amyloid toxicity, increasing neuronal activity, survival, and burst frequency, and enhancing synaptic health in vitro and in vivo.

[0328] The above data indicate that all of these antibodies bind to a region of UNC5C shared by netrin binding (as do the other UNC5C-binding antibodies described herein, including ATL_5262 and ATLX-1282). The binding site for netrin on UNC5C was previously unknown, but was thought to be somewhere within the first two immunoglobulin domains of UNC5C. This data provides updated information suggesting that the binding site may be within the first immunoglobulin domain of UNC5C and may at least partially overlap with where the antibodies described herein bind to UNC5C.

[0329] Taken together, the data described herein demonstrate that the UNC5C antibodies described herein exhibit therapeutic efficacy in the treatment and / or prevention of neurodegenerative diseases such as PD, FTD, AD and ALS, and provide antibodies that can achieve this by binding to the same binding site as netrin-1.

[0330] References All documents cited herein are incorporated by reference in their entirety. 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Claims

1. An isolated antibody or antibody fragment thereof that specifically binds to UNC5C protein or a fragment thereof, wherein said antibody mimics and / or competes with netrin-1 for binding to UNC5C, and optionally wherein competition with netrin-1 is determined by ELISA.

2. 2. The isolated antibody of claim 1, wherein the antibody binds to human and / or mouse UNC5C, and optionally the antibody binds to human UNC5C with an EC50 of up to 9.81E-08M, or up to 1E-08M, or up to 5.5E-09M, or up to 9.7E-09M when assessed by ELISA (such as binding of plated rhUNC5C).

3. 3. The isolated antibody of claim 1 or 2, wherein the antibody reduces UNC5C-mediated apoptosis, optionally wherein the antibody reduces UNC5C-mediated neuronal apoptosis, and / or in induced pluripotent stem cell (iPSC)-derived neurons, and / or in neuroblastoma cells, optionally SH-SY5Y cells, optionally wherein apoptosis is measured by measuring caspase 3 / 7.

4. 4. The isolated antibody of any one of claims 1 to 3, wherein the antibody selectively binds to UNC5C over one or more other netrin receptors, and optionally the antibody selectively binds to UNC5C over neogenin, DCC, and one or more (or all) of DSCAM, UNC5A, UNC5B, UNC5.

5. 5. The isolated antibody of any one of claims 1 to 4, wherein the antibody binds to UNC5C according to SEQ ID NO: 99 and to the UNC5C T835M variant according to SEQ ID NO:

110.

6. 6. The isolated antibody of any one of claims 1 to 5, wherein the antibody reduces beta-amyloid toxicity in neurons, optionally wherein the antibody reduces cell death of beta-amyloid-treated neurons in vitro, and / or wherein the antibody reduces caspase 3 / 7 signaling in response to beta-amyloid-induced cytotoxicity.

7. wherein the antibody enhances synaptic health, and optionally the enhanced synaptic health is increased neuronal expression of one or more synaptic proteins selected from PSD95, synaptophysin, synapsin-1, synaptotagmin-1, neurofilament-L, and GAP-43; and / or increased neuronal growth and / or branching, 7. The isolated antibody of any one of claims 1 to 6, wherein the antibody increases the synchrony index and firing rate of motor neurons harboring the TDP43 Q331K mutation, as assessed by increased neuronal firing synchrony and / or firing rate, and optionally wherein the antibody increases the synchrony index and firing rate of motor neurons harboring the TDP43 Q331K mutation.

8. 8. The isolated antibody of any one of claims 1 to 7, wherein the antibody improves dopaminergic neuron function and / or survival, and optionally the improved dopaminergic neuron function and / or survival is assessed by at least partial rescue by the antibody of the effect of UNC5C or netrin-1 haploinsufficiency on dopaminergic neuron innervation to and / or dopamine content in the medial prefrontal cortex.

9. 9. The isolated antibody of any one of claims 1-8, wherein the antibody reduces neurodegeneration of dopaminergic neurons, optionally assessed by measuring neurodegeneration upon exposure to a neurotoxin such as 6-hydroxydopamine (6-OHDA).

10. 10. An isolated antibody according to any one of claims 1 to 9, wherein the antibody binds to the extracellular region of UNC5C, and / or the antibody binds to one or more residues of UNC5C involved in the binding of netrin-1 to UNC5C, and / or the antibody binds to an epitope in the N-terminal immunoglobulin domain of UNC5C.

11. 11. The isolated antibody of any one of claims 1 to 10, wherein the antibody binds to an epitope in UNC5C comprising one or more or all of the following residues of UNC5C when numbered according to SEQ ID NO:99: Thr89, Gln90, Gln102, Lys103, Val107, Asp108, Glu109, Arg110, Val111, Ile118, Arg120, optionally wherein the antibody binds to residues Thr89, Gln90, Gln102, Lys103, Val107, Glu109, Ile118, and Arg120 of UNC5C when numbered according to SEQ ID NO:

99.

12. The antibody comprises the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 20; a CDRH2 comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 23; or 12. The isolated antibody of any one of claims 1 to 11, comprising a heavy chain variable domain (VH) having a set of CDRs comprising one or two amino acid mutations, optionally substitutions, compared to the set of CDRs described above.

13. 10. The antibody comprising the following framework sequence: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 51, HFWR3 of SEQ ID NO: 55, and HFWR4 of SEQ ID NO: 62, or 13. The isolated antibody of any one of claims 1 to 12, having a heavy chain variable domain (VH) with a framework sequence having 1 to 5 mutations, optionally substitutions, compared to the framework sequences described above.

14. The mutations in the framework sequences are at the following positions according to standard IMGT numbering: HFWR2: 40th, 49th, HFWR3: selected from positions 80, 82, 86 and / or wherein said substitution in said framework sequences is at the following positions according to standard IMGT numbering: HFWR2: 40th place: T40S, 49th place: R49G, 14. The isolated antibody of claim 13, selected from HFWR3: position 80: I80V, position 82: T82K, position 86: H86Q.

15. 15. The isolated antibody of claim 13 or 14, wherein the mutations in the framework sequences do not include a mutation at position 67 in the standard IMGT numbering.

16. 10. The antibody comprising the following framework sequence: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 51, 52, 53, or 54; HFWR3 of SEQ ID NO: 55, 56, 57, 58, or 61; HFWR4 of SEQ ID NO: 62, Optionally, the antibody comprises the following framework sequence: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 52, HFWR3 of SEQ ID NO: 61, and 16. The isolated antibody of any one of claims 1 to 15, having a heavy chain variable domain (VH) having SEQ ID NO: 62, HFWR4.

17. the antibody has a heavy chain variable domain (VH) comprising a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH) having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 1 (ATL_5262 VH), 5 (ATL_0006036_VH), 8 (ATL_0006039_VH), 91 (ATL_0006177_VH), and 93 (ATL_0006178_VH); 17. The isolated antibody of any one of claims 1-16, optionally wherein the antibody has a heavy chain variable domain (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 91 (ATL_6177 VH) and SEQ ID NO: 93 (ATL_6178 VH).

18. 18. The isolated antibody of any one of claims 1 to 17, wherein the antibody has a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3 in a germline framework, with the proviso that position 67 according to standard IMGT numbering is Q.

19. The antibody comprises the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 115 or 116; a CDRH2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and a heavy chain variable domain (VH) having a CDRH3 comprising the amino acid sequence of SEQ ID NO: 119 or 120; 11. The isolated antibody of any one of claims 1-10, optionally wherein the antibody comprises a heavy chain variable domain (VH) having the CDRs of ATL_6187 VH (SEQ ID NOs: 115, 117, and 119) or the CDRs of ATL_6191 VH (SEQ ID NOs: 116, 118, and 120).

20. 10. The antibody comprising the following framework sequence: HFWR1 of SEQ ID NO: 50, HFWR2 of SEQ ID NO: 52 or 195, HFWR3 of SEQ ID NO: 196 or 197, HFWR4 of SEQ ID NO: 198 or 62, or a heavy chain variable domain (VH) having framework sequences that contain 1 to 5 mutations, e.g., substitutions, compared to these framework sequences; 20. The isolated antibody of claim 19, optionally wherein the antibody comprises a heavy chain variable domain (VH) having HFWR of ATL_6187 VH (SEQ ID NOs: 50, 52, 196, 198) or HFWR of ATL_6191 VH (SEQ ID NOs: 50, 195, 197, 62).

21. 21. The isolated antibody of claim 19 or 20, wherein the antibody has a heavy chain variable domain (VH) comprising a selected sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 112 (ATL_0006187 VH) and 113 (ATL_0006191 VH).

22. The antibody comprises the following CDRs: CDRL1 comprising the amino acid sequence of SEQ ID NO: 24, 27, 30, 31 or 32; CDRL2 comprising the amino acid sequence of SEQ ID NO: 34, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 40, 43, 46, 47; or 22. The isolated antibody of any one of claims 1 to 21, comprising a light chain variable domain (VL) having a set of CDRs comprising 1, 2, or 3 amino acid mutations, optionally substitutions, compared to the set of CDRs described above.

23. 23. The isolated antibody of claim 22, wherein the CDRL1, CDRL2 and CDRL3 of the VL domain are in a germline framework.

24. 10. The antibody comprising the following framework sequence: LFWR1 of SEQ ID NOs: 63 and 66; LFWR2 of SEQ ID NOs: 71, 73, 75; LFWR3 of SEQ ID NOs: 77 and 80, and LFWR4 of SEQ ID NOs: 85, 87, 89, or 24. The isolated antibody of claim 22 or 23, having a light chain variable domain (VL) having a set of FWRs that comprises 1 to 5 amino acid substitutions compared to the set of FWRs described above.

25. the antibody has a light chain variable domain (VL) comprising a sequence selected from SEQ ID NOs: 9 (ATL_5262 VL), 12 (ATL_5998 VL), 15 (ATL_6001 VL), 16 (ATL_6002 VL), 17 (ATL_6003 VL) having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 9 (ATL_5262 VL), 12 (ATL_5998 VL), 15 (ATL_6001 VL), 16 (ATL_6002 VL), 17 (ATL_6003 VL); 25. The isolated antibody of any one of claims 1-24, optionally wherein the antibody has a light chain variable domain (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 9 (ATL_5262 VL), SEQ ID NO: 16 (ATL_6002 VL), and SEQ ID NO: 17 (ATL_6003 VL).

26. one or more (or all) of VH residues Tyr55, Gly63, Thr64, Thr65, Asn66, Ser74, Arg108, Met110 interact with one or more residues of UNC5C, and / or one or more (or all) of VL residues Tyr38, Ser107, Tyr108, ​​Ser109, Thr114 interact with one or more residues of UNC5C, and optionally the antibody and / or a VH having a sequence with conservative amino acid substitutions at one or more of the following positions: Tyr at position 55, Gly at position 63, Thr at position 64, Thr at position 65, Asn at position 66, Ser at position 74, Arg at position 108, and Met at position 110; The antibody 26. The isolated antibody of any one of claims 1 to 25, comprising a VL having a sequence with conservative amino acid substitutions at position 38: Tyr, position 107: Ser, position 108: Tyr, position 109: Ser, position 114: Thr, or one or more of these positions.

27. the antibody has a VH having at least 80% sequence identity to the VH of SEQ ID NO: 1 (ATL_5262 VH) and a VL having at least 80% sequence identity to the VL of SEQ ID NO: 9 (ATL_5262 VL); (a) VH residue 66 is Asn, and optionally Asn66 interacts with Val107 of UNC5C; and / or (b) VH residues 55, 66, 108, and 110 are Tyr, Asn, Arg, and Met, respectively, and optionally Tyr55, Asn66, Arg108, and / or Met110 interact with Glu109 of UNC5C; and / or (c) VH residue 108 is Arg and / or residue 110 is Met, and optionally Arg108 and / or Met110 interact with Ile118 of UNC5C; and / or (d) VH residues 55, 64, 65 are Tyr, Thr, and Thr, respectively, and optionally Tyr55, Thr64, and / or Thr65 interact with Arg120 of UNC5C; and / or (e) VH residue 64 is Thr, and optionally Thr64 interacts with Thr89 and / or Arg120 of UNC5C; and / or (f) VH residue 63 is Gly, and optionally Gly63 interacts with Gln90 of UNC5C; and / or (g) VH residue 65 is Thr, and optionally Thr65 interacts with Gln102 and / or Arg120 of UNC5C; and / or (h) VH residue 74 is Ser, and optionally Ser74 interacts with Lys103 of UNC5C; and / or (i) VL residue 114 is Thr, and optionally Thr114 interacts with Val107 and / or Glu109, and / or Arg110 of UNC5C; and / or (j) VL residue 109 is Ser, and optionally Ser109 interacts with Glu109 and / or Arg110 of UNC5C; and / or (k) VL residue 108 is Tyr, and optionally Tyr108 interacts with Arg110 and / or Val111 of UNC5C; and / or (l) VL residue 107 is Ser, and optionally Ser107 interacts with Val111 of UNC5C; and / or (m) VL residue 38 is Tyr, and optionally Tyr38 interacts with Val111 of UNC5C; 27. The isolated antibody of any one of claims 1 to 26, wherein the UNC5C residues are numbered according to SEQ ID NO:

99.

28. The antibody comprises the following CDRs: CDRH1 comprising the amino acid sequence of SEQ ID NO: 142-149; a CDRH2 comprising the amino acid sequence of SEQ ID NOs: 150-158; and a heavy chain variable domain (VH) having a CDRH3 comprising the amino acid sequence of SEQ ID NO: 159-168; Optionally, the antibody is selected from the group consisting of antibody ATL_6530 (SEQ ID NOs: 142, 150, 159); ATL_6531 (SEQ ID NOs: 143, 151, 160); ATL_6532 (SEQ ID NOs: 144, 152, 161); ATL_6533 (SEQ ID NOs: 145, 153, 162); ATL_6534 (SEQ ID NOs: 144, 152, 163); ATL_6535 (SEQ ID NO: 143 11. The isolated antibody of any one of claims 1 to 10, comprising a VH having CDRs of any of the following: ATL_6536 (SEQ ID NOs: 146, 155, 165); ATL_6537 (SEQ ID NOs: 147, 156, 166); ATL_6538 (SEQ ID NOs: 148, 157, 167); ATL_6539 (SEQ ID NOs: 149, 158, 168).

29. 10. The antibody comprising the following framework sequence: HFWR1 of SEQ ID NOs: 199, 202, 205, 209, 212, 213, 216, 220, 224, 227; HFWR2 of SEQ ID NOs: 200, 203, 206, 210, 214, 217, 221, 225, 228; HFWR3 of SEQ ID NOs: 201, 204, 207, 211, 215, 218, 222, 226, 229; having a heavy chain variable domain (VH) having HFWR4 of SEQ ID NO: 62, 208, 219, 223; Optionally, the antibody is selected from the group consisting of antibody ATL_6530 (SEQ ID NOs: 199, 200, 201, 62); ATL_6531 (SEQ ID NOs: 202, 203, 204, 62); ATL_6532 (SEQ ID NOs: 205, 206, 207, 208); ATL_6533 (SEQ ID NOs: 209, 210, 211, 208); ATL_6534 (SEQ ID NOs: 212, 206, 207, 208); ATL_6535 (SEQ ID NOs: 213, 214, 29. The isolated antibody of claim 28, having a heavy chain variable domain (VH) having a framework sequence of any of the following: ATL_6536 (SEQ ID NOs: 216, 217, 218, 219); ATL_6537 (SEQ ID NOs: 220, 221, 222, 223); ATL_6538 (SEQ ID NOs: 224, 225, 226, 208); ATL_6539 (SEQ ID NOs: 227, 228, 229, 208).

30. 30. The isolated antibody of claim 28 or 29, wherein the antibody has a heavy chain variable domain (VH) having the CDR and / or framework sequences of ATL_6532 or ATL6533.

31. the antibody comprises a heavy chain variable domain (VH) comprising a selected sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 122 (ATL_0006530 VH), 123 (ATL_0006531 VH), 124 (ATL_0006532 VH), 125 (ATL_0006533 VH), 126 (ATL_0006534 VH), 127 (ATL_0006535 VH), 128 (ATL_0006536 VH), 129 (ATL_0006537 VH), 130 (ATL_0006538 VH); 31. The isolated antibody of any one of claims 28-30, optionally wherein the antibody has a heavy chain variable domain (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 124 (ATL_6532 VH), SEQ ID NO: 125 (ATL_6533 VH).

32. The antibody comprises the following CDRs: CDRL1 comprising the amino acid sequence of SEQ ID NO: 169-176; CDRL2 comprising the amino acid sequence of SEQ ID NO: 177-183; a light chain variable domain (VL) having a CDRL3 comprising the amino acid sequence of SEQ ID NO: 184-193; Optionally, the antibody is selected from the group consisting of antibody ATL_6530 (SEQ ID NOs: 169, 177, 184); ATL_6531 (SEQ ID NOs: 170, 178, 185); ATL_6532 (SEQ ID NOs: 171, 177, 186); ATL_6533 (SEQ ID NOs: 169, 177, 187); ATL_6534 (SEQ ID NOs: 169, 177, 188); ATL_6535 (SEQ ID NOs: 172, 32. The isolated antibody of any one of claims 28 to 31, comprising a VL having any of the CDRs of: ATL_6536 (SEQ ID NOs: 173, 180, 190); ATL_6537 (SEQ ID NOs: 174, 181, 191); ATL_6538 (SEQ ID NOs: 175, 182, 192); ATL_6539 (SEQ ID NOs: 176, 183, 193).

33. 10. The antibody comprising the following framework sequence: LFWR1 of SEQ ID NOs: 230 to 237 and 68; LFWR2 of SEQ ID NOs: 239 to 246; LFWR3 of SEQ ID NOs: 247 to 255, and LFWR4 of SEQ ID NOs: 256-258, Optionally, the antibody is selected from the group consisting of antibody ATL_6530 (SEQ ID NOs: 230, 238, 247, 256); ATL_6531 (SEQ ID NOs: 231, 239, 248, 257); ATL_6532 (SEQ ID NOs: 232, 240, 249, 258); ATL_6533 (SEQ ID NOs: 230, 241, 247, 256); ATL_6534 (SEQ ID NOs: 233, 238, 250, 256); ATL_6535 (SEQ ID NOs: 234 33. The isolated antibody of any one of claims 28 to 32, comprising a VL having a FWR of any of the following: ATL_6536 (SEQ ID NOs: 68, 243, 252, 257); ATL_6537 (SEQ ID NOs: 235, 244, 253, 256); ATL_6538 (SEQ ID NOs: 236, 245, 254, 256); ATL_6539 (SEQ ID NOs: 237, 246, 255, 256).

34. 34. The isolated antibody of any one of claims 28-33, wherein the antibody has a light chain variable domain (VL) comprising a sequence selected from SEQ ID NOs: 132-141 (ATL6530-6539 VL) having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity, and optionally the antibody has a light chain variable domain (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NO: 134 (ATL_0006532) or SEQ ID NO: 135 (ATL_0006533).

35. 35. The isolated antibody of any one of claims 1 to 34, wherein the antibody comprises an scFv antibody molecule, a nanobody, an antibody constant region, or a whole antibody.

36. 36. The isolated antibody of any one of claims 1 to 35, wherein the antibody is a monoclonal antibody, and / or the antibody is a whole antibody, and / or the antibody is an IgG1 or a variant thereof, optionally the antibody is the IgG1 variant L234A / L235A (LALA).

37. An isolated VH domain of an antibody according to any one of claims 1 to 36.

38. 38. An isolated nucleic acid comprising a nucleotide sequence encoding an antibody or antibody fragment thereof comprising the VH or VL domain of any one of claims 1 to 37.

39. 39. A vector or set of vectors comprising the nucleic acid of claim 38.

40. 40. A host cell comprising the vector or set of vectors of claim 39, or a host cell transformed in vitro with a nucleic acid of claim 38.

41. A composition comprising an antibody or fragment thereof comprising the antibody VH domain or antibody VL domain of any one of claims 1 to 36, and at least one additional component, and optionally a pharmaceutically acceptable excipient, vehicle or carrier.

42. 20. An antibody or fragment thereof that binds to UNC5C for use in the treatment of a neurodegenerative disease or disorder, optionally wherein the antibody is as defined in any one of claims 1 to 36 and / or the neurodegenerative disorder is selected from the group consisting of frontotemporal dementia (FTD), Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), human immunodeficiency virus (HIV)-induced encephalitis, chronic traumatic encephalopathy (CTE), vascular dementia, prion diseases, Lewy body diseases, spinal muscular atrophy (SMA), and / or rheumatoid arthritis (RH). ), Motor Neuron Disease (MND), such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), Spinocerebellar Ataxia (SCA) types 1, 2, 6, 7 and 17, Machado-Joseph disease (MJD / SCA3), Dentatorubral-Pallidoluysian atrophy (DRPLA), Spinal-bulbar muscular atrophy type 1 linked (SMAX1 / SBMA), Anderson-Fabry (X-linked Fabry disease), and DNAJB6 myopathy, and optionally said neurodegenerative disease is selected from FTD, AD, HD, and PD.

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