Method of treating chronic pain using inhibitor of neuropilin-1 (NRP1) and g alpha interacting protein (GAIP) interacting protein c-terminus 1 (GIPC1)

Abstract

The application provides isolated peptides that bind to neuropilin-1 (NRP-1) and compositions thereof. The application also provides methods for treating chronic pain comprising administering to a subject in need thereof a therapeutically effective amount of an NRP1 inhibitor and / or a G Alpha Interacting Protein (GAIP) Interacting Protein C-terminus 1 (GIPC1) inhibitor.

Description

Attorney Docket No: 243735.000408 METHOD OF TREATING CHRONIC PAIN USING INHIBITOR OF NEUROPILIN-1 (NRP1) AND G ALPHA INTERACTING PROTEIN (GAIP) INTERACTING PROTEIN C-TERMINUS 1 (GIPC1) CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims priority to U.S. Provisional Application No. US63 / 605,825, filed December 4, 2023, the disclosure of which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under NS102722 awarded by theNational Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submittedelectronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 2, 2024, is named 243735_000408_SL.xml and is 115,743 bytes in size. FIELD OF THE INVENTION

[0004] The application relates to isolated peptides that bind to neuropilin-1 (NRP-1) andcompositions thereof. The application further relates to methods for treating chronic pain comprising administering to the subject a therapeutically effective amount of an inhibitor of NRP1 and / or an inhibitor of G Alpha Interacting Protein (GAIP) Interacting Protein C-terminus 1 (GIPC1). BACKGROUND

[0005] Nerve growth factor (NGF) was discovered for its ability to stimulate growth ofsympathetic neurons1. Tropomyosin-related kinase A (TrkA), a receptor tyrosine kinase (RTK), mediates the neurotrophic actions of NGF2. NGF / TrkA signalosomes are retrogradely transported from peripheral nerve terminals to the soma, where they regulate transcription and neuronal development3,4. NGF and TrkA are also implicated in pain5. p75 neurotrophin receptor (p75NTR) 1 304870344v1Attorney Docket No: 243735.000408 is a co-receptor for NGF and the immature pro-peptide6,7that activates opposing pro-apoptotic signaling pathways8. Although NGF and its receptors have studied in the context of neuronal development and pain, mechanistic gaps in understanding have hampered the successful development of NGF-directed therapeutics.

[0006] Chronic pain afflicts twenty percent of the global population at some point in life, yet isinadequately treated by opioids and non-steroidal anti-inflammatory drugs, which lack efficacy and have life-threatening side effects. NGF / TrkA are one of the few non-opioid targets for chronic pain validated in patients. NGF, which is produced by injured and diseased tissues, activates TrkA on peripheral nociceptors to evoke hyperexcitability and the expression of neuropeptides and ion channels that mediate pain5. The central role TrkA and NGF in pain is evident in patients with Hereditary Sensory and Autonomic Neuropathy (HSAN) Type IV and V, where pathological insensitivity to pain results from loss-of-function mutations in TrkA9and NGF10, respectively. Although monoclonal antibodies (mAbs) are analgesic in osteoarthritic patients11, mAbs lack FDA approval due to worsening joint damage in some individuals12. The identification of nociceptor- enriched mediators of NGF-induced pain may facilitate development of non-opioid analgesics, avoiding the adverse effects of systemic NGF sequestration with mAbs.

[0007] Transcriptomics analyses have identified proteins that are conserved between rodent andhuman nociceptors, including neuropilin-1 (NRP1)13. NRP1 is a type I transmembrane protein discovered for its role in axon guidance14. Lacking a catalytic domain, NRP1 does not transduce signals per se but rather acts as a co-receptor for unrelated families of proteins, including vascular endothelial growth factor A (VEGF-A)15. NRP1 acts as a co-receptor with the RTK VEGF receptor 2 (VEGFR2) to enhance VEGF-A-induced blood vessel development. NRP1 overexpression in cancers spurred the development of a human anti-NRP1 mAb, vesencumab / MNRP1685A, to inhibit tumorigenesis16. SUMMARY OF THE INVENTION

[0008] In view of the lack of availability of sufficient non-opioid analgesic-based therapies forchronic pain, the present disclosure provides, among other things, a method of treating chronic pain by targeting NRP1, a co-receptor for NGF and TrkA, in nociception and / or its associated adaptor protein G Alpha Interacting Protein (GAIP) Interacting Protein C-terminus 1 (GIPC1), which are essential for NGF-evoked pain. 2 304870344v1Attorney Docket No: 243735.000408

[0009] In one aspect, provided herein is an isolated peptide comprising a fragment of nervegrowth factor (NGF) comprising two motifs each having the amino acid sequence R / KXXR / K wherein X is any amino acid, or a pharmaceutically acceptable salt, fragment or derivative thereof.

[0010] In some embodiments, the isolated peptide comprises the amino acid sequenceQAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO: 26) or QAAWRFIRIDTACVCVLSRKAVRR (SEQ ID NO: 124), or a sequence having at least 80% identity thereto.

[0011] In some embodiments, the isolated peptide consists essentially of the amino acidsequence of SEQ ID NOs: 26 or 124.

[0012] In some embodiments, the isolated peptide consists of the amino acid sequence of SEQID NOs: 26 or 124.

[0013] In some embodiments, the isolated peptide comprises one or more of the followingcharacteristics: a. binds to neuropilin-1 (NRP1);b. inhibits the interaction between NRP1 and NGF; andc. inhibits NGF-induced sensitization of transient receptor potential vanilloid 1(TRPV1).

[0014] In one aspect, provided herein is an isolated peptide comprising the amino acid sequenceLRGLGELRNLTIVKSGLRFVAPDAF (SEQ ID NO: 28), KSGLRFVAPDAFHFTPRLSRLNLSF (SEQ ID NO: 29), or WLFNGSVLNETSFIFTEFLEPAANE (SEQ ID NO: 30), or an amino acid sequence having at least 80% identity to any of SEQ ID NOs: 28-30, or a pharmaceutically acceptable salt, fragment or derivative thereof.

[0015] In some embodiments, the isolated peptide consists essentially of an amino acid sequenceof any one of SEQ ID NOs: 28-30.

[0016] In some embodiments, the isolated peptide consists of an amino acid sequence of anyone of SEQ ID NOs: 28-30.

[0017] In some embodiments, the isolated peptide comprises one or more of the followingcharacteristics: a. binds to neuropilin-1 (NRP1);b. inhibits the interaction between NRP1 and tropomyosin receptor kinase A (TrkA);3 304870344v1Attorney Docket No: 243735.000408 c. prevents NGF activation of TrkA;d. inhibits NGF-induced sensitization of transient receptor potential ion channelsincluding TRPV1, vanilloid 1 (TRPV1); e. prevents NGF-induced sensitization of pain sensing nerves; andf. inhibits NGF-evoked mechanical allodynia and thermal hyperalgesia.

[0018] In some embodiments, the isolated peptide comprises one or more modification toimprove affinity and / or metabolic stability.

[0019] In some embodiments, the isolated peptide comprises one or more D amino acids orunnatural amino acids.

[0020] In one aspect, provided herein is a pharmaceutical composition comprising one or moreisolated peptides described herein, and a pharmaceutically acceptable carrier or excipient.

[0021] In one aspect, provided herein is a method for treating chronic pain in a subject in needthereof, comprising administering to the subject a therapeutically effective amount of an inhibitor of neuropilin-1 (NRP1).

[0022] In some embodiments, the inhibitor of NRP1 inhibits expression or function of NRP1protein.

[0023] In some embodiments, the inhibitor of NRP1 inhibits the interaction between NRP1 andtropomyosin receptor kinase A (TrkA). In some embodiments, the inhibitor of NRP1 inhibits the direct interaction between NRP1 and TrkA. In some embodiments, the inhibitor of NRP1 inhibits the interaction between NRP1 and TrkA mediated by nerve growth factor (NGF).

[0024] In some embodiments, the inhibitor of NRP1 inhibits the interaction between NRP1 andNGF. In some embodiments, the inhibitor of NRP1 inhibits the interaction between a1a2b1b2 domains of NRP1 and NGF.

[0025] In some embodiments, the inhibitor of NRP1 reduces NGF-evoked nociception.

[0026] In some embodiments, the inhibitor of NRP1 is a small molecule, an siRNA, an shRNA,an antisense oligonucleotide, an antibody or antigen-binding fragment thereof, a peptide inhibitor, or a site-specific nuclease.

[0027] In some embodiments, the peptide inhibitor comprises an isolated peptide describedherein, or a combination thereof.

[0028] In some embodiments, the small molecule that inhibits NRP1 is N2-[[3-[(2,1,3-Benzothiadiazol-4-ylsulfonyl)amino]-2-thienyl]carbonyl]-L-arginine (EG00229), 3-((5-(4- 4 304870344v1Attorney Docket No: 243735.000408 (Aminomethyl)phenyl)-2,3-dihydrobenzofuran)-7-sulfonamido)-thiophene-2-carbonyl)-L- arginine (EG01377), or the like, or an analog or derivative thereof.

[0029] In some embodiments, the antibody or antigen-binding fragment is a human antibody, ahumanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a single chain antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a VH domain, or a nanobody.

[0030] In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody.

[0031] In some embodiments, the bispecific antibody comprises a first antigen-binding domainthat binds to NRP1 and a second antigen-binding domain that binds to TrkA. In some embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to NRP1 and a second antigen-binding domain that binds to NGF.

[0032] In some embodiments, the antibody or antigen-binding fragment that inhibits NRP1 isVesencumab (or MNRP1685A), or an antigen-binding fragment thereof.

[0033] In some embodiments, the siRNA that inhibits NRP1 comprises a nucleotide sequenceGAAUUGCUGUGGAUGAUAU (SEQ ID NO: 1), AGUAAGAGGUGUCAUCAUU (SEQ ID NO: 2), CCACAAGGUUCAUCAGGAU (SEQ ID NO: 3), GGAAUGUUCUGUCGC UAUG (SEQ ID NO: 4), CGAUAAAUGUGGCGAUACU (SEQ ID NO: 22), GGACAG AGACUGCAAGUAU (SEQ ID NO: 23), GUAUACGGUUGCAAGAUAA (SEQ ID NO: 24), AAGACUGGAUCACCAUAAA (SEQ ID NO: 25), or a modified version, a fragment, or a combination thereof.

[0034] In some embodiments, the siRNA that inhibits mouse NRP1 comprises a nucleotidesequence GAAUUGCUGUGGAUGAUAU (SEQ ID NO: 1), AGUAAGAGGUGUCAUCAUU (SEQ ID NO: 2), CCACAAGGUUCAUCAGGAU (SEQ ID NO: 3), GGAAUGUUCUGUCGC UAUG (SEQ ID NO: 4), or a modified version, a fragment, or a combination thereof.

[0035] In some embodiments, the siRNA that inhibits human NRP1 comprises a nucleotidesequence CGAUAAAUGUGGCGAUACU (SEQ ID NO: 22), GGACAGAGACUGCAAGUAU (SEQ ID NO: 23), GUAUACGGUUGCAAGAUAA (SEQ ID NO: 24), AAGACUGG AUCAC CAUAAA (SEQ ID NO: 25), or a modified version, a fragment, or a combination thereof. 5 304870344v1Attorney Docket No: 243735.000408

[0036] In another aspect, provided herein is a method for treating chronic pain in a subject inneed thereof, comprising administering to the subject a therapeutically effective amount of an inhibitor of G Alpha Interacting Protein (GAIP) Interacting Protein C-terminus 1 (GIPC1).

[0037] In some embodiments, the inhibitor of GIPC1 inhibits expression or function of GIPC1protein.

[0038] In some embodiments, the inhibitor of GIPC1 reduces NRP1-induced plasma membraneexpression of TrkA.

[0039] In some embodiments, the inhibitor of GIPC1 reduces NGF-evoked nociception.

[0040] In some embodiments, the inhibitor of GIPC1 is a small molecule, an siRNA, an shRNA,an antisense oligonucleotide, a peptide inhibitor, or a site-specific nuclease.

[0041] In some embodiments, the siRNA that inhibits GIPC1 comprises a nucleotide sequenceGCACUCGGGCUCACCAUCA (SEQ ID NO: 5), GGCCGUACCUUCACGCUGA (SEQ ID NO: 6), GCAAGGCCUUCGACAUGAU (SEQ ID NO: 7), CUGGAGA GUUACAUGGGUA (SEQ ID NO: 8), GCAUCGAGGGCUUCACUAA (SEQ ID NO: 9), CGUCGGCCUUUGAGGAGAA (SEQ ID NO: 10), GUGGAUGACUUGCUAGAGA (SEQ ID NO: 11), GCUGAGGCCUUCCGACUAC (SEQ ID NO: 12), or a modified version, a fragment, or a combination thereof.

[0042] In some embodiments, the siRNA that inhibits human GIPC1 comprises a nucleotidesequence GCACUCGGGCUCACCAUCA (SEQ ID NO: 5), GGCCGUACCUU CACGCUGA (SEQ ID NO: 6), GCAAGGCCUUCGACAUGAU (SEQ ID NO: 7)), or a modified version, a fragment, or a combination thereof.

[0043] In some embodiments, the siRNA that inhibits mouse GIPC1 comprises a nucleotidesequence CUGGAGAGUUACAUGGGUA (SEQ ID NO: 8), GCAUCGA GGGCUUCACUAA (SEQ ID NO: 9), CGUCGGCCUUUGAGGAGAA (SEQ ID NO: 10), GUGGAUGACUUGCUAGAGA (SEQ ID NO: 11), GCUGAGGCCUUCCGACUAC (SEQ ID NO: 12)), or a modified version, a fragment, or a combination thereof.

[0044] In some embodiments, the peptide inhibitor that inhibits GIPC comprises the amino acidsequence N-myristoyl-PSQSSSEA (SEQ ID NO: 13) (also known as CR1023), or a modified version or a fragment thereof.

[0045] In some embodiments, the chronic pain is an inflammatory pain, a neuropathic pain, acancer pain, or a postoperative pain. 6 304870344v1Attorney Docket No: 243735.000408

[0046] In some embodiments, the inflammatory pain is inflammatory bowel disease, irritablebowel syndrome, pancreatitis, arthritis, or migraine.

[0047] In some embodiments, the neuropathic pain is neuropathic pain secondary to nerve injuryand trauma, diabetic neuropathy, viral neuropathy (e.g., trigeminal neuralgia), or chemotherapy- induced peripheral neuropathy.

[0048] In some embodiments, the cancer pain is associated with oral cancer, non-small cell lungcancer, mesothelioma, melanoma, head and neck cancer, breast cancer, ovarian cancer, prostate cancer, renal cancer, liver cancer, or colorectal cancer.

[0049] In some embodiments, the administration of the inhibitor is via systemic or local delivery.

[0050] In some embodiments, the administration of the inhibitor is via systemic delivery.

[0051] In some embodiments, the administration of the inhibitor is via local delivery.

[0052] In some embodiments, the local delivery is via oral, intraocular, intrathecal, intranasal,intracolonical, intraluminal, intraintestinal, intracisternal, intraventricular, epidural, intratumoral, or intraarticular delivery, or any combination thereof.

[0053] In some embodiments, the inhibitor is formulated in a nanoparticle or a liposome.

[0054] In some embodiments, the method for treating chronic pain further comprises monitoringa pain level experienced by the subject before, during, and / or after the administering of the inhibitor. In some embodiments, the method for treating chronic pain further comprises monitoring a pain level experienced by the subject before the administering of the inhibitor. In some embodiments, the method for treating chronic pain further comprises monitoring a pain level experienced by the subject during the administering of the inhibitor. In some embodiments, the method for treating chronic pain further comprises monitoring a pain level experienced by the subject after the administering of the inhibitor.

[0055] In some embodiments, the monitoring a pain level experienced by the subject comprisesmeasuring one or more pain parameters associated with chronic pain in the subject.

[0056] In some embodiments, the one or more pain parameters are selected from pain duration,pain radiation pattern, pain severity, pain quality, degree of pain level fluctuation, frequency of pain remissions, and level of function of the subject.

[0057] In some embodiments, the measuring pain parameters comprises assessing the pain levelexperienced by the subject using a pain intensity scale and / or a pain questionnaire. 7 304870344v1Attorney Docket No: 243735.000408

[0058] In some embodiments, the inhibitor of NRP1 is administered either alone or incombination with an inhibitor of GIPC1.

[0059] In some embodiments, the inhibitor of GIPC1 is administered either alone or incombination with an inhibitor of NRP1.

[0060] In some embodiments, the inhibitor of NRP1 is administered before, simultaneouslywith, or after the administration of the inhibitor of GIPC1. In some embodiments, the inhibitor of NRP1 is administered before the administration of the inhibitor of GIPC1. In some embodiments, the inhibitor of NRP1 is administered simultaneously with the administration of the inhibitor of GIPC1. In some embodiments, the inhibitor of NRP1 may be present in the same composition as the inhibitor of GIPC1. In some embodiments, the inhibitor of NRP1 is administered after the administration of the inhibitor of GIPC1.

[0061] In some embodiments, the method for treating pain further comprises administering anadditional treatment to the subject.

[0062] In some embodiment, the additional treatment comprises administering a nonsteroidalanti-inflammatory drug (NSAID), acetaminophen, a local anesthetic, a benzodiazepine, capsaicin, an antidepressant, an anti-seizure medication, an anti-epileptic medication, a Cox-2 inhibitor, an opioid, a muscle relaxant, a steroid, an anticonvulsant, a triptan, dihydroergotamine, a beta blocker, a calcium channel blocker, a calcitonin gene-related peptides antagonist, a serotonin and norepinephrine reuptake inhibitor (SNRI), onabotulinumtoxinA, Lasmiditan, or an anti-nausea drug, or a combination thereof.

[0063] In some embodiments, the subject is a mouse.

[0064] In some embodiments, the subject is a human.BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figs. 1A-1E show that NGF directly interacts with NRP1. Fig. 1A shows carboxy-terminal sequences of NGF, highlighting prospective CendR motifs (R / KXXR / K). Amino acids numbered according to the mature βNGF sequence (1-120 equivalent to proNGF 122-241). h, Homo sapiens (UniProt Accession No. P01138: MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIA ARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRS KRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETK 8 304870344v1Attorney Docket No: 243735.000408 CRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO: 18)); r, Rattus norvegicus (UniProt Accession No. P25427:MSMLFYTLITAFLIGVQAEPYTDSNVPEGDSVPEAHWTKLQHSLDTALRRARSAPAEPIA ARVTGQTRNITVDPKLFKKRRLRSPRVLFSTQPPPTSSDTLDLDFQAHGTISFNRTHRSKR SSTHPVFHMGEFSVCDSVSVWVGDKTTATDIKGKEVTVLGEVNINNSVFKQYFFETKCR APNPVESGCRGIDSKHWNSYCTTTHTFVKALTTDDKQAAWRFIRIDTACVCVLSRKAAR RG (SEQ ID NO: 19)); m, Mus musculus (UniProt Accession No. P01139: MSMLFYTLITAFLIGVQAEPYTDSNVPEGDSVPEAHWTKLQHSLDTALRRARSAPTAPIA ARVTGQTRNITVDPRLFKKRRLHSPRVLFSTQPPPTSSDTLDLDFQAHGTIPFNRTHRSKR SSTHPVFHMGEFSVCDSVSVWVGDKTTATDIKGKEVTVLAEVNINNSVFRQYFFETKCR ASNPVESGCRGIDSKHWNSYCTTTHTFVKALTTDEKQAAWRFIRIDTACVCVLSRKATR RG (SEQ ID NO: 20)). Fig. 1A discloses SEQ ID Nos: 128-130, respectively, in order of appearance. Fig.1B shows prospective binding site between human beta-NGF (βNGF) and NRP1 modelled using protein docking PIPER software. Fig.1C shows interaction between purified His- tagged human NRP1 (residues 22-644) and unlabeled human βNGF using MST. Data from 4 independent experiments. Mean±SEM. Figs. 1D-1E show bioluminescence resonance energy transfer (BRET) to measure proximity (<10 nm) at full-length NRP1 in living HEK293T cells at 37ºC. Supernatant was collected from cells secreting growth factor (VEGF165a or NGF) tagged with HiBiT, a small portion of nanoluciferase with high affinity. When reconstituted with recombinant LgBiT and luciferase substrate, this acts as a bioluminescent donor for SnapTag- NRP1 labeled with SNAPTag-Alexa Fluor®488 (AF488). BRET was compared to negative control (HiBiT / LgBiT only lacking AF488). Cells were pre-incubated with vehicle or 10 nM unlabeled VEGF165a (30 minutes), followed by luminescent growth factor (15 minutes, 37ºC). BRET with HiBiT-VEGF165a was measured between HiBiT-VEGF165a and either SnapTag-NRP1 (WT) or the known VEGF165a binding-dead mutant (Y297A), as well as HiBiT-tagged NGF. Fig. 1E shows data from 4 independent experiments with triplicate wells. Fig. 1E, 1-way ANOVA, Šídák's multiple comparisons. *P<0.05, **P<0.01, *** P<0.001, ****P<0.0001.

[0066] Figs. 2A-2E show that Ntrk1 / TrkA and Nrp1 / NRP1 are coexpressed in dorsal rootganglia (DRG). Fig. 2A shows TrkA and NRP1 immunofluorescence in mouse DRG, showingsubcellular TrkA (dashed arrows), and cells coexpressing TrkA and NRP1 (solid arrows). Scalebar, 50 μm. Fig. 2B shows RNAScope® localization of Ntrk1 and Nrp1 mRNA in mouse DRG,9 304870344v1Attorney Docket No: 243735.000408 showing mRNA expression in the same neuron using NeuN immunofluorescence (arrowheads).Scale bar, 500 μm. Fig. 2C shows RNAScope® localization of Ntrk1 and Nrp1 mRNA in humanDRG, showing mRNA expression in the same cell (arrowheads). Fluorescence due to lipofuscin (*). Nuclei are shown. Figs.2D and 2E show quantified co-expression of Ntrk1 and Nrp1 mRNA in mouse DRG (Fig. 2D) or human DRG (Fig. 2E). Representative images, n=4-5 mice; n=3 human sections. Hybridized positive cells (%) were normalized to the total number of cells.

[0067] Figs. 3A-3P show that NRP1 inhibition prevents NGF-mediated increases in neuronalaction potential firing and ion channel currents. Effect of NRP1 inhibitor EG00229 (30 µM, 30minutes) on responses of dissociated human (Figs. 3A-3D) or mouse DRG neurons (Figs. 3E-3P)to NGF (50 nM, 30 minutes). Figs. 3A-3B and Figs. 3E-3F show representative action potentialfiring evoked by a depolarizing ramp stimulus (Fig.3A, Fig.3E), with summary of the number of evoked action potentials (Fig. 3B, Fig. 3F). n=7-10 cells. Figs. 3C-3D and Figs. 3G-3H showresting membrane potential (Fig. 3C, Fig. 3G, millivolts, mV) and rheobase (Fig. 3D, Fig. 3H,picoamperes, pA). Figs.3I-3L show a representative family of Ca2+current traces recorded from small diameter DRG neurons in response to depolarization steps from -70 to +70 mV from aholding potential of -90 mV (Fig.3I), with double Boltzmann fits for current density-voltage curve(Fig. 3J), summary of peak calcium current densities (Fig. 3K; pA / pF) and Boltzmann fits forvoltage-dependence of activation and inactivation (Fig. 3L). n=7-10 cells. Figs. 3M-3P show arepresentative family of Na+current traces, where currents were evoked by 150 ms pulses between−70 and +60 mV (Fig. 3M), with double Boltzmann fits for current density-voltage curve (Fig.3N), summary of peak sodium current densities (Fig. 3O, pA / pF) and Boltzmann fits for voltage-dependence of activation and inactivation (Fig. 3P). n=10-14 cells. Mean±SEM. *P<0.05, **P<0.01. Figs 3B, 3C, 3D, 3G, and 3K, Kruskal-Wallis, Dunn’s multiple comparisons.

[0068] Figs. 4A-4P show that NRP1 inhibition abrogates NGF- and CFA-induced nociceptionin mice. Figs.4A-4G and 4M-4N show NGF-induced nociception. Effects of an antibody againstthe b1 domain of NRP1 (Fig. 4B, Fig. 4E; 7 µg / 10 µl intraplantar, i.pl.) or NRP1 inhibitorsEG00229 (Fig. 4C, Fig. 4F; 30 µM / 10 µl i.pl.), CendR (Figs. 4M, 4N; 0.2, 2 and 10 µM / 10 µli.pl.), or Compound 5 (Fig.4D, Fig. 4G; Cpd5, Fig.4C, Fig.4F; 30 µM / 10 µl i.pl.). After baseline(“B”) measurements, inhibitors were co-injected with murine NGF (50 ng / 10 µl, i.pl.). Mechanicalallodynia (Figs. 4B-4D) and thermal hyperalgesia (Figs. 4E-4G) were measured. n=5-8 mice pergroup. Figs.4H-4L show complete Freund's adjuvant (CFA)-induced inflammatory pain. Effects 10 304870344v1Attorney Docket No: 243735.000408of anti-NRP1 (Fig. 4I, Fig. 4K) or EG00229 (Fig. 4J, Fig. 4L). Inhibitors were injected (i.pl.) 48h (hours) after CFA (i.pl.). Mechanical allodynia (Fig. 4I, Fig. 4J) and thermal hyperalgesia (Fig.4K, Fig. 4L) were measured. n=8-9 mice per group. Mean±SEM. *P<0.05, **P<0.01,***P<0.001, ****P<0.0001 vs. PBS or IgG Ctrl. Figs 4B-4G, 4I-4L, 2-way ANOVA, Sídák’smultiple comparisons. Dose-response curve of NRP1 inhibitors CendR (Fig. 4O, Fig. 4P; 0.2, 2and 10 µM / 10 µl i.pl.). After baseline (“B”) measurements, inhibitors were co-injected with mouseNGF (50 ng / 10 µl, i.pl.). Mechanical allodynia (Fig. 4O) and thermal hyperalgesia (Fig. 4P) weremeasured 1 hour after injection. N=5-8 male mice per group. Mean±SEM. *P<0.05, ***P<0.001, ****P<0.0001 vs.0 µM.1-way ANOVA, Dunnett multiple comparisons.

[0069] Figs. 5A-5L show that NRP1 modulates TrkA-mediated kinase signaling. Figs. 5A-5Bshow effect of murine NGF (15 minutes) and NRP1 inhibitor EG00229 (30 minute pre-incubation)on TrkA Y785 staining in mouse DRG neurons. Scale bar, 20 µm. RFU, relative fluorescence units. n=34-44 neurons from 3 independent experiments. Figs. 5C-5I show NGF-induced extracellular signal-regulated kinase (ERK) signaling measured using FRET-based EKARbiosensors (Figs. 5D-5I) or a downstream luciferase reporter (Fig. 5J). Figs. 5D-5I show NGF-induced modulation of ERK using biosensors localized to the cytosol (Fig.5D, Fig.5E) or nucleus(Fig. 5D, Fig. 5F) in neuron-like CAD.a cells expressing human TrkA. Kinetics of NGF-inducedERK monitored in CAD.a cells (Fig. 5D), comparing increasing NGF concentrations after pre-incubation with EG00229 (30 µM, 30 minutes) (Figs. 5E-5F). Figs. 5G-5I show ERK signalingin HEK293T cells expressing TrkA alone or transfected with both TrkA and NRP1 (Fig. 5H, Fig.5I). Fig. 5J shows the effect of increasing NGF concentrations on ERK transcription in cellsexpressing TrkA, NRP1 or both (% positive control, 10 µM PDBu). AUC, area under curve. RLU, relative luminescence units. Mean±SEM. Data from 4-8 independent experiments with triplicate wells. *P<0.05, ***P<0.001, ****P<0.0001. Figs.5B, 5I, 5J, 1-way ANOVA, Sídák’s multiple comparisons. Effect of mouse NGF (100 nM) and NRP1 inhibitors EG00229 (30 µM, 30 minutespre-incubation) (Fig. 5K) and CendR (1 µM, 30 minutes pre-incubation) (Fig. 5L) onphosphorylated ERK Thr202 / Tyr204 staining in mouse DRG neurons. N=15-432 neurons from 4 independent experiments. Scale bar, 20 µm.

[0070] Figs. 6A-6J show that TrkA and NRP1 form a heteromeric complex. Fig. 6A showsHEK293T or CAD.a cells expressing SnapTag-TrkA and HaloTag-NRP1 simultaneously labeled with membrane-impermeant substrate (SNAPTag-Alexa Fluor® 488, HaloTag-Alexa Fluor®660). 11 304870344v1Attorney Docket No: 243735.000408 Representative images from n=5 independent experiments. Figs. 6B-6C show BRET assays to monitor proximity between NanoLuc-NRP1 or NanoLuc-p75NTR(10 ng) and increasing SnapTag- TrkA DNA. Negative control, NanoLuc-TrkA and SnapTag-CALCRL. Representative replicate(Fig. 6C) plotting BRET against relative fluorescence units (RFU) to quantify protein expression.Figs.6D-6G show isolating cell-surface TrkA, imaged in HEK293T overlayed with phase-contrastimaging in the absence or presence of NRP1 coexpression (Fig. 6E). Figs. 6F-6G showquantification comparing fluorescence without receptor (-), SnapTag-TrkA alone or SnapTag-TrkA co-transfected with NRP1 in HEK293T (Fig. 6F) or CAD.a (Fig. 6G) cells. Figs. 6H-6Jshow BRET between TrkA tagged with Renilla luciferase (Rluc8) and Renilla green fluorescent protein (RGFP) tagged markers of the plasma membrane (PM, RGFP-CAAX), early endosome (EE, tdRGFP-Rab5a), recycling endosomes (RE, tdRGFP-Rab4a) or the cis-Golgi apparatus(tdRGFP-Giantin). HEK293T cells (Fig. 6I) or CAD.a cells (Fig. 6J) were transfected with TrkA-Rluc8 in the absence (-) or presence (+) of NRP1 co-expression. BRET was normalized relative to TrkA-Rluc8 alone (100%). Data from 5-6 independent experiments with triplicate wells. Mean±SEM. Scale bar, 20 µm. Fig.6F, Fig.6G. Paired t-test. Figs.6I, 6J, 1-way ANOVA with Šídák's multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0071] Figs. 7A-7K show that NRP1 modulates NGF-induced TrkA trafficking and RTKoligomerization. Figs.7A-7G show that BRET was measured at 37ºC between Rluc8-tagged TrkA and a fluorescent marker of the plasma membrane (RGFP-CAAX). Decreased BRET indicatesremoval from the plasma membrane (Fig. 7A). Figs. 7B-7D show trafficking in HEK293T cellsincubated with increasing NGF concentrations (Fig. 7B), or pre-incubated for 30 with hypertonicsucrose (0.45 M), clathrin inhibitor pitstop 2 (30 μM) or vehicle (Veh) (Fig. 7C). Effect of NRP1co-expression (Fig. 7D). Figs. 7E-7G show trafficking in CAD.a cells expressing human TrkAwith increasing NGF concentrations (Fig. 7E), pre-incubation with endocytic inhibitors (Fig. 7F)with transfected with control or murine NRP1 siRNA (Fig. 7G). Figs. 7H-7K show BRETmeasured between NanoLuc- and SnapTag- TrkA to measure RTK oligomerization in HEK293T cells. Fig.7I shows BRET with increasing SnapTag-TrkA and fixed NanoLuc-TrkA (10 ng), after 30 minute incubation with vehicle or NGF. Figs.7J-7K show using a fixed donor:acceptor ratio(1:2.5), oligomerization kinetics in response to increasing NGF concentrations (Fig. 7J) in theabsence and presence of NRP1 co-expression (Fig.7K). Kinetic measurements were made at 37ºC.Data from 4-6 independent experiments with triplicate wells. AUC, area under curve. Mean±SEM. 12 304870344v1Attorney Docket No: 243735.000408 *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Fig. 7G, Unpaired t-test. Figs. 7D, 7I, 7K, 1- way ANOVA with Sídák’s multiple comparisons.

[0072] Figs. 8A-8O show GIPC1 modulates TrkA trafficking, signaling, and NGF-inducednociception. Figs. 8A-8C show RNAScope®localization of Gipc1 mRNA in mouse DRG (Fig.8A) or Ntrk1 and Gipc1 mRNA in human DRG (Fig. 8B, Fig. 8C). Arrows indicate mRNAexpression within the same cell. Representative images, n=5 mice; n=3 human sections. Scale bar,500 μm. Fig. 8D shows the effect of GIPC1 siRNA on increased TrkA at the plasma membraneunder basal conditions upon NRP1 co-expression in HEK293T cells measured using BRET. Figs. 8E-8F show the effect of 30 minute pre-incubation of GIPC1 antagonist (300 µM CR1023 or negative control) or myosin VI inhibitor (50 µM 2,4,6-Triiodophenol, TIP) on NGF-induced TrkA-Rluc8 trafficking from a marker of the plasma membrane (RGFP-CAAX) in CAD.a cells.Fig. 8G shows the effect of GIPC1 siRNA on NGF-induced downstream ERK transcription inCAD.a cells. RLU, relative luminescence units. Data from 5-6 independent experiments with triplicate wells. Figs.8H-8I show sample traces of action potential firing in mouse DRG neuronsevoked by injecting a 1 s (second) ramp pulse from 0 to 250 pA (Fig. 8H), with the number ofevoked action potentials (Fig. 8I). n=7-10 cells. Figs. 8J-8O show effects of GIPC1 siRNA(intrathecal, i.t.) on NGF (50 nM / 10 µl, i.pl.)-induced (Figs. 8J-8L) or CFA-induced (Figs. 8M-8O) mechanical allodynia (Fig. 8J, Fig. 8L, Fig. 8M, Fig.-8O) and thermal hyperalgesia (Fig.8K, Fig. 8N) in mice, including mechanical allodynia tested in the contralateral hind paw afterNGF (Fig. 8L) or CFA injection (Fig. 8O). n=6-8 mice per group. AUC, area under curve. B,basal. Mean±SEM. Fig. 8C, Fig. 8E, 1-way ANOVA, Sídák’s multiple comparisons. Fig. 8H, Tukey’s multiple comparison. Figs. 8I-8O, 2-way ANOVA, Sídák’s multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0073] Fig. 9 illustrates a proposed mechanism for NRP1- and GIPC1-mediated NGF / TrkAsignaling. NGF released from sites of injury, inflammation, or the tumor microenvironment (TME) act on TrkA expressed by primary afferent neurons. NRP1 and GIPC1 potentiate kinase signaling, neuronal excitability, and nociception. As a co-receptor, NRP1 can directly bind NGF and act as a chaperone, mediated by interactions with adaptor protein GIPC1.

[0074] Figs. 10A-10B show specificity of SnapTag- and HaloTag- labeling in live cells.HEK293T cells (Fig. 10A) or CAD.a cells (Fig. 10B) were transfected with SnapTag-TrkA,HaloTag-NRP1 or both (1:2 TrkA to NRP1 ratio). Tags were simultaneously labeled with 13 304870344v1Attorney Docket No: 243735.000408 membrane-impermeant substrate (SNAPTag-Alexa Fluor®488, HaloTag-Alexa Fluor®660). Representative from n=5 independent experiments. Scale bar, 20 µm.

[0075] Figs. 11A-11B show characterization of receptor expression in neuron-like CAD.a cells.Fig. 11A shows quantification of expression of Ntrk1 mRNA (TrkA) or Ngfr (p75NTR) inHEK293T or CAD.a cells determined by quantitative RT-PCR. Data from 3 independent experiments with triplicate wells. Fig. 11B shows immunofluorescent (IF) staining of NRP1 in HEK293T or CAD.a cells using vesencumab. Scale bar, 20 µm. Representative from 4 independent experiments. Mean±SEM. Fig. 11A, 1-way ANOVA, Dunnett’s multiple comparisons. * P<0.05.

[0076] Figs. 12A-12F show the effect of co-expression or siRNA on TrkA and NRP1expression. Figs. 12A-12D show HEK293T (Fig. 12A, Fig. 12C) or CAD.a (Fig. 12B, Fig. 12D)cells expressed TrkA-Rluc8 in the absence or presence of HaloTag-NRP1. Cells were labeled with membrane-impermeant HaloTag-Alexa Fluor®660 and emissions were measured forAlexaFluor660 as relative fluorescence units (RFU) (Fig. 12A, Fig. 12B). Alternatively, TrkA-Rluc8 emissions were measured after incubation with coelenterazine purple to infer TrkAexpression as relative luminescence units (RLU) (Fig. 12C, Fig. 12D). Figs. 12E-12F show theeffect of NRP1 siRNA on expression of NRP1 (Fig. 12E) or TrkA (Fig. 12F) quantified using IFstaining normalized to nuclei count (DAPI). Data expressed as percentage change over control (100%). Mean±SEM. A-D, G, H. Unpaired t-test. **P<0.01, *** P<0.001.

[0077] Figs. 13A-13D demonstrate confirmation of GIPC1 knockdown using siRNA. Figs.13A-13C show quantification of expression of Gipc1 mRNA in HEK293T (Fig. 13A), CAD.acells (Fig. 13B), or cultured dissociated DRG neurons (Fig. 13C) in the presence of control (Ctrl)or GIPC1 siRNA determined by quantitative RT-PCR. Data from 3-4 independent experiments with triplicate wells. Fig. 13D shows RNAScope®localization of Gipc1 mRNA in murine DRG after intrathecal injection of control or GIPC1 siRNA. Representative from n=5 mice. Figs.13A- 13D, unpaired t-test. *P<0.05, **P<0.01, *** P<0.001, **** P<0.0001.

[0078] Figs. 14A-14G show that NGF promotes interactions between NRP1 and TRKA. NGFinteracted with His-tagged extracellular TrkA with nanomolar affinity (Kd = 5.49 nM). Fig.14B shows that the TrkA extracellular domain interacted with the His-tagged NRP1 extracellular domain with low affinity. The highest concentrations of TrkA were 6 µM (left) and 30 µM (right). Even at the highest concentration, no saturation was reached (Kds ~ 10 µM). Figs.14C-14D show 14 304870344v1Attorney Docket No: 243735.000408 that the NRP1 extracellular domain interacted with the His-tagged TrkA extracellular domain with moderate affinity (~30-300 nM) after 2 h incubation at room temperature (14°C) and overnight incubation at 4°C (Fig.14D). Figs.14E-14F show that the TrkA extracellular domain interacted with the His-tagged NRP1 extracellular domain with high affinity (Kd= 30.2-114 nM) in the presence of 0.5 µM NGF after 2 h incubation at room temperature (Fig.14E) and of 1 µM NGF after overnight incubation at 4°C (Fig.14F). Fig.14G shows that the NRP1 extracellular domain interacted with the His-tagged TrkA extracellular domain with moderate affinity (Kd= 30.2-114 nM) in the presence of NGF (0.5 µM or 1 µM) 2 h incubation at room temperature.

[0079] Figs. 15A-15D show that endosomal TrkA signaling mediates NGF-inducedsensitization of nociceptors and nociception. Fig. 15A shows detection of phosphorylated-TrkA (P-TrkA) in endosomes of nociceptors. Fig.15B shows NGF sensitization of nociceptors showinginhibition by dyngo4a. Fig. 15C and Fig. 15D show NGF (intraplantar) mechanical allodyniashowing inhibition by pitstop2 (i.pl., Fig. 15C) and by Dnm siRNA (i.t., Fig. 15D). n=5-8, ANOVA, Šídák’s, *P<0.05, **P<0.01, ***P<0.001.

[0080] Figs. 16A-16D show that nanoparticles (NPs) target early endosomes of nociceptors andNP-encapsulated NRP1 antagonist blocks NGF-induced nociception Fig.16A shows transmission electron microscopy (TEM) image of Q-dot NPs. TEM revealed Q-dot clustering in the NP core. Fig. 16B shows Q-dot NP uptake to Rab5a-GFP early endosomes of NeuN positive (NeuN+ve) mouse DRG neurons. Small Q-dot NPs (50 nm) rapidly internalized (30-240 minutes) into Rab5a- GFP (CellLights) early endosomes of NeuN+ve mouse DRG neurons in culture. Fig.16C shows inhibition of NGF mechanical allodynia by intraplanar PEG-00229 NPs. NPs had a dynamic light scattering (DLS) intensity weighted diameter of 88 nm (polydispersity 0.14), drug loading of 7.1 wt% EG00229, and drug encapsulation efficiency of 35%. After intraplantar injection, PEG- EG00229 NPs prevented NGF (intraplantar)-induced mechanical allodynia for 2 h. Empty PEG- ^ NPs had no effect. Fig.16D shows percent inhibition by NPs of NGF mechanical allodynia after 3 h. n=5-7, ANOVA, Šidák’s, **P<0.01, ****P<0.0001. The duration and magnitude of the antinociceptive action of PEG-EG00229 exceeded that of free EG00229 (% inhibition at 3 h after administration: PEG-EG00229, 76%, free EG00229, 46%).

[0081] Figs. 17A-17D show properties of PEG-DNase-1 NPs. Fig. 17A illustrates a two-stepinverse flash nanoprecipitation (iFNP) process in which PEGylated NPs were loaded with the therapeutic protein deoxyribonuclease-1 (DNase-1). NPs had an intensity weight size of 115 nm 15 304870344v1Attorney Docket No: 243735.000408 (DLS). Fig. 17B shows DNase-1 encapsulation efficiency was 96±5% by bicinchoninic acid (BCA) assay. Fig. 17C shows zymography gel assay results. The zymography gel assay demonstrated retention of DNase-1 activity after NP release. Figs. 17D-17E show a neutrophil extracellular trap assay demonstrating degradation by PEG-DNase-1 NP at 4 and 6 h. n=5, t test, *P<0.05. Incubation with Dnase-1 NPs resulted in decreased fluorescence indicating DNA.

[0082] Figs. 18A-18F show NRP1 inhibition prevents NGF-induced sensitization of TRPV1.Figs. 18A, 18C, and 18E show time course of responses of mouse DRG neurons to repeatedchallenge with capsaicin (Cap, 100 nM) expressed as ΔF / Fo ratio. Figs.18B, 18D, and 18F show summary of responses to capsaicin expressed as the ΔF / Fo of the second capsaicin Ca2+responseover the ΔF / Fo of the first capsaicin Ca2+ response (2nd peak / 1st peak). Figs. 18A, 18B show effectof NRP1 inhibitor EG00229 (3, 10, 30 µM, 30 minutes pre-incubation) or vehicle (Veh). Fig.18A. N=64-119 cells per trace. Fig. 18B. Summary from N=5 independent experiments. Figs. 18C, 18D. Effect of NRP1 inhibitor CendR or control (Ctrl) (0.1, 0.3, 1 µM). Fig. 18C. N=142-535 cells per trace. Fig.18D. Summary from N=5 independent experiments. Figs.18E, 18F. Effect of a human mAb against the b1b2 domain of NRP1 (Vesencumab) or control (Ctrl) IgG (0.7 µg / ml). Fig.18E. N=97-199 cells per trace. Fig.18F. Summary from N=5 independent experiments. A.U., arbitrary units. Mean±SEM. *P<0.05, **P<0.01, ****P<0.0001, ns not significant. Two-way ANOVA with Tukey’s multiple comparisons test.

[0083] Figs.19A-19B show TrkA interacts with extracellular domains of full-length NRP1. Fig.19A shows interactions of TrkA-GFP with extracellular domains of NRP1. NRP1 ECD constructs were transfected alone (right panel) or with a full-length TrkA-GFP fusion (left panel) into HEK cells as shown. Proteins in lysates and immunoprecipitations were visualized using the indicated primary antibodies. NRP1-ECD1: amino acid residues 22-850. NRP1-ECD2: amino acid residues 22-644. “S” after ECD2 refers to the STREP-tagged version (see also Material and Methods in the Examples section). Fig.19B shows interactions of TrkA-GFP with full-length NRP1. Full-length NRP1-mCherry fusion was transfected alone or with full-length TrkA-GFP as indicated. Proteins in lysates and immunoprecipitations were visualized using the indicated primary antibodies. (*) after NRP1-mCherry (middle lane) refers to a ten-fold lower amount of transfected plasmid (0.1 µg instead of 1 µg).

[0084] Figs. 20A-20D show heat map and binding hotspots of peptide microarray studies. Figs.20A-20C show heatmap diagram for the 91 overlapping TrkA peptides (SEQ ID NOs: 32-122). 16 304870344v1Attorney Docket No: 243735.000408 Fluorescence intensities are displayed with strongest binding to peptides (units > 60,000), followed by a group of peptides with 50,000 – 60,000 units, a group of peptides with 30,000 – 50,000 units, a group of peptides with 20,000 – 30,000 units, a group of peptides with 10,000 – 20,000 units, a group of peptides with 1,000 – 10,000 units and a group of peptides with no binding (<1,000 units). The peptides covering the two hotspots are designated with SEQ ID Nos: 46-47, and 49-51. Hotspots are defined as regions with at least two consecutive peptides that showed moderately strong binding (>30,000 units) at two or three dilutions of the NRP1-His antigen (10 µg / ml, 1 µg / ml and 0.1 µg / ml). Fig.20D shows the extracellular domain of TrkA with binding hotspots to NRP1. The ECD of TrkA with known domains are amino acid residues 90-113, 116-137, 148-193, 194-283, and 299-365. The two hotspots identified by peptide microarray analysis are the peptide # 15-16 and peptide # 17-19. Peptide numbering refers to Fig.20A. SP: signal peptide. LRR1 / 2: leucine rich repeat 1 / 2. LCCRT: leucine rich repeat C-terminal domain. IGc2-1 / 2: immunoglobulin c2-type domain 1 and 2. Numbers refer to amino acid residues.

[0085] Figs. 21A-21C show selection of peptide fragments of TrkA speculated to block TrkAand NRP1 interactions. Fig.21A depicts amino acid sequences having SEQ ID NOs: 32-57, Fig. 21B depicts amino acid sequences having SEQ ID NOs: 61-87, and Fig.21C depicts amino acid sequences having SEQ ID NOs: 40-43. Residues implicated in NGF binding are indicated with bold and underline letters in SEQ ID NO: 30.

[0086] Figs. 22A-22D show effect of peptides #15-16, #18-20, and #72-73 on NGF-inducedsensitization of TRPV1. Fig. 22A shows summary of responses to capsaicin. expressed as the ΔF / Fo of the second capsaicin Ca2+response over the ΔF / Fo of the first capsaicin Ca2+response (2ndpeak / 1stpeak). Effect of coadministration of NRP1 inhibitors peptides #15-16, #18-20, and #72-73 (1 µM) or control peptide (CTR) and NGF (100 nM) or vehicle (PBS). n=2-4 independent experiments. A.U., arbitrary units. Mean±SEM. ns not significant. Two-way ANOVA with Tukey’s multiple comparisons test. Figs.22B-22D show time course of responses of mouse DRG neurons to repeated challenges with capsaicin (Cap, 100 nM) expressed as ΔF / Fo ratio. PBS CTR and NGF CTR traces are identical across all three graphs. Fig.22B.160-256 neurons. Fig.22C. 160-294 neurons. Fig.22D.72-198 neurons.

[0087] Figs. 23A-23B show effect of peptides #15-16 and #18-20 on NGF-induced nociception.Peptides #15-16, #18-20 or CTR (2 μM / 10 μl) were pre-treated intraplantar (i.pl) 30 minutes before 17 304870344v1Attorney Docket No: 243735.000408 NGF (50 ng / 10 μl, i.pl.). Mechanical allodynia (Fig. 23A) and thermal hyperalgesia (Fig. 23B) were measured for 3 hours after NGF injection. N=2-4 mice.

[0088] Fig. 24 depicts an amino acid sequence of TrkA peptides #72 and #73 (SEQ ID NO:123). Residues implicated in NGF binding are highlighted with bolded and underlined letters64. Numbering refers to TrkA protein (UniProt #P04629-1).

[0089] Figs. 25A-25D show NGF / TrkA / NRP1 docked models in the context of glycosylatedTrkA extracellular domains and ternary complex of human NGF / TrkA / NRP1 generated using constraint-driven computational docking. Fig.25A shows side view of structural superposition of NGF / TrkA / NRP1 docked model and NGF / TrkA crystal structure (PDB 2IFG) through common NGF / TrkA component (only monomers are shown for NRP1 and TrkA for clarity). Asn-linked glycosylations on TrkA in NGF / TrkA crystal structure are shown as space-filling representation. Individual domains are labeled and described in text. In cellular context, this comparison shows a glycosylated TrkA compatible binding mode of NRP1 in the docked NGF / TrkA / NRP1 model. Fig.25B shows other docked poses of NRP1 observed in NGF / TrkA / NRP1 docking calculations. Onlyone monomer of NRP1 bound to NGF / TrkA (surface) is shown. For context, TrkA Asn-linked glycosylations are shown in space-filling representations. Individual domains are labeled and described in the text. In contrast to NGF / TrkA / NRP1 docked model described in text, one NRP1 molecule binds to two TrkA monomers at once thereby bridging TrkA dimers in these docked poses. Although NRP1 domains appear to interfere with TrkA glycosylations, domain flexibility in the cellular context (not accounted for in docking calculations) may allow these scenarios to exist. It is important to note that in different docked poses from docking calculations, NRP1 b1 domain still binds to NGF C-terminus in a similar manner while NRP1 a1, a2 and b2 domains areoriented differently. Figs. 25C-25D show cartoon and surface representation of TrkA, NGF, andNRP1 are shown. Fig. 25C shows an NGF / TrkA / NRP1 model and conserved interactions at the NGF / NRP1 interface suggest a 2:2:2 stoichiometry with one NRP1 molecule interacting with one TrkA molecule and the NGF dimer. Views of the NRP1 / TrkA complex in surface representation represent complementarity between NRP1 and TrkA. The inset shows binding of NGF C-terminal R118 to conserved residues (Y297, D320, S346, Y353) in C-terminal arginine binding pocket ofthe NRP1 b1 domain. Fig. 25D shows proposed cell surface NGF / TrkA / NRP1 complex.Membrane proximal MAM NRP1 domains are included to propose a sterically feasible membrane- 18 304870344v1Attorney Docket No: 243735.000408 tethered NGF / TrkA / NRP1 complex. Membrane linkers and transmembrane regions are not derived from structures and are not to scale. DETAILED DESCRIPTION

[0090] Nerve growth factor (NGF) monoclonal antibodies are one of the few patient-validatednon-opioid treatments for chronic pain, despite failing to gain FDA approval. The present disclosure describes that the neuropilin-1 (NRP1) is a co-receptor for NGF that potentiates tropomyosin-related kinase A (TrkA) signaling. NGF binds NRP1 with nanomolar affinity. NRP1 and its associated adaptor protein GIPC1 are essential for NGF-evoked and inflammatory pain. NRP1 and GIPC1 are coexpressed with TrkA in nociceptors, particularly in human tissue. As demonstrated in the Examples section described below, inhibiting NRP1 prevented NGF-evoked action potential firing of both murine and human nociceptors. NRP1 knockdown blunted NGF- stimulated TrkA phosphorylation, kinase signaling and transcription, whereas NRP1 overexpression enhanced signaling. As well as interacting with the NGF, NRP1 can form a heteromeric complex that chaperones TrkA from the biosynthetic pathway to the plasma membrane and signaling endosomes. NRP1 thereby can enhance NGF-induced TrkA dimerization, endocytosis and signaling. Knockdown of GIPC1, a PDZ-binding protein that scaffolds NRP1 and TrkA cargo to myosin VI, abrogated NGF-mediated nociception. Attributed to both direct NGF binding and chaperoning TrkA to the plasma membrane and signaling endosomes via adaptor protein GIPC1, NRP1 was identified as a previously unrecognized co-receptor necessary for NGF / TrkA pain signaling, providing a long-awaited alternative to block NGF-evoked pain. Definitions

[0091] The term “about” or “approximately” means within a statistically meaningful range ofa value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0092] The terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denotethe presence of “at least one” of the referenced item. 19 304870344v1Attorney Docket No: 243735.000408

[0093] The terms “polypeptide” and “protein” used interchangeably herein encompass nativeor artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide or protein may be monomeric or polymeric. The terms encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.). Small polypeptides of less than 100 amino acids, preferably less than 50 amino acids, may be referred to as “peptides”.

[0094] The term “isolated peptide”, “isolated polypeptide”, or “isolated protein” is a peptide,polypeptide, or protein that by virtue of its origin or source of derivation has one to four of the following: (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other peptides, polypeptides, proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a peptide, polypeptide, or protein that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A peptide, polypeptide, or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

[0095] The term “fragment” in regard to peptides or polypeptides refers to a peptide or apolypeptide that has an amino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the full-length naturally- occurring sequence. Also, fragments according to the invention may be made by truncation, e.g., by removal of one or more amino acids from the N and / or C-terminal ends of a polypeptide. Up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and / or C terminal in this way. Fragments may also be generated by one or more internal deletions. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, 200, or 400 amino acids long.

[0096] The term “derivative” as used herein refers to a peptide, polypeptide, or polynucleotide,or a variant or analog thereof, comprising one or more mutations and / or chemical modifications as compared to a reference peptide, polypeptide or polynucleotide. Mutations and / or chemical 20 304870344v1Attorney Docket No: 243735.000408 modifications are further detailed below and can include, for example, insertions, substitutions, deletions, transversions, and / or inversions at one or more locations in the amino acid or nucleotide sequence.

[0097] In certain embodiments, amino acid substitutions of a peptide or a protein or portionthereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, or (4) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the normally-occurring sequence.

[0098] A conservative amino acid substitution should not substantially change the structuralcharacteristics of the parent sequence. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), which are each incorporated herein by reference.

[0099] As used herein, the twenty naturally occurring amino acids and their abbreviationsfollow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0100] The term “polynucleotide” as referred to herein means a polymeric form of nucleotidesof at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.

[0101] The term “percent sequence identity” in the context of nucleic acid sequences meansthe percent of residues when a first contiguous sequence is compared and aligned for maximum correspondence to a second contiguous sequence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. 21 304870344v1Attorney Docket No: 243735.000408 FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol.183:63-98 (1990); Pearson, Methods Mol. Biol.132:185- 219 (2000); Pearson, Methods Enzymol.266:227-258 (1996); Pearson, J. Mol. Biol.276:71-84 (1998); herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

[0102] A reference to a nucleotide sequence encompasses its complement unless otherwisespecified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

[0103] The term “percent sequence identity” means a ratio, expressed as a percent of thenumber of identical residues over the number of residues compared.

[0104] The term “substantial similarity” or “substantial sequence similarity,” when referringto a nucleic acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0105] The terms “inhibit” or “inhibition” as used herein refer to reducing a function or activityto an extent sufficient to achieve a desired biological or physiological effect. Inhibition may be complete or partial.

[0106] The terms “treat” or “treatment” of a state, disorder or condition include: (1)preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or sub-clinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing, delaying or reversing the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or 22 304870344v1Attorney Docket No: 243735.000408 condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0107] As used herein, the term “therapeutically effective amount” refers to the amount of acompound (e.g., an inhibitor of NRP1 and / or inhibitor of GIPC1) or a composition that, when administered to a subject for treating (e.g., preventing, ameliorating, or reversing) a state, disorder or condition, is sufficient to effect such treatment. The “therapeutically effective amount” will vary depending, e.g., on the compound, or analogues administered as well as the disease, its severity, and physical conditions and responsiveness of the subject to be treated.

[0108] The phrase “pharmaceutically acceptable”, as used in connection with compositionsdescribed herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0109] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptableexcipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0110] The term “simultaneous administration,” as used herein, means that a first agent andsecond agent in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the first and second agents are administered simultaneously, the first and second agents may be contained in the same composition (e.g., a composition comprising both a first and second agent) or in separate compositions (e.g., a first agent in one composition and a second agent is contained in another composition).

[0111] As used herein, the term “sequential administration” means that the first agent andsecond agent in a combination therapy are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the first 23 304870344v1Attorney Docket No: 243735.000408 agent or the second agent may be administered first. The first and second agents are contained in separate compositions, which may be contained in the same or different packages or kits.

[0112] As used herein, the term “concurrent administration” means that the administration ofthe first agent and that of a second agent in a combination therapy overlap with each other.

[0113] The terms “patient”, “individual”, “subject”, and “animal” are used interchangeablyherein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0114] The practice of the present invention employs, unless otherwise indicated, conventionaltechniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual.3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U.S. Patent No.7,912,698 and U.S. Patent Appl. Pub. Nos.2011 / 0202322 and 2011 / 0307437, each of which is herein incorporated by reference in its entirety for all intended purposes. Isolated Peptides

[0115] In one aspect, the present disclosure provides an isolated peptide, or a pharmaceuticallyacceptable salt, fragment or derivative thereof.

[0116] In some embodiments, the isolated peptide descried herein inhibits NRP1. In someembodiments, a fragment or derivative of isolated peptides of the present disclosure includes a functional fragment or derivative that can inhibit NRP1.

[0117] NRP1 was first identified as a transmembrane glycoprotein expressed in growth conesof growing axons. In some embodiments, the NRP1 described herein refers to human NRP1 (NCBI 24 304870344v1Attorney Docket No: 243735.000408 Gene ID: 8829). In some embodiments, the NRP1 described herein refers to mouse NRP1 (NCBI Gene ID: 18186).

[0118] TrkA is the high affinity catalytic receptor for the neurotrophin, NGF. The binding ofNGF to TrkA leads to a ligand-induced dimerization, and a proposed mechanism by which this receptor and ligand interact is that two TrkA receptors associate with a single NGF ligand. In some embodiments, the TrkA described herein refers to human TrkA (NCBI Gene ID: 4914). In some embodiments, the TrkA described herein refers to mouse TrkA (NCBI Gene ID: 18211).

[0119] In some embodiments, amino acids of TrkA-derived peptides are numbered accordingto the UniProt Accession No. P04629-1 having the amino acid sequence: MLRGGRRGQLGWHSWAAGPGSLLAWLILASAGAAPCPDACCPHGSSGLRCTRDGALD SLHHLPGAENLTELYIENQQHLQHLELRDLRGLGELRNLTIVKSGLRFVAPDAFHFTPRL SRLNLSFNALESLSWKTVQGLSLQELVLSGNPLHCSCALRWLQRWEEEGLGGVPEQKL QCHGQGPLAHMPNASCGVPTLKVQVPNASVDVGDDVLLRCQVEGRGLEQAGWILTEL EQSATVMKSGGLPSLGLTLANVTSDLNRKNVTCWAENDVGRAEVSVQVNVSFPASVQ LHTAVEMHHWCIPFSVDGQPAPSLRWLFNGSVLNETSFIFTEFLEPAANETVRHGCLRLN QPTHVNNGNYTLLAANPFGQASASIMAAFMDNPFEFNPEDPIPVSFSPVDTNSTSGDPVE KKDETPFGVSVAVGLAVFACLFLSTLLLVLNKCGRRNKFGINRPAVLAPEDGLAMSLHF MTLGGSSLSPTEGKGSGLQGHIIENPQYFSDACVHHIKRRDIVLKWELGEGAFGKVFLAE CHNLLPEQDKMLVAVKALKEASESARQDFQREAELLTMLQHQHIVRFFGVCTEGRPLL MVFEYMRHGDLNRFLRSHGPDAKLLAGGEDVAPGPLGLGQLLAVASQVAAGMVYLA GLHFVHRDLATRNCLVGQGLVVKIGDFGMSRDIYSTDYYRVGGRTMLPIRWMPPESILY RKFTTESDVWSFGVVLWEIFTYGKQPWYQLSNTEAIDCITQGRELERPRACPPEVYAIM RGCWQREPQQRHSIKDVHARLQALAQAPPVYLDVLG (SEQ ID NO: 125).

[0120] In some embodiments, the isolated peptide inhibits an interaction between NRP1 andNGF via binding to NRP1.

[0121] In some embodiments, the isolated peptide mimics NGF. In some embodiments, theisolated peptide comprises a fragment of NGF comprising a motif having an amino acid sequence of R / KXXR / K wherein X is any amino acid, or a sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the isolated peptide comprises a fragment of NGF comprising a motif having an amino acid 25 304870344v1Attorney Docket No: 243735.000408 sequence of R / KXXR / K wherein X is any amino acid, or a sequence having at least 80% identity thereto.

[0122] In some embodiments, the isolated peptide comprises a fragment of NGF comprisingtwo motifs having an amino acid sequence of R / KXXR / K wherein X is any amino acid, or a sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the isolated peptide comprises a fragment of NGF comprising two motifs having an amino acid sequence of R / KXXR / K wherein X is any amino acid, or a sequence having at least 80% identity thereto.

[0123] In some embodiments, the isolated peptide comprises an amino acid sequence of SEQID NO: 26 or 124, or a sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0124] In some embodiments, the isolated peptide comprises an amino acid sequence of SEQID NO: 26 or 124, or a sequence having at least 80% identity thereto.

[0125] In some embodiments, the isolated peptide consists essentially of an amino acidsequence of SEQ ID NO: 26 or 124, or a sequence having at least 80% identity thereto.

[0126] In some embodiments, the isolated peptide consists of an amino acid sequence of SEQID NO: 26 or 124, or a sequence having at least 80% identity thereto.

[0127] In some embodiments, the isolated peptide the peptide comprises one or more of thefollowing characteristics: a. binds to neuropilin-1 (NRP1); b. inhibits the interaction between NRP1 and NGF; and c. prevents NGF-induced sensitization of transient receptor potential vanilloid 1 (TRPV1).

[0128] In some embodiments, the isolated peptide inhibits an interaction between NRP1 andTrkA via binding to NRP1.

[0129] In some embodiments, the isolated peptide mimics TrkA. In some embodiments, theisolated peptide is derived from an extracellular domain of the TrkA. In some embodiments, the isolated peptide comprises amino acid residues 87-111, 100-124, or 315-339 residues of SEQ ID NO: 125. In some embodiments, the isolated peptide comprises amino acid residues of SEQ ID NO: 125 as depicted in Figs.20A-20C.

[0130] In some embodiments, the isolated peptide comprises an amino acid sequence of anyone of SEQ ID NOs: 28-30 and 32-122, or a sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the 26 304870344v1Attorney Docket No: 243735.000408 NRP1 inhibitor comprises an amino acid sequence of SEQ ID NO: 28, 29, or 30, or a sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0131] In some embodiments, the isolated peptide comprises an amino acid sequence of SEQID NO: 28, 29, or 30, or a sequence having at least 80% identity thereto.

[0132] In some embodiments, the isolated peptide consists essentially of an amino acidsequence of SEQ ID NO: 28, 29, or 30, or a sequence having at least 80% identity thereto.

[0133] In some embodiments, the isolated peptide consists of an amino acid sequence of SEQID NO: 28, 29, or 30, or a sequence having at least 80% identity thereto.

[0134] In some embodiments, the isolated peptide comprises one or more of the followingcharacteristics: a. binds to neuropilin-1 (NRP1); b. inhibits the interaction between NRP1 and tropomyosin receptor kinase A (TrkA); c. prevents NGF-induced sensitization of transient receptor potential vanilloid 1 (TRPV1); and d. inhibits NGF-evoked mechanical allodynia and thermal hyperalgesia.

[0135] In some embodiments, the isolated peptides of the present disclosure can be furthermodified to improve affinity of the peptides and / or metabolic stability. In some embodiments, the isolated peptides of the present disclosure may also be synthesized with additional chemical groups present at their N- and / or C-termini, to enhance the affinity, bioavailability, and / or stability of the peptides.

[0136] In some embodiments, N-terminal modifications can include, but not limited toacetylation (e.g., with acetic acid or a halogenated derivative thereof such as ^-chloroacetic acid, ^-bromoacetic acid, or ^-iodoacetic acid), methylation (e.g., —NHCH3or —N(CH3)2), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO— or sulfonyl functionality defined by R—SO2—, where R is selected from alkyl, aryl, heteroaryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the peptide. Alternatively or additionally, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups can be added to the N-terminus. Similarly, an acetyl group or a 9-fluorenylmethoxy-carbonyl group can be placed at the N-terminus. 27 304870344v1Attorney Docket No: 243735.000408

[0137] In some embodiments, C-terminal modifications can include but not limited toreplacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. The peptide inhibitors of the present disclosure can be cyclized, or a desamino or descarboxy residue can be incorporated at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. C-terminal functional groups of the peptide inhibitors of the present disclosure include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof. In some embodiments, the hydrophobic group, t-butyloxycarbonyl, or an amido group can be added to the C-terminus of the peptide inhibitors.

[0138] Non-limiting examples of non-natural modifications include incorporation of non-encoded α-amino acids, retroinversion by using D-amino acids, β-amino acids, backbone reduction, photoreactive cross-linking amino acids, N-methylated amino acids, and C-terminal amidation and PEGylation.

[0139] Non-limiting examples of amino acid replacements include stereoisomers (e.g., D-amino acids) and unnatural amino acids such as, L-citrulline, L-homocysteine, L-homoserine, L- ornithine, 3-sulfino-L-alanine, N-(L-arginino)succinate, 3,4-dihydroxy-L-phenylalanine, 3-iodo- L-tyrosine, 3,5-diiodo-L-tyrosine, triiodothyronine, L-selenocysteine, L-thyroxine, N-(L- arginino)taurine, 4-aminobutylate, (R,S)-3-amino-2-methylpropanoate, a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid, β-alanine, 3-pyridylalanine, 4-hydroxyproline, O- phosphoserine, N-acetylserine, N-formylmethionine, N-methylglycine, 3-methylhistidine, 5- hydroxylysine, nor-leucine, and other similar amino acids and imino acids.

[0140] In some embodiments, the isolated peptides of the present disclosure may comprise oneor more amino acid substitutions and / or insertions and / or deletions. Amino acid substitution encompasses a substitution of the amino acid residue for a replacement amino acid residue at the same position. Inserted amino acid residues can be inserted at any position and can be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or can be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue. One or more amino acids may be substituted and / or inserted and / or deleted from the sequence of any one of SEQ ID NOs: 26, 28-30, and 124. 28 304870344v1Attorney Docket No: 243735.000408

[0141] In some embodiments, the isolated peptides of the present disclosure may compriseadditional amino acid(s) at the C-terminal end and / or at the N-terminal end of the sequence of any one SEQ ID NOs: 26, 28-30, and 124. An isolated peptide of the disclosure may comprise the amino acid sequence of any one of SEQ ID NOs: 26, 28-30, and 124 except for one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, insertions or deletions.

[0142] Inserted amino acids and substituted amino acids can be naturally occurring aminoacids or can be non-naturally occurring amino acids. Non-limiting examples of non-naturally occurring amino acids may contain a non-natural side chain, and / or be linked together via non- native peptide bonds. A few examples of altered peptide ligands are discussed further in Douat- Casassus et al., J. Med. Chem, 2007; 50(7):1598-609 and Hoppes et al., J. Immunol 2014; 193(10):4803-13. In some embodiments, if more than one amino acid residue is substituted and / or inserted, the replacement / inserted amino acid residues may be the same as each other or different from one another. The replacement amino acid may have a different side chain to the amino acid being replaced.

[0143] In some embodiments, D-amino acids may be substituted for the L-amino acids in theantigenic peptides of the disclosure. In some embodiments, non-standard amino acids (i.e., other than the common naturally occurring proteinogenic amino acids) for example, β-γ-δ-amino acids, or many derivatives of L-α-amino acids can also be used for substitutions or additions to produce peptides of the present disclosure.

[0144] Amino acid substitutions can be conservative, for example, the substituted amino acidhas similar chemical properties to the original amino acid. The non-limiting example of such substitutions are the following groups of amino acids that share similar chemical properties such as size, charge, and polarity: Group 1: Ala, Ser, Thr, Pro, Gly; Group 2: Asp, Asn, Glu, Gln; Group 3: His, Arg, Lys; Group 4: Met, Leu, Ile, Val, Cys; Group 5: Phe, Thy, Trp.

[0145] In some embodiments, the naturally occurring side chains of the 20 genetically encodedamino acids (or the stereoisomeric D-amino acids) can be replaced with other side chains, for instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic. One of the non-limiting examples is proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed. Cyclic groups may be saturated or unsaturated, 29 304870344v1Attorney Docket No: 243735.000408 The unsaturated cyclic groups may be aromatic or non-aromatic. Preferred heterocyclic groups contain one or more nitrogen, sulfur, and / or oxygen heteroatoms. Non-limiting examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. If a group is substituted, the substituent can be halogen, oxygen, alkyl, alkoxy, or substituted or unsubstituted phenyl.

[0146] Other non-limiting examples of amino acid replacements include stereoisomers (e.g.,D-amino acids) and unnatural amino acids such as, for example, L-citrulline, L-homocysteine, L- homoserine, L-ornithine, 3-sulfino-L-alanine, N-(L-arginino)succinate, 3,4-dihydroxy-L- phenylalanine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, triiodothyronine, L-thyroxine, L- selenocysteine, N-(L-arginino)taurine, β-alanine, 3-pyridylalanine, 4-hydroxyproline, O- phosphoserine, N-methylglycine, 4-aminobutylate, (R,S)-3-amino-2-methylpropanoate, a,a- disubstituted amino acids, N-alkyl amino acids, lactic acid, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, nor-leucine, and other similar amino acids and imino acids.

[0147] In some embodiments, the isolated peptides of the present disclosure may besynthetically produced or produced by hydrolysis. Non-limiting examples of synthetically produced peptides can include randomly generated peptides, designed peptides, and peptides where at least some of the amino acid positions of the peptides are conserved among several generated peptides and the remaining positions are random. In some embodiments, the peptide inhibitors of the present disclosure may be produced by expression in a heterologous host cell.

[0148] In some embodiments, the isolated peptides of the present disclosure may be about 10-50 amino acids in length. For example, the isolated peptides of the present disclosure may be 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, or 30 amino acids in length. In some embodiments, the isolated peptides of the present disclosure are 25 amino acids in length. In some embodiments, the isolated peptides of the present disclosure are 24 amino acids in length. 30 304870344v1Attorney Docket No: 243735.000408

[0149] In some embodiments, the peptide inhibitors of the present disclosure may compriseone or more chemical modifications, one or more D-isomers of amino acids, one or more non- peptide bonds, one or more reverse peptide bonds, or any combination thereof.

[0150] In some embodiments, the isolated peptides of the present disclosure are fused orconjugated to one or more heterologous moieties. Non-limiting examples of heterologous moieties suitable for chemical conjugation and / or genetical fusion with the isolated peptides of the disclosure include, but are not limited to, nucleic acids, lipids, sugars, peptides, polypeptides, small molecules, polymers, etc. The heterologous moieties may be fused at the N- and / or C-terminus of the peptide.

[0151] In some embodiments, the isolated peptide of the present disclosure is fused tocarbohydrate, protein, and / or lipid moiety. In some embodiments, the isolated peptide of the present disclosure is fused to lipid moiety. The fusion of the isolated peptide of the present disclosure to lipids may enhance concentration of said isolated peptide at the cell surface.

[0152] Non-limiting examples of heterologous peptides and polypeptides include, but are notlimited to, an epitope (such as FLAG) or a tag sequence (such as hexa histidine, and the like) to allow for the detection and / or isolation of a fusion protein; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region (e.g., an Fc domain); a half-life- extending sequence comprising a combination of two or more naturally occurring or non-naturally occurring charged and / or uncharged amino acids designed to form hydrophilic or hydrophobic fusion partner for a fusion protein; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, for example, a leucine zipper domain; a transmembrane receptor protein or a portion thereof, for example, an extracellular domain or a transmembrane and intracellular domain; a functional or non-functional antibody (e.g., an antibody that is specific for dendritic cells), or a heavy or light chain thereof; and a polypeptide which has an activity, such as a therapeutic activity, different from fusion proteins of the present disclosure. In some embodiments, the one or more heterologous moieties enhances an isolated peptide-specific immune response in a subject. In some embodiments, the one or more heterologous moieties mediates isolated peptide delivery to a specific site within a subject.

[0153] In some embodiments, fusion proteins of the disclosure may comprise one or moreaffinity tags, e.g., to allow for affinity purification or coupling to another molecule. Examples of 31 304870344v1Attorney Docket No: 243735.000408 affinity tags include, but are not limited to, an Avi-tag, a biotin, a calmodulin tag, a hexahistidine tag, a hemagglutinin (HA) tag, a Myc tag, a GST tag, a green fluorescent protein (GFP), YFP, RFP, CFP, a MBP tag, a chitin binding protein tag, a FLAG tag, a V5 tag, a streptavidin binding tag, mCherry, tdTomato, SUMO tag, and Ubiquitin tag.

[0154] In one aspect, the present disclosure provides an isolated polynucleotide encoding theisolated peptide described herein. For expression of a polynucleotide described herein, a promoter sequence may be included to position the start site for RNA synthesis. The promoter may be a constitutive promoter or inducible promoter. The polynucleotide may also be operably linked to one or more additional regulatory sequences, such as terminators or enhancers. In one embodiment, the isolated polynucleotide is an mRNA.

[0155] In one aspect, the present disclosure provides a vector comprising the isolatedpolynucleotide encoding the isolated peptide described herein.

[0156] In order to assess the expression of an isolated peptide described herein, apolynucleotide or vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

[0157] In one aspect, the present disclosure provides a host cell comprising the isolatedpolynucleotide or the vectors described herein. Pharmaceutical Compositions

[0158] Pharmaceutical compositions comprising the isolated peptides, polynucleotides, orvectors described herein are within the scope of the present invention. Such pharmaceutical compositions can comprise a therapeutically effective amount of an isolated peptide, polynucleotide, or vector, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. Acceptable formulation agents preferably are nontoxic to recipients at the dosages and concentrations employed. 32 304870344v1Attorney Docket No: 243735.000408

[0159] The pharmaceutical composition can contain formulation agent(s) for modifying,maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation agents include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, methionine or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris- HCl, histidine, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).

[0160] The optimal pharmaceutical composition will be determined by a skilled artisandepending upon, for example, the intended route of administration, delivery format, and desired dosage (see, e.g., Remington's Pharmaceutical Sciences, supra). Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the isolated peptides, polynucleotides, or vectors of the present disclosure.

[0161] The primary vehicle or carrier in a pharmaceutical composition can be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other 33 304870344v1Attorney Docket No: 243735.000408 materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Histidine or Tris buffer of about pH 6.0-8.5, which can further include sorbitol or a suitable substitute. In one embodiment of the present invention, isolated peptide compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of an aqueous solution.

[0162] The pharmaceutical compositions can be selected for parenteral delivery. Alternatively,the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 6 to about 8.

[0163] When parenteral administration is contemplated, the therapeutic compositions for usein this invention can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired isolated peptide, polynucleotide, or vector, in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which an isolated peptide, polynucleotide, or vector, is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

[0164] In one embodiment, a pharmaceutical composition can be formulated for inhalation.For example, the pharmaceutical composition can be formulated as a dry powder for inhalation. Inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized. Pulmonary administration is further described in International Publication No. WO1994020069 (incorporated herein by reference in its entirety), which describes the pulmonary delivery of chemically modified proteins. 34 304870344v1Attorney Docket No: 243735.000408

[0165] It is also contemplated that certain formulations can be administered orally. In oneembodiment of the present invention, formulations that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0166] Another pharmaceutical composition can involve an effective quantity of the isolatedpeptide in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0167] Additional pharmaceutical compositions include formulations involving isolatedpeptides, polynucleotides, or vectors, in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means include the use of liposome carriers, bio-erodible microparticles or porous beads and depot injections (see, e.g., International Publication No. WO1993015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions, and Wischke & Schwendeman, 2008, Int. J. Pharm.364: 298-327, and Freiberg & Zhu, 2004, Int. J. Pharm.282: 1-18, which discuss microsphere / microparticle preparation and use; each reference incorporated herein by reference in its entirety). As described herein, a hydrogel is an example of a sustained- or controlled-delivery formulation.

[0168] Additional examples of sustained-release preparations include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919 and European Patent No. 0058481, each incorporated herein by reference in its entirety), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et ah, 1983, Biopolymers 22: 547-56), poly(2- hydroxyethyl-methacrylate) (Langer et ah, 1981, J. Biomed. Mater. Res.15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al, supra) or poly-D(−)-3- 35 304870344v1Attorney Docket No: 243735.000408 hydroxybutyric acid (European Patent No. 0133988). Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Epstein et ah, 1985, Proc. Natl. Acad. Sci. U.S.A.82: 3688-92; and European Patent Nos.0036676, 0088046, and 0143949, each incorporated herein by reference in its entirety.

[0169] The pharmaceutical composition to be used for in vivo administration typically shouldbe sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The parenteral composition can be diluted into parenteral acceptable diluents (e.g., saline and 5% Dextrose).

[0170] Once the pharmaceutical composition has been formulated, it can be stored in sterilevials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0171] In one embodiment, the present invention is directed to kits for producing a single-doseadministration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and dual chamber syringes).

[0172] In one embodiment, the present invention is directed to a pharmaceutical compositioncomprising an isolated peptide formulated as a powder for injection after reconstitution to a solution for injection.

[0173] Selecting an administration regimen for a therapeutic depends on several factors,including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, Fc fusion therapeutic proteins, cytokines, 36 304870344v1Attorney Docket No: 243735.000408 and small molecules are available (see, e.g., Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003, New Engl. J. Med. 348:601-608; Milgrom, et al., 1999, New Engl. J. Med. 341:1966-1973; Slamon, et al., 2001, New Engl. J. Med.344:783-792; Beniaminovitz, et al., 2000, New Engl. J. Med.342:613- 619; Ghosh, et al., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000, New Engl. J. Med. 343:1594-1602, each incorporated herein by reference in its entirety).

[0174] Determination of the appropriate dose is made by the clinician, e.g., using parametersor factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., increased serum phosphate or decreased phosphate excretion.

[0175] Actual dosage levels of the active ingredients in the pharmaceutical compositions ofthe present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0176] Compositions comprising the isolated peptides of the disclosure can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, one week, 1-7 times per week, or one month. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose may be at least 0.05 μg / kg body weight, at least 0.2 μg / kg, at least 0.5 μg / kg, at least 1 μg / kg, at least 10 μg / kg, at least 100 μg / kg, at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at 37 304870344v1Attorney Docket No: 243735.000408 least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, or at least 50 mg / kg (see, e.g., Yang, et al., 2003, New Engl. J. Med.349:427-434; Herold, et al., 2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J. Neurol. Neurosurg. Psych.67:451-456; Portielji, et al., 2003, Cancer. Immunol. Immunother.52: 133-144). The dose may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg. The doses administered to a subject may number at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.

[0177] For therapeutic peptides of the disclosure, the dosage administered to a patient may be0.0001 mg / kg to 100 mg / kg of the patient's body weight. The dosage may be between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg or 0.01 to 0.10 mg / kg of the patient's body weight.

[0178] The dosage of the therapeutic peptides of the disclosure may be calculated using thepatient's weight in kilograms (kg) multiplied by the dose to be administered in mg / kg. The dosage of the proteins of the disclosure may be 150 μg / kg or less, 125 μg / kg or less, 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less, 85 μ / kg or less, 80 μ / kg or less, 75 μ / kg or less, 70 μ / kg or less, 65 μ / kg or less, 60 μ / kg or less, 55 μ / kg or less, 50 μ / kg or less, 45 μ / kg or less, 40 μ / kg or less, 35 μ / kg or less, 30 μ / kg or less, 25 μ / kg or less, 20 μ / kg or less, 15 μ / kg or less, 10 μ / kg or less, 5 μ / kg or less, 2.5 μ / kg or less, 2 μ / kg or less, 1.5 μ / kg or less, 1 μ / kg or less, 0.5 μ / kg or less, or 0.1 μ / kg or less of a patient's body weight.

[0179] Unit dose of the therapeutic peptides of the disclosure may be 0.1 mg to 20 mg, 0.1 mgto 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 m g, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0180] The dosage of the therapeutic peptides of the disclosure may achieve a serum titer ofat least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 v, at least 175 μg / ml, at least 200 μg / ml, at least 38 304870344v1Attorney Docket No: 243735.000408 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml / ml, or at least 400 μg / ml / ml in a subject. Alternatively, the dosage of the antibodies of the disclosure may achieve a serum titer of at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least, 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml, or at least 400 μg / ml in the subject.

[0181] Doses of therapeutic peptides of the disclosure may be repeated and the administrationsmay be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

[0182] An effective amount for a particular patient may vary depending on factors such as thecondition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard, et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

[0183] The route of administration may be by, e.g., topical or cutaneous application, injectionor infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et al., 1983, Biopolymers 22:547-556; Langer, et al., 1981, J. Biomed. Mater. Res.15: 167-277; Langer, 1982, Chem. Tech.12:98-105; Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688- 3692; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference their entirety. In one embodiment, an engineered antibody or engineered antibody conjugate, combination therapy, or a composition of the disclosure is administered using Alkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). 39 304870344v1Attorney Docket No: 243735.000408

[0184] The frequency of dosing will depend upon the pharmacokinetic parameters of theisolated peptides in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.

[0185] The route of administration of the pharmaceutical composition is in accord with knownmethods, e.g., orally; through injection by subcutaneous, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems (which may also be injected); or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.

[0186] Alternatively or additionally, the composition can be administered locally viaimplantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. In order to deliver drug, e.g., an isolated peptide as disclosed herein, at a predetermined rate such that the drug concentration can be maintained at a desired therapeutically effective level over an extended period, a variety of different approaches can be employed. In one example, a hydrogel comprising a polymer such as a gelatin (e.g., bovine gelatin, human gelatin, or gelatin from another source) or a naturally- occurring or a synthetically generated polymer can be employed. Any percentage of polymer (e.g., gelatin) can be employed in a hydrogel, such as 5, 10, 15 or 20%. The selection of an appropriate concentration can depend on a variety of factors, such as the therapeutic profile desired and the pharmacokinetic profile of the therapeutic molecule.

[0187] Examples of polymers that can be incorporated into a hydrogel include polyethyleneglycol (“PEG”), polyethylene oxide, polyethylene oxide-co-polypropylene oxide, co-polyethylene oxide block or random copolymers, polyvinyl alcohol, poly(vinyl pyrrolidinone), poly(amino acids), dextran, heparin, polysaccharides, polyethers and the like. 40 304870344v1Attorney Docket No: 243735.000408

[0188] Another factor that can be considered when generating a hydrogel formulation is thedegree of crosslinking in the hydrogel and the crosslinking agent. In one embodiment, cross- linking can be achieved via a methacrylation reaction involving methacrylic anhydride. In some situations, a high degree of cross-linking may be desirable while in other situations a lower degree of crosslinking is preferred. In some cases a higher degree of crosslinking provides a longer sustained release. A higher degree of crosslinking may provide a firmer hydrogel and a longer period over which drug is delivered. Any ratio of polymer to crosslinking agent (e.g., methacrylic anhydride) can be employed to generate a hydrogel with desired properties. For example, the ratio of polymer to crosslinker can be, e.g., 8:1, 16:1, 24:1, or 32:1. For example, when the hydrogel polymer is gelatin and the crosslinker is methacrylate, ratios of 8:1, 16:1, 24:1, or 32:1 methyacrylic anhydride:gelatin can be employed.

[0189] One skilled in the art recognizes that different methods of delivery may be utilized toadminister a polynucleotide (e.g., an mRNA) or vector into a cell. Examples include: (1) methods utilizing physical means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein the vector is complexed to another entity, such as a liposome, aggregated protein or transporter molecule.

[0190] Furthermore, the actual dose and schedule can vary depending on whether thecompositions are administered in combination with other compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular cell line utilized (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line). Furthermore, the amount of polynucleotide or vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the polynucleotide or vector, as well as also the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis). One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

[0191] The polynucleotide molecule may also contain a suicide gene i.e., a gene whichencodes a product that can be used to destroy the cell. In many gene therapy situations, it is desirable to be able to express a gene for therapeutic purposes in a host cell but also to have the 41 304870344v1Attorney Docket No: 243735.000408 capacity to destroy the host cell at will. The therapeutic agent can be linked to a suicide gene, whose expression is not activated in the absence of an activator compound. When death of the cell in which both the agent and the suicide gene have been introduced is desired, the activator compound is administered to the cell thereby activating expression of the suicide gene and killing the cell. Examples of suicide gene / prodrug combinations which may be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

[0192] In some embodiments, the pharmaceutical compositions of the present disclosurecomprise two or more isolated peptides described herein. In some embodiments, the two or more peptides are selected from SEQ ID NO: 26, SEQ ID NO: 124, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30, or a pharmaceutically acceptable salt, fragment or derivative thereof. In some embodiments, the pharmaceutical compositions of the present disclosure comprise two isolated peptides described herein. In some embodiments, the pharmaceutical compositions of the present disclosure comprise three or more isolated peptides described herein. In some embodiments, the pharmaceutical compositions of the present disclosure comprise three isolated peptides described herein. In some embodiments, the pharmaceutical compositions of the present disclosure comprise four or more isolated peptides described herein. In some embodiments, the pharmaceutical compositions of the present disclosure comprise four isolated peptides described herein. In some embodiments, the pharmaceutical compositions of the present disclosure comprise five or more isolated peptides described herein. In some embodiments, the pharmaceutical compositions of the present disclosure comprise five isolated peptides described herein. Methods of Treatment

[0193] The present application in one aspect provides a method for treating pain in a subject,comprising administering to the subject a therapeutically effective amount of an inhibitor of NRP1 and / or GIPC1.

[0194] In some embodiments, the method for treating chronic pain in a subject in need thereof,comprising administering to the subject a therapeutically effective amount of an inhibitor of NRP1. 42 304870344v1Attorney Docket No: 243735.000408

[0195] In some embodiments, the method for treating chronic pain in a subject in need thereof,comprising administering to the subject a therapeutically effective amount of an inhibitor of GIPC1.

[0196] In some embodiments, the method for treating chronic pain in a subject in need thereof,comprising administering to the subject a therapeutically effective amount of an inhibitor of NRP1 and an inhibitor of GIPC1.

[0197] GIPC1 (synectin), GIPC2 and GIPC3 are cytoplasmic adaptor proteins that regulateprotein trafficking and signaling. The GIPC1 PDZ domain interacts with transmembrane receptors, coreceptors, channels, adhesion molecules and proteins involved in endocytosis and organelle trafficking. The GH2 domain interacts with myosin VI, an actin-based retrograde motor, to mediate internalization of endocytic vesicles. Thus, GIPC1 regulates assembly of receptor signaling complexes and controls multiple steps of receptor trafficking (clustering, endocytosis, recycling). GIPC1 is a critical node in signaling networks that underlie tumor proliferation, growth, metastasis, and angiogenesis. GIPC1 interacts with neuropilin 1 and tropomyosin receptor kinase A and likely serves to cluster signaling molecules in transport vesicles. In some embodiments, the GIPC1 described herein refers to human GIPC1 (NCBI Gene ID: 10755). In some embodiments, the GIPC1 described herein refers to mouse GIPC1 (NCBI Gene ID: 67903).

[0198] Within the context of the present disclosure, the term “pain” includes chronicinflammatory pain (e.g. pain associated with rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, gouty arthritis and juvenile arthritis); musculoskeletal pain, lower back and neck pain, sprains and strains, neuropathic pain, sympathetically maintained pain, myositis, pain associated with cancer and fibromyalgia, pain associated with migraine, pain associated with cluster and chronic daily headache, pain associated with influenza or other viral infections such as the common cold, rheumatic fever, pain associated with functional bowel disorders such as non-ulcer dyspepsia, non-cardiac chest pain and irritable bowel syndrome, pain associated with myocardial ischemia, post operative pain, headache, toothache, dysmenorrhea, neuralgia, fibromyalgia syndrome, complex regional pain syndrome (CRPS types I and II), neuropathic pain syndromes (including diabetic neuropathy, chemotherapeutically induced neuropathic pain, sciatica, non-specific lower back pain, multiple sclerosis pain, HIV-related neuropathy, post-herpetic neuralgia, trigeminal neuralgia) and pain resulting from physical trauma, amputation, cancer, toxins or chronic inflammatory conditions. 43 304870344v1Attorney Docket No: 243735.000408

[0199] In various embodiments, the pain is a chronic pain.

[0200] In some embodiments, the chronic pain is inflammatory pain, such as the painassociated with inflammatory bowel disease, irritable bowel syndrome, pancreatitis, arthritis, postoperative pain, migraine, or cancer pain.

[0201] In some embodiments, the chronic pain is neuropathic pain, such as neuropathic painsecondary to nerve injury and trauma, diabetic neuropathy, viral neuropathy (e.g., trigeminal neuralgia), chemotherapy-induced peripheral neuropathy, migraine, or cancer pain. In some embodiments, the cancer pain is associated with oral cancer, non-small cell lung cancer, mesothelioma, melanoma, head and neck cancer, breast cancer, ovarian cancer, prostate cancer, renal cancer, liver cancer, or colorectal cancer.

[0202] In some embodiments, the inhibitor that antagonize or inhibit NRP1 results in areduction of NGF-induced nociception. In some embodiments, the inhibitor inhibits expression or function of the NRP1 protein.

[0203] In some embodiments, the inhibitor that antagonize or inhibit GIPC1 results in areduction of NGF-induced nociception. In some embodiments, the inhibitor inhibits expression or function of the GIPC1 protein.

[0204] The NRP1 and / or GIPC1 proteins described in the present application include anynaturally occurring proteins or variants thereof that have function of the wild-type protein, preferably in human. Also included are orthologs of the NRP1 and / or GIPC1 proteins found in other species, such as in horse, bull, chimp, chicken, zebrafish, dog, pig, cow, sheep, rat, mouse, guinea pig or a primate. The NRP1 and / or GIPC1 proteins may also be targeted at the gene or mRNA level to reduce expression of the respective proteins.

[0205] In some embodiments, the inhibitor of NRP1 and / or GIPC1 is a small molecule, ansiRNA, an shRNA, an antisense oligonucleotide, a miRNA, an antibody or antibody fragment, a peptide, a polypeptide, a peptide analog, a fusion peptide, a polynucleotide, a peptidomimetic, a natural product, a carbohydrate, an aptamer, an avimer, an anticalin, a speigelmer, or a site-specific nuclease. Non-limiting examples of the inhibitor are described below.

[0206] In some embodiments, the inhibitor is a peptide, a fragment or derivative thereof thatinhibits NRP1 and / or GIPC1. In some embodiments, the fragment or derivative of peptides or any compounds of the present disclosure includes a functional fragment or derivative that can inhibit NRP1 and / or GIPC1. 44 304870344v1Attorney Docket No: 243735.000408

[0207] In some embodiments, the inhibitor is a peptide, a fragment or derivative thereof thatinhibits NRP1. NRP1 peptide inhibitors that can be used in the methods of the present disclosure include any of the isolated peptides described herein, or any combination thereof.

[0208] In some embodiments, the inhibitor is a small molecule that inhibits NRP1 and / orGIPC1.

[0209] In some embodiments, the inhibitor is a small molecule that inhibits NRP1. Non-limiting examples of small molecules that inhibit NRP1 include N2-[[3-[(2,1,3-Benzothiadiazol- 4-ylsulfonyl)amino]-2-thienyl]carbonyl]-L-arginine (EG00229), 3-((5-(4-(Aminomethyl)phenyl)- 2,3-dihydro benzofuran)-7-sulfonamido)-thiophene-2-carbonyl)-L-arginine (EG01377), or the like.

[0210] In some embodiments, the inhibitor is a small molecule that inhibits GIPC1.

[0211] In some embodiments, the inhibitor is a small interfering RNAs (siRNA), also knownas short interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Such siRNA molecules typically include a region of sufficient homology to the target region, and are of sufficient length in terms of nucleotides, such that the siRNA molecules down-regulate target nucleic acid. It is not necessary that there be perfect complementarity between the siRNA molecule and the target, but the correspondence must be sufficient to enable the siRNA molecule to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA. In some embodiments, the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double- strand character of the molecule.

[0212] In some embodiments, the inhibitor is an siRNA that inhibits NRP1. In someembodiments, the siRNA that inhibits NRP1 comprises GAAUUGCUGUGGAUGAUAU (SEQ ID NO: 1), AGUAAGAGGUGUCAUCAUU (SEQ ID NO: 2), CCACAAGGUUCAUCA GGAU (SEQ ID NO: 3), GGAAUGUUCUGUCGCUAUG (SEQ ID NO: 4), CGAUAAAUGUGGCGAUACU (SEQ ID NO: 22), GGACAG AGACUGCAAGUAU (SEQ ID NO: 23), GUAUACGGUUGCAAGAUAA (SEQ ID NO: 24), AAGACUGGAUCACCAUAAA (SEQ ID NO: 25), or a modified version, a fragment, or a combination thereof.

[0213] In some embodiments, the siRNA that inhibits mouse NRP1 comprises a nucleotidesequence GAAUUGCUGUGGAUGAUAU (SEQ ID NO: 1), AGUAAGAGGUGUCAUCAUU 45 304870344v1Attorney Docket No: 243735.000408 (SEQ ID NO: 2), CCACAAGGUUCAUCAGGAU (SEQ ID NO: 3), GGAAUGUUCUGUCGC UAUG (SEQ ID NO: 4), or a modified version, a fragment, or a combination thereof.

[0214] In some embodiments, the siRNA that inhibits human NRP1 comprises a nucleotidesequence CGAUAAAUGUGGCGAUACU (SEQ ID NO: 22), GGACAGAGACUGCAAGUAU (SEQ ID NO: 23), GUAUACGGUUGCAAGAUAA (SEQ ID NO: 24), AAGACUGG AUCAC CAUAAA (SEQ ID NO: 25), or a modified version, a fragment, or a combination thereof.

[0215] In some embodiments, the inhibitor is an siRNA that inhibits GIPC1. In someembodiments, the siRNA that inhibits GIPC1 comprises GCACUCGGGCUCACCAUCA (SEQ ID NO: 5), GGCCGUACCUUCACGCUGA (SEQ ID NO: 6), GCAAGGCCUUCGACAUGAU (SEQ ID NO: 7), CUGGAGAGUUACAUGGGUA (SEQ ID NO: 8), GCAUCGAGGGCUUC ACUAA (SEQ ID NO: 9), CGUCGGCCUUUGAGGAGAA (SEQ ID NO: 10), GUGGA UGA CUUGCUAGAGA (SEQ ID NO: 11), GCUGAGGCCUUCCGACUAC (SEQ ID NO: 12), or a modified version, a fragment, or a combination thereof.

[0216] In some embodiments, the siRNA that inhibits human GIPC1 comprises a nucleotidesequence GCACUCGGGCUCACCAUCA (SEQ ID NO: 5), GGCCGUACCUUCA CGCUGA (SEQ ID NO: 6), GCAAGGCCUUCGACAUGAU (SEQ ID NO: 7) ), or a modified version, a fragment, or a combination thereof.

[0217] In some embodiments, the siRNA that inhibits mouse GIPC1 comprises a nucleotidesequence CUGGAGAGUUACAUGGGUA (SEQ ID NO: 8), GCAUCGAG GGCUUCACUAA (SEQ ID NO: 9), CGUCGGCCUUUGAGGAGAA (SEQ ID NO: 10), GUGGAUGACUUGCUAGAGA (SEQ ID NO: 11), GCUGAGGCCUUCCGACUAC (SEQ ID NO: 12) ), or a modified version, a fragment, or a combination thereof.

[0218] In some embodiments, the inhibitor is a short hairpin RNA (shRNA). A “small hairpinRNA” or “short hairpin RNA” or “shRNA” described herein may include a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure may be cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).

[0219] Non-limiting examples of shRNAs include a double-stranded polynucleotide moleculeassembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide 46 304870344v1Attorney Docket No: 243735.000408 molecule with a hairpin secondary structure having self-complementary sense and antisense regions. In some embodiments, the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

[0220] In some embodiments, the siRNAs or shRNAs of the present disclosure comprise anortholog version (e.g., human) of the sequences described herein.

[0221] Specificity of siRNA molecules may be measured via the binding of the antisensestrand of the molecule to its target RNA. Effective siRNA molecules are often fewer than 30 to 35 base pairs in length, e.g., to prevent stimulation of non-specific RNA interference pathways in the cell by way of the interferon response, however longer siRNA may also be effective. In various embodiments, the siRNA molecules are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In various embodiments, the siRNA molecules are about 35 to about 70 more base pairs in length. In some embodiments, the siRNA molecules are more than 70 base pairs in length. In some embodiments, the siRNA molecules are 8 to 40 base pairs in length, 10 to 20 base pairs in length, 10 to 30 base pairs in length, 15 to 20 base pairs in length, 19 to 23 base pairs in length, 21 to 24 base pairs in length. In some embodiments, the sense and antisense strands of the siRNA molecules are each independently about 19 to about 24 nucleotides in length. In some embodiments, the sense strand of an siRNA molecule is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, both the sense strand and the antisense strand of an siRNA molecule are 21 nucleotides in length.

[0222] In various embodiments, an siRNA molecule can comprise a 3' overhang at one end ofthe molecule. In some embodiments, the other end can be blunt-ended or may also comprise an overhang (e.g., 5' and / or 3'). When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be different or the same. In some embodiments, an siRNA molecule described herein may comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule. In some embodiments, the siRNA molecule comprises 3’ overhangs of about 1 to about 3 nucleotides on both the sense strand and the antisense strand. In some embodiments, the siRNA molecule comprises 3’ overhangs of about 1 to about 3 nucleotides on 47 304870344v1Attorney Docket No: 243735.000408 the antisense strand. In some embodiments, the siRNA molecule may comprise 3’ overhangs of about 1 to about 3 nucleotides on the sense strand.

[0223] In various embodiments, the siRNA molecule comprises one or more modifiednucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more). In some embodiments, all of the nucleotides of the sense strand and / or the antisense strand of the siRNA molecule are modified. In certain embodiments, the siRNA molecule can comprise one or more modified nucleotides and / or one or more modified internucleotide linkages. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand and at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand.

[0224] In some embodiments, the modified nucleotide may comprise a modified sugar moiety(e.g., a 2' modified nucleotide). In some embodiments, the siRNA molecule can comprise one or more 2’ modified nucleotides, e.g., a 2'-deoxy, 2'-fluoro (2’-F), 2'-O-methyl (2’-O-Me), 2'-O- methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA). In various embodiments, each nucleotide of the siRNA molecule can a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the siRNA molecule may comprise one or more phosphorodiamidate morpholinos. In some embodiments, each nucleotide of the siRNA molecule consists of a phosphorodiamidate morpholino.

[0225] In various embodiments, the siRNA molecule may comprise a phosphorothioate orother modified internucleotide linkage. In various embodiments, the siRNA molecule may comprise, e.g., a phosphorothioate internucleoside linkage(s). In some embodiments, the siRNA molecule may comprise a phosphorothioate internucleoside linkage(s) between two or more nucleotides. In some embodiments, the siRNA molecule may comprise a phosphorothioate internucleoside linkage(s) between all nucleotides. In some embodiments, the siRNA molecule 48 304870344v1Attorney Docket No: 243735.000408 may comprise modified internucleotide linkages at the first, second, and / or third internucleoside linkage at the 5' or 3' end of the siRNA molecule. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ and / or 3′ end of the siRNA molecule. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first and second internucleoside linkages at the 5′ end of the siRNA molecule sense strand and at the first and second internucleoside linkages at the 5′ and 3′ ends of the siRNA molecule antisense strand. In some embodiments, the siRNA molecule may comprise modified internucleotide linkages at the first internucleoside linkage at the 5′ and 3′ ends of the siRNA molecule sense strand, at the first, second, and third internucleoside linkages at the 5′ end of the siRNA molecule antisense strand, and at the first internucleoside linkage at the 3′ end of the siRNA molecule antisense strand.

[0226] A “variant” of a molecule is a sequence that is substantially similar to the sequence ofthe referenced molecule. Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis. Generally, nucleotide sequence variants of the disclosure will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%- 84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the referenced nucleotide sequence.

[0227] The present application also provides an expression cassette containing an isolatednucleic acid sequence encoding an siRNA or shRNA targeted against NRP1 and / or GIPC1. The expression cassette may further contain a pol II promoter. Examples of pol II promoters include regulatable promoters and constitutive promoters. For example, the promoter may be a CMV or RSV promoter. The expression cassette may further contain a polyadenylation signal, such as a synthetic minimal polyadenylation signal. The nucleic acid sequence may further contain a marker gene. The expression cassette may be contained in a viral vector. An appropriate viral vector for 49 304870344v1Attorney Docket No: 243735.000408 use in the present disclosure may be an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, herpes simplex virus (HSV) or murine Maloney-based viral vector.

[0228] In some embodiments, the inhibitor of NRP1 and / or inhibitor of GIPC1 reduces theexpression of NRP1 and / or GIPC1. In some embodiments, the inhibitor of NRP1 and / or inhibitor of GIPC1 reduces the expression of NRP1 and / or GIPC1 protein described herein by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the level without the inhibitor. In some embodiments, the inhibitor renders the expression of NRP1, and / or GIPC1 proteins comparable as a reference level. In some embodiments, the reference level is the level of gene expression in a subject or group of subjects that do not have the disease or condition.

[0229] In some embodiments, the inhibitor of NRP1 and / or GIPC1 is an antibody or anantigen-binding fragment. In some embodiments, an antigen-binding fragment may be selected from the group consisting of a bi-epitopic antibody or antigen-binding fragment thereof, a single- chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, a Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a tribody, and a tetrabody. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a Fab or Fab’. In some embodiments, the antibody is chimeric, human, partially humanized, fully humanized, or semi-synthetic. Antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0230] In some embodiments, the inhibitor of NRP1 is an antibody or antigen-bindingfragment that inhibits NRP1. In some embodiments, the antibody or antigen-binding fragment that inhibits NRP1 is Vesencumab, or an antigen-binding fragment thereof.

[0231] In some embodiments, the inhibitor of GIPC1 is an antibody or antigen-bindingfragment that inhibits GIPC1.

[0232] In some embodiments, the inhibitor of NRP1 and / or GIPC1 is a polypeptide. Theinhibitory polypeptide may be about 50 to about 1000 amino acids in length, such as about 50- 800, 50-500, 50-400, 50-300 or 50-200 amino acids in length. In some embodiments, the inhibitory polypeptide may be about 50 to about 100 amino acids, about 100 to about 150 amino acids, or about 150 amino acids to about 200 amino acids in length.

[0233] In some embodiments, the inhibitor of NRP1 is the polypeptide that inhibits NRP1.50 304870344v1Attorney Docket No: 243735.000408

[0234] In some embodiments, the inhibitor of GIPC1 is the polypeptide that inhibits GIPC1.In some embodiments, the polypeptide that inhibits GIPC1 comprises the amino acid sequence N- myristoyl-PSQSSSEA (SEQ ID NO: 13), or a modified version or a fragment thereof.

[0235] In some embodiments, the peptide, according to the present disclosure is a derivativeor a fragment of NRP1 and / or GIPC1. In some embodiments, the peptide analog, according to the present disclosure is an analog of NRP1 and / or GIPC1.

[0236] In some embodiments, the antibody, an antigen-binding fragment, or inhibitorypolypeptide may further comprise a stabilizing domain. The stabilizing domain can be any domain that stabilizes the inhibitory polypeptide (for example, extending half-life of the inhibitory polypeptide in vivo). In some embodiments, the stabilizing domain is an Fc domain.

[0237] In some embodiments, the inhibitor of NRP1 and / or GIPC1 (e.g., antibody, antigen-binding fragment, polypeptide) comprises an Fc fragment. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from a human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or a combination or hybrid IgG.

[0238] In some embodiments, the inhibitor of NRP1 and / or GIPC1 described herein may be amulti-specific molecule. Multi-specific molecules are molecules that have binding specificities for at least two different antigens or epitopes (e.g., bispecific antibodies have binding specificities for two antigens or epitopes). Multi-specific molecules with more than two valences and / or specificities are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al. J. Immunol.147: 60 (1991)).

[0239] In some embodiments, the inhibitor of NRP1 and / or GIPC1 comprises a multi-specific(e.g., bispecific) molecule comprising a first binding moiety (such as a first antibody) specifically recognizing NRP1 and / or GIPC1, and a second binding moiety (such as a second antibody) specifically recognizing a second antigen. In some embodiments, the multi-specific molecule is, for example, a diabody (Db), a single-chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a di-diabody, a tandem scFv, a tandem di-scFv (e.g., a bispecific T cell engager), a tandem tri-scFv, a tri(a)body, a bispecific Fab2, a di-miniantibody, a tetrabody, an scFv-Fc-scFv fusion, a dual-affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, an IgG-scFab, an scFab-ds-scFv, an Fv2-Fc, an 51 304870344v1Attorney Docket No: 243735.000408 IgG-scFv fusion, a dock and lock (DNL) antibody, a knob-into-hole (KiH) antibody (bispecific IgG prepared by the KiH technology), a DuoBody (bispecific IgG prepared by the Duobody technology), a heteromultimeric antibody, or a heteroconjugate antibody.

[0240] In some embodiments, the bispecific antibody comprises a first antigen-bindingdomain that binds to NRP1 and a second antigen-binding domain that binds to TrkA. In some embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to NRP1 and a second antigen-binding domain that binds to NGF.

[0241] Bispecific antibodies can be produced by a variety of methods including fusion ofhybridomas or linking of Fab′ fragments., See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990) or by the “knob-in-hole” strategy (see, e.g., PCT Publ. No. WO2006 / 028936) to generate full length bispecific antibodies.

[0242] In some embodiments, the inhibitor of NRP1 and / or GIPC1 comprises a site-specificnuclease that targets NRP1 and / or GIPC1. In some embodiments, the site-specific nuclease comprises a DNA nuclease such as an engineered (e.g., programmable or targetable) DNA nuclease to induce genome editing of a target DNA sequence of NRP1 and / or GIPC1. Any suitable DNA nuclease can be used including, but not limited to, CRISPR-associated protein (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof. In some embodiments, the genome editing comprises modifying a gene encoding NRP1, and / or GIPC1 so that no functional protein is produced, or the modified protein no longer mediates NGF-evoked nociception or mediates NGF- evoked nociception to a less extent than wildtype protein. In some embodiments, the genome editing comprises modifying a gene encoding NRP1, and / or GIPC1 so that no functional protein is produced, or the modified protein no longer mediates NGF-evoked endocytosis of TrkA or mediates NGF-evoked endocytosis of TrkA to a less extent than wildtype protein.

[0243] In some embodiments, the method comprises administering to the subject acombination of the inhibitors of NRP1 and / or GIPC1 described herein. Two or more inhibitors of NRP1 and / or GIPC1 described herein may be administered sequentially, simultaneously, and / or concurrently.

[0244] The methods may also involve multiple rounds of administration of the inhibitors ofNRP1 and / or GIPC1 described herein. In some embodiments, following an initial round of 52 304870344v1Attorney Docket No: 243735.000408 administration, the level and / or symptoms of pain, in the subject may be evaluated, and, if needed, an additional round of administration can be performed. In this way, multiple rounds of the inhibitors of NRP1 and / or GIPC1 administration can be performed.

[0245] In some embodiments, the inhibitor of NRP1 and / or GIPC1 is administered locally toan area affected by the chronic pain. In some embodiments, the inhibitor of NRP1 and / or GIPC1 is administered intrathecally, intranasally, intracolonically, intraluminally, intraintestinally, intracisternally, intraventricularly, intratumorally, or intraarticularly.

[0246] In some embodiments, the method comprising monitoring a pain level experienced bythe subject before, during, and / or after the administering of the inhibitor. The monitoring pain level according to the present invention includes measuring one or more pain parameters associated with chronic pain in the subject. In some embodiments, the pain parameters are selected from pain duration, pain radiation pattern, pain severity, pain quality, degree of pain level fluctuation, frequency of pain remissions, and level of function of the subject. In some embodiments, the measuring pain comprises assessing the pain level experienced by the subject using a pain intensity scale and / or a pain questionnaire.

[0247] Any of the inhibitors of NRP1 and / or GIPC1 described herein can be present in acomposition (such as a formulation) that includes other agents, excipients, or stabilizers.

[0248] It is understood that the compounds of the present disclosure can be present in one ormore stereoisomers (e.g., diastereomers). The disclosure includes, within its scope, all of these stereoisomers, either isolated (e.g., in enantiomeric isolation) or in combination (including racemic and diastereomeric mixtures). The present disclosure uses amino acids independently selected from L and D forms (e.g., the peptide may contain two serine residues, each serine residue having the same or opposite absolute stereochemistry), etc., are intended for the use of both L- and D- form amino acids.

[0249] Accordingly, the compounds of the present disclosure also include substantially purestereoisomeric form of the specific compound with respect to the asymmetric center of the amino acid residue, for example about 90% de, such as greater than about 95% to 97% de, or 99% de. For larger compounds, as well as mixtures thereof (such as racemic mixtures). Such diastereomers may be prepared, for example, by asymmetric synthesis using chiral intermediates, or the mixture may be divided by conventional methods, such as chromatography or the use of dividing agents. 53 304870344v1Attorney Docket No: 243735.000408

[0250] If the compounds of the disclosure require purification, chromatographic techniquessuch as high-performance liquid chromatography (HPLC) and reverse phase HPLC can be used. Peptides may be characterized by mass spectrometry and / or other suitable methods.

[0251] If the compound contains one or more functional groups that can be protonated ordeprotonated (e.g., at physiological pH), the compound can be prepared and / or isolated as a pharmaceutically acceptable salt. It will be appreciated that the compound can be zwitterion at a given pH. As used herein, the expression "pharmaceutically acceptable salt" refers to a salt of a given compound, which salt is suitable for pharmaceutical administration. Such salts can be formed, for example, by reacting an acid or base with an amine or carboxylic acid group, respectively.

[0252] Pharmaceutically acceptable acid addition salts can be prepared from inorganic andorganic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvate, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartrate acid, citrate, benzoic acid, cinnamic acid, mandelic acid, Examples thereof include methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.

[0253] Pharmaceutically acceptable base addition salts can be prepared from inorganic andorganic bases. Corresponding counterions derived from inorganic bases include salts of sodium, potassium, lithium, ammonium, calcium and magnesium. Organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, prokine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, Substituted amines such as primary, secondary and tertiary amines such as N-alkylglucamine, theobromine, purines, piperazine, piperazine and N-ethylpiperidine, substituted amines such as natural substituted amines and cyclic amines can be mentioned.

[0254] Acid / base addition salts tend to be more soluble in aqueous solvents than thecorresponding free acid / base forms.

[0255] In some embodiments, it is envisioned that two or more combinations of thecompounds of the disclosure will be administered to the subject. It is believed that the compound (s) may also be administered in combination with one or more additional therapeutic agents. This 54 304870344v1Attorney Docket No: 243735.000408 combination can allow separate, continuous or simultaneous administration with the other active ingredients of the above compounds. This combination may be provided in the form of a pharmaceutical composition.

[0256] In some embodiments, the inhibitor of NRP1 is administered either alone or incombination with an inhibitor of GIPC1. In some meebodiments, the inhibitor of GIPC1 is administered either alone or in combination with an inhibitor of NRP1. In some embodiments, the inhibitor of NRP1 is administered before, simultaneously with, or after the administration of the inhibitor of GIPC1.

[0257] In some embodiments, the method of treatment further comprising administering anadditional treatment to the subject. In some embodiments, the additional treatment comprises administering a nonsteroidal anti-inflammatory drug (NSAID), acetaminophen, a local anesthetic, a benzodiazepine, capsaicin, an antidepressant, an anti-seizure medication, an anti-epileptic medication, a Cox-2 inhibitor, an opioid, a muscle relaxant, a steroid, an anticonvulsant, a triptan, dihydroergotamine, a beta blocker, a calcium channel blocker, a calcitonin gene-related peptides antagonist, a serotonin and norepinephrine reuptake inhibitor (SNRI), onabotulinumtoxinA, Lasmiditan, or an anti-nausea drug, or a combination thereof.

[0258] As used herein, the term "combination" is used by the combination agents as definedabove dependently or independently, or by the use of different fixed combinations with different amounts of combination agents, i.e., simultaneously or at different times. Refers to a kit of compositions or parts that can be administered. The combination agents can then be administered, for example, simultaneously or staggered in time (i.e., at different times and at equal or different time intervals for any part of the kit). The ratio of the total amount of combination agents administered in a combination can vary, e.g., to address the needs of a subpopulation of patients to be treated or the needs of a single patient, and different needs are the age of the patient, it can be due to gender, weight, etc.

[0259] The route of administration and the type of pharmaceutically acceptable carrier willdepend on the condition being treated and the type of mammal. Formulations containing the active compound may be prepared such that the activity of the compound is not disrupted during the process and the compound can reach its site of action without disruption. In some cases, it may be necessary to protect the compound by means known in the art, such as microencapsulation. Similarly, the route of dosing selected should be such that the compound reaches its site of action. 55 304870344v1Attorney Docket No: 243735.000408

[0260] In some embodiments, the composition further comprises a targeting agent or a carrierthat promotes the delivery of the inhibitors of NRP1 and / or GIPC1 to an area affected by the chronic pain. Exemplary carriers include liposomes, micelles, nanodisperse albumin and its modifications, polymer nanoparticles, dendrimers, inorganic nanoparticles of different compositions. In some embodiments, the inhibitor is formulated in a nanoparticle or a liposome. In some embodiments, the inhibitor formulated in a nanoparticle or a liposome is NRP1 inhibitor. In some embodiments, the NRP1 inhibitor formulated in nanoparticle or liposome as described herein, is administered for the treatment of pain.

[0261] The appropriate formulation for the compound of the disclosure can be adjusted for pH.Buffer systems are routinely used to provide pH values in the desired range and include carboxylic acid buffers such as acetates, citrates, lactates and succinates. In some embodiments, the composition is formulated to have a pH range of about 4.5 to about 9.0, including for example pH ranges of about any of 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. In some embodiments, the pH of the composition is formulated to no less than about 6, including for example no less than about any of 6.5, 7, or 8 (such as about 8). The composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.

[0262] The formulation may also include suitable excipients, such as antioxidants. Examplesof antioxidants include phenolic compounds such as BHT or Vitamin E, reducing agents such as methionine or sulfites, and metal chelating agents such as EDTA.

[0263] The compounds or pharmaceutically acceptable salts thereof described herein can beprepared in parenteral dosage forms such as those suitable for intravenous, intrathecal, and intracerebral or epidural delivery. Suitable pharmaceutical forms for injectable use include sterile injectable or dispersions and sterile powders for the immediate preparation of sterile injectable solutions. They must be stable under manufacturing and storage conditions and protected from reduction or oxidation and the contaminating effects of microorganisms such as bacteria or fungi.

[0264] The solvent or dispersion medium for the injectable solution or dispersion may includeeither conventional solvents or carrier systems for the active compound, e.g., water, ethanol, polyols (e.g., glycerol, propylene glycol and). Liquid polyethylene glycol, etc., suitable mixtures thereof, and vegetable oils may be included. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. Prevention of the action of microorganisms can be 56 304870344v1Attorney Docket No: 243735.000408 performed as needed by incorporating various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it may be preferable to include agents that regulate osmotic pressure, such as sugar or sodium chloride. Preferably, the injectable formulation is isotonic with blood. Sustained absorption of the injectable composition can be brought about by the use of agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition. In some embodiments, the administration of the compounds, preferably inhibitor is via systemic or local delivery. Suitable pharmaceutical forms for injection can be delivered by any suitable route, including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

[0265] Sterilized injectable solutions are prepared by adding the required amount of thecompounds of the disclosure to a suitable solvent containing various other components, such as those listed above, as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from those described above. For sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying or lyophilization of the pre-sterile filtered solution of the active ingredient plus any additional desired ingredients.

[0266] Other pharmaceutical forms include the oral and enteral formulations, where the activecompound can be formulated with an inert diluent or an assimilated edible carrier, or encapsulated in hard or softshell gelatin capsules. The formulations can also be tableted, or it can be incorporated directly into diet foods. For oral therapeutic administration, the active compound is taken up with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. The amount of active compound in such a therapeutically useful composition is such that an appropriate dose can be obtained.

[0267] Tablets, lozenges, pills, capsules, etc. may also contain the ingredients listed below:binders such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; corn starch, Disintegrants such as potato starch, arginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin, or flavors such as peppermint, winter green oil, or cherry flavor may be added. If the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above types of materials. Various other materials may be present as a coating or in other ways to alter the physical form of the dosage unit. For example, tablets, pills, or capsules 57 304870344v1Attorney Docket No: 243735.000408 can be coated with shellac, sugar, or both. The syrup or elixir may contain active compounds, sucrose as a sweetener, methyl and propylparabens as preservatives, pigments and flavors such as cherry or orange flavors. Of course, any substance used to prepare the dosage unit form must be pharmaceutically pure and substantially non-toxic in the amount used. In addition, the compounds of the disclosure may be incorporated into sustained release formulations and formulations comprising those that specifically deliver the active peptide to a particular region of the intestine.

[0268] Liquid formulations can also be administered enterally via the stomach or esophagealcanal. The enteral preparation can be prepared in the form of a suppository by mixing with a suitable base such as an emulsifying base or a water-soluble base. It is possible, but not necessary, to administer the compound of the present disclosure topically, intranasally, intravaginally, intraocularly or the like.

[0269] Pharmaceutically acceptable vehicles and / or diluents include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarders, and the like. The use of such vehicles and agents for pharmaceutically active substances is well known in the art. Its use in therapeutic compositions is intended unless any conventional vehicle or agent is incompatible with the active ingredient. Auxiliary active ingredients can also be incorporated into the composition.

[0270] It is particularly advantageous to formulate the composition in unit dosage form forease of administration and uniformity of dosage. As used herein, a dosage unit form means a physically distinct unit suitable as a unit dosage for a mammalian subject to be treated; each unit is a required pharmaceutically acceptable vehicle. Contains a predetermined amount of active substance calculated to produce the desired therapeutic effect in connection with. Details of the novel dosage unit forms of the disclosure include (a) the unique properties of the active substance and the particular therapeutic effect to be achieved, and (b) physical health as disclosed in detail herein. It is determined by and directly dependent on the technology-specific limitations of the active substances formulated for the treatment of the disease in living subjects with impaired disease states.

[0271] As mentioned above, the main active ingredient may be formulated for convenient andeffective administration in therapeutically effective amounts using a suitable pharmaceutically acceptable vehicle in the form of a dosage unit. The unit dosage form can contain, for example, the major active compound in an amount ranging from 0.25 μg to about 2000 mg. Expressed in 58 304870344v1Attorney Docket No: 243735.000408 proportion, the active compound may be present in a carrier of about 0.25 μg to about 2000 mg / mL. In the case of a composition containing an auxiliary active ingredient, the dose is determined with reference to the usual dosage and mode of administration of the ingredient.

[0272] In some embodiments, the composition is suitable for administration to a human. Insome embodiments, the composition is suitable for administration to a mammal such as, in the veterinary context, domestic pets and agricultural animals. There are a wide variety of suitable formulations of the composition comprising the inhibitor of NRP1 and / or GIPC1. The following formulations and methods are merely exemplary and are in no way limiting. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0273] Examples of suitable carriers, excipients, and diluents include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. In some embodiments, the composition comprising the inhibitor of NRP1 and / or GIPC1 with a carrier as discussed herein is present in a dry formulation (such as lyophilized composition). The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.

[0274] Formulations suitable for parenteral administration include aqueous and non-aqueous,isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and 59 304870344v1Attorney Docket No: 243735.000408 solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. EXAMPLES

[0275] The following examples are provided to further describe some of the embodimentsdisclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. Example 1. NRP1 binds human β-NGF

[0276] NRP1 is a co-receptor for unrelated families of proteins including neuronal guidancemolecules (semaphorins)19, growth factors (VEGF-A)15, cell-penetrating peptides20, and viruses (SARS-CoV-2)14. NRP1 ligands share a C-terminal basic motif (C-end rule, “CendR” motif, R / KXXR / K), which interacts with extracellular “b” domains of NRP115,19. Inspection of the amino acid sequence of mature NGF identified prospective ‘CendR’ R / KXXR / K motifs within the C- terminus that are conserved between rodents and humans and known to mediate the interaction of other growth factors with NRP120(Fig. 1A). Molecular modeling of ^NGF and human NRP1 identified a potential interaction between the CendR motif at the C-terminus of NGF and the b1b2 domain of NRP1. To examine the potential for productive interactions between NGF and NRP1, restrained protein-protein docking of a human NGF structure terminating in Arg118 to the AlphaFold-predicted structure of soluble human a1a2b1b2 domains of NRP1 was used (PDB 4GZ921; Fig. 1B). The molecular modeling used in this experiment supports the hypothesis that these proteins could interact via the canonical CendR binding mechanism, similar to the interactions observed for structures of VEGF165a CendR bound to the b1 domain22. To directly investigate whether NGF binds purified NRP1, microscale thermophoresis (MST) was used to analyze interactions between human NGF and His-tagged human NRP1. NGF interacted with extracellular NRP1 a1a2b1b2 domains with nanomolar affinity (Kd = 65.64 ± 6.66 nM, n=4, Fig. 60 304870344v1Attorney Docket No: 243735.000408 1C). This is comparable to known affinities between purified VEGF165a and NRP1 (Kd9 nM23to 120 nM24).

[0277] NGF binding to NRP1 in living cells at 37ºC was analyzed using a modifiedbioluminescence resonance energy transfer (BRET)-based assay (Fig. 1D). VEGF165a (positive control growth factor known to interact with NRP1) or NGF was genetically fused to an 11 amino acid fragment (HiBiT) of nanoluciferase (NanoLuc). HiBiT has a high affinity for the complementary LgBiT fragment. HiBiT-tagged growth factors were expressed in HEK293T cells, secreted into supernatant, and conjugated to recombinant LgBiT to form full length and catalytically active NanoLuc. Upon addition of furimazine substrate, complemented NanoLuc emitted bioluminescence. The supernatant containing HiBiT-tagged growth factor was incubated with cells expressing SnapTag-NRP1 labeled with SNAP-Surface®Alexa Fluor®488 fluorophore (SnapTag-AF488, +) or lacking fluorophore (-). If proteins are in close proximity (<10 nm), luminescent growth factor can act as a BRET donor to NRP1. As a positive control, BRET was detected between luminescent VEGF165a and SnapTag-NRP1 (Fig.1E). Pre-incubation of cells with an excess of VEGF165a (10 nM) abolished the BRET signal. No BRET signal was detected when luminescent VEGF165a was incubated with cells expressing a binding-dead NRP1 mutant in the b1 domain25,26. Importantly, BRET was detected between NGF-HiBiT and SnapTag-NRP1, and pre-incubation with unlabeled VEGF165a inhibited this response.

[0278] The results from molecular modeling, analysis of interactions between recombinantproteins, and ligand binding studies in intact cells, provided evidence that NGF directly binds NRP1 with nanomolar affinity. Example 2. Ntrk1 / TrkA and Nrp1 / NRP1 are co-expressed in mouse and human DRG neurons

[0279] To determine whether NRP1 could function as an NGF / TrkA co-receptor in nociceptors,TrkA and NRP1 were localized in mouse and human dorsal root ganglia (DRG) by immunofluorescence (protein) and RNAScope®in situ hybridization (mRNA). In mice, immunoreactive TrkA was detected in vesicles and immunoreactive NRP1 was localized at the plasma membrane of the same DRG neurons (Fig.2A). Ntrk1 (TrkA) and Nrp1 (NRP1) mRNAs colocalized in both mouse and human DRG neurons, which were identified by NeuNimmunostaining (Fig.2B, Fig.2C). In mice, Ntrk1 mRNA was expressed in 27% and Nrp1 mRNA61 304870344v1Attorney Docket No: 243735.000408 was expressed in 30% of the neurons; Nrp1 was co-expressed in approximately one third of Ntrk1-positive neurons (Fig. 2D). In humans, Nrp1 was co-expressed in approximately 93% of Ntrk1-positive neurons (Fig. 2E). Thus, TrkA and NRP1 were co-expressed in the same neurons in DRGin mouse and human, where NRP1 was appropriately located to control NGF / TrkA signaling. Example 3. NRP1 antagonism abrogates NGF-induced ionic currents in mouse and human DRG neurons

[0280] NGF / TrkA signaling promotes pain by kinase-mediated phosphorylation andsensitization of ion channels, leading to enhanced sensitivity to mechanical, thermal and chemical stimuli, thus activating voltage-gated ion channels in DRG neurons5. Patch-clamp recordings under current-clamp mode were made to determine whether NRP1 is necessary for NGF-induced excitability and ion channel activity in dissociated human DRG neurons. Human NGF (50 nM) increased the number of action potentials elicited by a depolarizing ramp pulse from 0 to 250 pA in 1 s (Fig.3A, Fig.3B). Pre-incubation with the NRP1 inhibitor EG00229 (30 µM)25prevented NGF-induced hyperexcitability (Fig. 3A, Fig. 3B). NGF or EG00229 did not affect the resting membrane potential (Fig.3C) or the rheobase, the minimum current necessary to elicit an action potential (Fig. 3D). Murine NGF similarly stimulated action potential firing in mouse DRG nociceptors (Figs.3E-3G). Pre-incubation with EG00229 blocked NGF-induced excitability (Fig. 3E, Fig.3F), while NGF had no effect on resting membrane potential or rheobase (Fig.3G, Fig. 3H).

[0281] Since both Ca2+ and Na+ channels have critical roles in controlling the excitability ofsensory neurons, whether NGF increased the activity of voltage-gated ion channels was tested by making patch-clamp recordings under the voltage-clamp mode in dissociated mouse DRG. NGF (50 nM) increased total Ca2+currents (Fig. 3I) and current density (Fig. 3J, Fig. 3K) by ~50% when compared to vehicle (DMSO)-treated cells. EG00229 prevented NGF-induced activation of Ca2+currents, but had no effect on Ca2+currents in unstimulated DRGs (Fig.3J, Fig.3K). Half- maximal activation and inactivation (V1 / 2) potential, as well as slope factor values (k) for activation and inactivation, were not different between the conditions tested, except for an ∼8-mV hyperpolarizing shift in Ca2+channel activation induced by EG00229 when compared to DMSO- and NGF-treated DRGs (Fig. 3L, Table 1). Similarly, EG00229 normalized NGF-induced increases in Na+ currents (Fig.3M). NGF caused a two-fold increase in Na+ current density (Fig. 62 304870344v1Attorney Docket No: 243735.000408 3N) and peak current density (Fig.3O) when compared to vehicle (DMSO)-treated DRG neurons. EG00229 abolished these effects of NGF but had no effects on Na+currents in unstimulated DRGs (Fig.3N, Fig.3O). There were no detectable changes in the voltage-dependence of activation and inactivation between the conditions tested, except for a ~10-mV depolarizing shift in the V1 / 2of inactivation of EG00229 and NGF-treated cells compared to control (Fig. 3P). These data demonstrate that NRP1 antagonism abrogates NGF-induced ionic currents in mouse and human DRG neurons, and implicate voltage-gated Ca2+and Na+ channels as downstream effectors of NRP1-mediated NGF signaling. Thus, NRP1 is necessary for NGF-induced sensitization of nociceptors in rodents to humans. Example 4. NRP1 antagonism inhibits NGF-induced nociception in mice

[0282] NGF injection is known to induce pain in humans and rodents5. In addition to sensitizingion channels, NGF / TrkA signaling also promotes chronic pain through the upregulation of genes that mediate pain (e.g., substance P, transient receptor potential vanilloid 1)5. To test whether NRP1 is necessary for NGF-induced nociception, murine NGF (50 ng) was administered to mice by intraplantar (i.pl., 10 µl) injection (Fig.4A). A human mAb against the b1b2 domain of NRP1 (vesencumab16, 7 µg / 10 µl) or control IgG (7 µg / 10 µl), or small molecule NRP1 inhibitor EG00229 (30 µM / 10 µl)25or compound 5 (Cpd-5; 30 µM / 10 µl)27or vehicle (PBS, 10 µl) was co- administered (i.pl.) with NGF. Withdrawal responses of NGF-injected (ipsilateral) hindpaws to stimulation with von Frey filaments (VFF) and radiant heat were assessed to evaluate mechanical allodynia and thermal hyperalgesia, respectively. In control experiments, NGF decreased the withdrawal threshold to VFF stimulation and the latency of withdrawal to thermal stimulation within 30 minutes for at least 4 h, indicating mechanical allodynia and thermal hyperalgesia (Figs. 4B-4G). Vesencumab and EG00229 strongly inhibited NGF-induced mechanical allodynia for 2 h (hour) and Cpd-5 inhibited mechanical allodynia for 1 h (Figs.4B-4D). Vesencumab, EG00229 and Cpd-5 also inhibited NGF-induced thermal hyperalgesia for 1-2 h (Figs.4E-4G). None of the inhibitors affected the withdrawal responses of the ipsilateral paw to mechanical or thermal stimuli in mice that did not receive NGF (Figs.4B-4G).

[0283] NGF mediates inflammatory pain5, providing an opportunity to determine whether NRP1is necessary for the pronociceptive actions of endogenous NGF. Complete Freund’s Adjuvant (CFA, 1 mg / ml, 10 µl, i.pl.) was injected into the hindpaw. Vesencumab mAb (7 µg / 10 µl) or IgG 63 304870344v1Attorney Docket No: 243735.000408 Ctrl (7 µg / 10 µl), or EG00229 (30 µM / 10 µl) or PBS (10 µl) (i.pl.) was injected into the inflamed paw 48 h after CFA (Fig. 4H). CFA induced sustained mechanical allodynia and thermal hyperalgesia (Figs.4I-4L). Vesencumab, but not EG00229, reduced thermal hyperalgesia 1 h after treatment (Fig. 4K, Fig. 4L). Vesencumab inhibited mechanical allodynia compared to IgG control, with a maximal effect at 1 h, lasting up to 2 h post-injection (Fig. 4I). EG00229 also partially reversed mechanical allodynia compared with vehicle, with a maximum inhibitory effect at 1 h, lasting for up to 4 h (Fig. 4J). Thus, NRP1 is necessary for the pronociceptive actions of exogenous and endogenous NGF in mice.

[0284] CendR (0.2, 2, 10 µM / 10 µl) also dose-dependently inhibited NGF-induced mechanicalallodynia and thermal hyperalgesia compared to control (Ctrl) peptide (Figs.4M-4P,). CendR at 10 µM strongly inhibited mechanical allodynia and thermal hyperalgesia for 2 h, 2 µM CendR inhibited mechanical allodynia for 4 h and thermal hyperalgesia for 1 h, whereas 0.2 µM CendR had no effect. Example 5. NRP1 controls NGF and TrkA kinase signaling

[0285] NGF primarily signals through TrkA28, a ‘crab claw’-like RTK where NGF binds toextracellular immunoglobulin-like domains to induce conformational changes throughout the RTK dimer that initiate auto- and trans-phosphorylation of intracellular tyrosine residues (e.g., Y490, Y785). To determine whether NRP1 controls NGF-stimulated phosphorylation of TrkA, a proximal and necessary component of TrkA signaling, dissociated mouse DRG neurons were stained with a phospho-specific antibody to TrkA phosphorylation at residue Y785 (equivalent to Y794 in mouse TrkA). NGF (100 nM, 15 minutes) stimulated a ~1.4-fold increase in the intensity TrkA phospho-Y785 immunofluorescence compared to vehicle (Fig.5A, Fig.5B). Pre-incubation with EG00229 (30 µM, 30 minutes) prevented NGF-stimulated phosphorylation of TrkA but had no effect on the basal unstimulated phosphorylation. These results support the hypothesis that NRP1 is necessary for NGF-induced activation of TrkA.

[0286] Activated TrkA stimulates extracellular signal-regulated kinase (ERK) phosphorylation,as well as AKT and phospholipase Cγ. In addition to triggering neuronal development, TrkA- induced ERK signaling contributes to NGF-induced sensitization of nociceptors and pain5. The contribution of NRP1 to ERK activation was monitored in CAD.a cells, a neuron-like cell line modified from the Cath.a catecholaminergic cell line obtained from a murine tumor29. While 64 304870344v1Attorney Docket No: 243735.000408 CAD.a cells lack TrkA expression, they contain p75NTRand NRP1 (Fig.10). To probe NGF / TrkA ERK signaling with high spatial and temporal resolution, Förster resonance energy transfer (FRET) EKAR biosensors targeted to the cytosol or nucleus were coexpressed with human TrkA in CAD.a cells. EKAR biosensors contain a reversible substrate sequence separated by two fluorophores (Fig. 5C). NGF activated ERK in the cytosol and nucleus within 5 minutes for at least 20 minutes (Fig. 5D). NGF-induced ERK activation was concentration-dependent, with a higher potency for nuclear than cytosolic ERK (Fig. 5E, Fig. 5F, Table 2). Pre-incubation with EG00229 (30 µM) did not affect NGF activation of cytosolic ERK (Fig. 5E), but significantly reduced the potency for NGF activation of nuclear ERK (Fig. 5F, Table 2; NGF EC50: DMSO control, ~8 pM; EG00229 ~98 pM; paired t-test, P=0.023). CendR (1 µM, 30 minutes) prevented NGF-stimulated phosphorylation of ERK1 / 2 in neurons (Figs.5K-5L).

[0287] The contribution of NRP1 to NGF-induced ERK signaling was also investigated byNRP1 overexpression with TrkA in HEK293T cells, which express low levels of these proteins. As in CAD.a cells, NGF activated ERK in the cytosol and nucleus of HEK293T cells (Fig. 5G). While NRP1 co-expression did not influence cytosolic ERK signaling (Fig.5H), NRP1 expression significantly enhanced activation of nuclear ERK in response to low (1 pM) NGF concentrations (Fig.5I). The outcomes of nuclear ERK signaling were studied by expression of a transcriptional luciferase reporter, where a luminescent protein is produced downstream of an ERK promoter (Fig. 5C). NGF stimulated concentration-dependent ERK transcriptional activity with significantly higher potency in cells overexpressing TrkA and NRP1 compared to cells expressing TrkA alone (Fig.5J, Table 2; NGF EC50: TrkA alone, 214 pM; TrkA + NRP1, 71 pM; paired t- test, P=0.0004). NGF did not stimulate ERK transcriptional activity in cells expressing NRP1 alone, confirming lack of inherent signaling capability of NRP1 (Fig. 5J). These results support the notion that NRP1 enhances NGF-induced TrkA activation and signaling by pathways that underpin nociception. Example 6. NRP1 is a chaperone that forms a complex with TrkA to control trafficking from the biosynthetic pathway to the plasma membrane

[0288] In addition to directly binding NGF, NRP1 can amplify NGF / TrkA signaling byreceptor / co-receptor interactions with TrkA, akin to its co-receptor function with VEGFR230. Cell surface expression of human TrkA and NRP1 was measured by specific substrate-based labeling 65 304870344v1Attorney Docket No: 243735.000408 of enzymatic tags genetically fused to the extracellular N-terminus of either receptor. SnapTag- TrkA and HaloTag-NRP1 were expressed in HEK293T and CAD.a cells and covalently labeled with membrane-impermeant AlexaFluor®substrates, thereby selectively labeling receptors transported to the cell surface. SnapTag-TrkA and HaloTag-NRP1 were highly co-localized at the cell surface of HEK293T and CAD.a cells, with the latter also showing a high level of co- localization in subcellular compartments (Fig. 6A). Labeling specificity was confirmed in cells expressing TrkA or NRP1 alone (Fig.11).

[0289] To investigate the formation of a heteromeric complex between TrkA and NRP1, BRETwas measured in HEK293T cells expressing NanoLuc-NRP1 and SnapTag-TrkA. Extracellular N- terminal NanoLuc acts as an energy donor to excite a nearby SnapTag. Measurement of BRET between NanoLuc-NRP1 and increasing levels of SnapTag-TrkA revealed a hyperbolic relationship, indicative of assembly of a heteromeric complex between NanoLuc-NRP1 and SnapTag-TrkA (Fig. 6B, Fig. 6C). As a positive control, a hyperbolic BRET signal relationship was also detected between NanoLuc-p75NTR, which is known to interact with TrkA5, and increasing levels of SnapTag-TrkA. In contrast, there was a linear BRET signal between NanoLuc- NRP1 and increasing levels of SnapTag-calcitonin-like receptor (CALCRL), an unrelated transmembrane receptor. To verify whether this relationship was observed with protein expression, data from a representative experiment quantifying cell surface SnapTag labeling also demonstrated a hyperbolic curve between TrkA and NRP1 (Fig.6C). These results suggest that NRP1 and TrkA co-localize at the plasma membrane as a heteromeric complex.

[0290] Recruiting TrkA to the plasma membrane would enable increased access to extracellulargrowth factor. The effect of NRP1 on the cell surface expression of TrkA was determined using the membrane-impermeant SnapTag fluorophore to selectively label cell surface TrkA (Fig.6D). NRP1 co-expression increased level of TrkA at the surface in HEK293T cells by 150 ^ 43% and of CAD.a cells by 109 ^ 4% (Fig.6E, Fig.6G). Enhanced bystander BRET, which capitalizes on the endogenous affinity of pairs of Renilla-tagged proteins to boost sensitivity31, was developed to quantify the effects of NRP1 expression on the localization of TrkA in different subcellular compartments of living cells. TrkA tagged on the C-terminus with Renilla luciferase (Rluc, BRET) was coexpressed in HEK293T or CAD.a cells with proteins resident of the plasma membrane (CAAX), early endosomes (Rab5a), recycling endosomes (Rab4a) and the cis-Golgi apparatus (Giantin) tagged with Renilla green fluorescent protein (RGFP) (Fig. 6H). In HEK293T cells, 66 304870344v1Attorney Docket No: 243735.000408 NRP1 expression significantly increased BRET between TrkA-Rluc8 and RGFP-CAAX and significantly decreased BRET between TrkA-Rluc8 and tdRGFP-Rab5a, tdRGFP-Rab4a and tdRGFP-Giantin (Fig.6I). In CAD.a cells, NRP1 expression did not affect BRET between TrkA- Rluc8 and RGFP-CAAX but significantly decreased BRET between TrkA-Rluc8 and tdRGFP- Rab5a, tdRGFP-Rab4a and tdRGFP-Giantin (Fig. 6J). These results indicate that NRP1 expression causes a redistribution of TrkA from subcellular regions involved in receptor recycling or de novo export to the plasma membrane, consistent with a chaperone function. Control studies confirmed NRP1 was successfully expressed using a HaloTag label, where NRP1 co-expression had no significant effect on overall TrkA expression (Figs.12A-12D). These results suggest that NRP1 acts as a chaperone for TrkA. Example 7. NRP1 enhances NGF-induced TrkA trafficking and dimerization

[0291] Upon NGF stimulation, TrkA traffics to endosomes by clathrin-dependent and -independent mechanisms, a process known to further enable TrkA signaling through NGF / TrkA signalosomes that are retrogradely transported in endosomes or multivesicular bodies to the soma of sympathetic neurons mediate the neurotrophic actions of NGF3,4. To determine the effects of NRP1 on NGF-evoked endocytosis of TrkA, BRET was measured between TrkA-Rluc8 and plasma membrane marker RGFP-CAAX (Fig. 7A). NGF caused a concentration-dependent decrease in TrkA-Rluc8 and RGFP-CAAX BRET (EC50 ~870 pM), with 10-100 nM NGF inducing maximal decrease within 5 minutes that was sustained for 20 minutes, consistent with TrkA endocytosis (Fig.7B, Table 2). Hypertonic (0.45 M) sucrose or the clathrin inhibitor pitstop 2 (30 µM) prevented NGF-induced endocytosis of TrkA (Fig.7C). Co-expression of NRP1 with TrkA enhanced NGF-stimulated endocytosis of TrkA (Fig.7D). This effect of NRP1 was observed at higher NGF concentrations (>10 nM), with minimal effect on the potency of NGF-induced endocytosis of TrkA (Table 2). NGF also stimulated concentration-dependent removal of TrkA from the plasma membrane of CAD.a cells (EC50 ~490 nM; pEC50 = 9.31 ± 0.18, n=4) that was inhibited by hypertonic sucrose and pitstop 2 (Figs. 7E-7G). Knockdown of endogenous NRP1 with siRNA significantly inhibited NGF-induced endocytosis of TrkA in CAD.a cells (Fig. 7G). NRP1 knockdown was confirmed at a protein level using immunofluorescence in CAD.a cells, while there was no effect on TrkA expression (Figs. 12E-12F). Thus, whereas NRP1 overexpression enhances agonist-stimulated endocytosis of TrkA in HEK293T cells, NRP1 67 304870344v1Attorney Docket No: 243735.000408 knockdown has the opposite effect in CAD.a cells. These results are consistent with a chaperone role for NRP1 in NGF-stimulated endocytosis of TrkA.

[0292] Dimerization of TrkA is pivotal for NGF / TrkA signaling5. The contribution of NRP1 toTrkA oligomerization was evaluated by measuring BRET between NanoLuc-TrkA and SnapTag- TrkA in HEK293T cells (Fig. 7H). Expression of increasing amounts of SnapTag-TrkA in HEK293T cells expressing a fixed amount of NanoLuc-TrkA produced a hyperbolic BRET signal, indicating oligomerization (Fig.7I). NGF (30 nM) significantly enhanced this signal, consistent with agonist-evoked TrkA oligomerization. In cells expressing a fixed ratio of SnapTag-TrkA and NanoLuc-TrkA, NGF stimulated a concentration-dependent increase in BRET that was maximal after 5 minutes and sustained for at least 20 minutes (EC5015 nM; Fig.7J, Table 2). While NGF had a lower potency with respect to dimerization than endocytosis, the kinetics of TrkA dimerization and endocytosis were similar. NRP1 overexpression enhanced TrkA oligomerization in response to higher NGF concentrations (>30 nM) (Fig.7K). As dimerization is the first step in RTK activation, with well-established evidence for TrkA signaling from endosomes, these results provide a mechanistic insight into the role of NRP1 in controlling TrkA signaling and trafficking. Example 8. GIPC1 mediates NRP1 / TrkA interactions and NGF-induced pain signaling

[0293] While NRP1 lacks intrinsic catalytic activity, the short cytoplasmic C-terminus interactswith G Alpha Interacting Protein (GAIP) Interacting Protein C-terminus 1 (GIPC1), or synectin, through a PDZ domain32. GIPC1 also interacts with the membrane-proximal regions of TrkA33. Thus, interaction with GIPC1 can underpin the co-receptor function of NRP1 in NGF / TrkA- evoked nociception. Analysis by RNAScope®in situ hybridization revealed that Gipc1 mRNA is expressed by a large proportion of neurons in DRG from mouse (Fig.8A). In humans, Gipc1 was expressed by ~85-100% of neurons expressing Ntrk1 mRNA (Fig.8B, Fig.8C).

[0294] GIPC1 is an intracellular adaptor protein that associates with receptors and channels toregulate their trafficking by interacting with the inwardly-directed myosin VI motor34. To determine whether GIPC1 is necessary for the TrkA chaperone function of NRP1, BRET between TrkA-Rluc8 and RGFP-CAAX was measured in HEK293T cells after GIPC1 knockdown. GIPC1 siRNA inhibited NRP1-induced plasma membrane expression of TrkA (Fig.8D). In CAD.a cells, preincubation with a GIPC1 inhibitor (300 µM CR102335) or a myosin VI inhibitor (50 µM TIP36) reduced NGF-induced TrkA trafficking to the plasma membrane (Fig. 8E, Fig. 8F). Similarly, 68 304870344v1Attorney Docket No: 243735.000408 GIPC1 siRNA inhibited the maximal response of NGF-induced ERK signaling quantified using the downstream transcriptional reporter in CAD.a cells, with no effect on potency (Fig.8G) (Ctrl siRNA pEC50 = 9.58 ± 0.03, GIPC1 siRNA pEC50 = 9.63 ± 0.09, n=5). GIPC1 siRNA also prevented NGF-induced action potential firing in mouse DRG nociceptors, determined by patch- clamp recordings (Fig.8H, Fig.8I). GIPC1 siRNA knockdown was confirmed at the mRNA level in HEK293T cells, CAD.a cells and DRG (Figs.13A-13C).

[0295] To evaluate the role of GIPC1 in NGF-induced nociceptive behavior, GIPC1 or controlsiRNA was administered to mice by intrathecal (i.t.) injection 48 h before NGF intraplantar injection. In mice treated with control siRNA, NGF (50 nM / 10 µl, i.pl.) caused mechanical allodynia and thermal hyperalgesia in the ipsilateral paw within 30 minutes for at least 24 h (Fig. 8J, Fig. 8K). GIPC1 siRNA prevented NGF-evoked mechanical allodynia and thermal hyperalgesia (Fig 8J, Fig.8K). NGF or siRNA administration did not affect withdrawal responses of the contralateral paw to mechanical stimuli (Fig.8L). The nociceptive role of GIPC1 was also investigated in a preclinical model of inflammatory pain in mice. Control or GIPC1 siRNA was administered (i.t.) 48 h after CFA (i.pl.). Knockdown of GIPC1 in DRG was confirmed using RNAScope®in situ hybridization (Fig. 13C, Fig. 13D). While no effect was observed in the contralateral paw (Fig. 8O), GIPC1 siRNA inhibited CFA-induced mechanical allodynia and thermal hyperalgesia after 24 and 48 h (Fig. 8M, Fig.8N). These results reveal a major role for GIPC1 in NGF-evoked pain. Example 9. Evidence that NGF promotes interactions between NRP1 and TrkA

[0296] Microscale thermophoresis (MST) was used to analyze interactions between recombinanthuman NGF and the extracellular domains of recombinant human TrkA and recombinant human NRP1. NGF interacted with His-tagged extracellular NRP1 a1a2b1b2 domains with nanomolar affinity (Kd = 65.64 ± 6.66 nM, n=4, Fig. 1C) which is comparable to known affinities between purified VEGF165a and NRP1 (Kd 9 nM to 120 nM). The data generated herein showed that NGF interacted with His-tagged extracellular TrkA with nanomolar affinity (Kd= 5.49 nM) (Fig.14A). The TrkA extracellular domain interacted with the His-tagged NRP1 extracellular domain with low affinity (Fig.14B). The highest concentrations of TrkA were 6 µM (Fig.14B, left) and 30 µM (Fig.14B, right). Even at the highest concentration, no saturation was reached (Kds ~ 10 µM). The NRP1 extracellular domain was shown to interact with the His-tagged TrkA extracellular domain 69 304870344v1Attorney Docket No: 243735.000408 with moderate affinity (~30-300 nM) (Figs.14C-14D). In the presence of NGF (0.5 µM or 1 µM), the TrkA extracellular domain interacted with the His-tagged NRP1 extracellular domain with high affinity (Kd = 30.2-114 nM) (Figs. 14E-14F). In the presence of NGF (0.5 µM or 1 µM)), the NRP1 extracellular domain interacted with the His-tagged TrkA extracellular domain with moderate affinity (Kd = 30.2-114 nM) (Fig.14G).

[0297] Findings of the present Example demonstrate that NGF directly interacts with NRP1 andTrkA with high affinity (nM). Extracellular domains of NRP1 and TrkA weakly interact with low affinity (> µM). In the presence of NGF, NRP1 and TrkA interact with high to moderate affinity. Inhibitors of NRP1 and TrkA interactions can block NGF-evoked pain. Example 10. Administration of nanoparticles (NPs) encapsulating an NRP1 antagonist for the treatment of pain Evidence that endosomal TrkA signaling mediates NGF-induced sensitization of nociceptors and nociception

[0298] Phospho-TrkA pY490 (i.e., activated TrkA) was detected in endosomes of NGF-treatedmouse dorsal toot ganglia (DRG) neurons (Fig.15A). The contribution of endosomal signaling to NGF-evoked nociception was studied with electrophysiologic and behavioral approaches. NGF increased action potential firing frequency of mouse DRG nociceptors to a current ramp, denoting sensitization (Fig. 15B). The dynamin (Dnm) inhibitor dyngo4a prevented NGF-induced sensitization. Intraplantar injection of NGF induced mechanical allodynia in mice. Intraplantar clathrin inhibitor pitstop2 abrogated NGF-induced mechanical allodynia (Fig. 15C). Intrathecal Dnm1+2+3 siRNA blocked NGF-induced mechanical allodynia (Fig.15D). These results suggest that endosomal signaling of NGF / TrkA contributes to nociception. Dnm-mediated endocytosis contributes to central transmission of NGF-induced nociception. Evidence that NPs target early endosomes of nociceptors and NP-encapsulated NRP1 antagonist blocks NGF-induced nociception

[0299] To study NP uptake, quantum (Q) dots were encapsulated into polymeric PEGylated NPs(50 nm and 200 nm, determined by dynamic light scattering, DLS). Transmission electron microscopy (TEM) revealed Q-dot clustering in the NP core (Fig. 16A, grey dots). Small Q-dot NPs (50 nm) rapidly internalized (30-240 minutes) into Rab5a-GFP (CellLights) early endosomes of NeuN+ve mouse DRG neurons in culture (Fig.16B); larger Q-dot (200 nm) NPs internalized 70 304870344v1Attorney Docket No: 243735.000408 more slowly. The NRP1 antagonist EG00229 was encapsulated into polymeric NPs by Flash NanoPrecipitation (FNP) with hydrophobic ion pairing (HIP). Pamoic acid was used as the counter ion to pair with the guanidino group on EG00229. To form NPs, EG00229, pamoic acid, poly(lactic acid) (PLA, 15kDa) and poly(lactic acid)-b-poly(ethylene glycol, PLA-PEG, 5kDa- 5kDa) were dissolved in a dimethylsulfoxide and tetrahydrofuran mixture to create the solvent stream. The solvent stream was rapidly mixed against three water anti-solvent streams in a multi- inlet vortex mixer to create the NPs. The resulting NP solution was dialyzed against water and sterile filtered. NPs had a DLS intensity weighted diameter of 88 nm (polydispersity 0.14), drug loading of 7.1 wt% EG00229, and drug encapsulation efficiency of 35%. After intraplantar injection, PEG-EG00229 NPs prevented NGF (intraplantar) -induced mechanical allodynia for 2 h (Fig.16C). Empty PEG-^ NPs had no effect. The duration and magnitude of the antinociceptive action of PEG-EG00229 exceeded that of free EG00229 (% inhibition at 3 h after administration: PEG-EG00229, 76%, free EG00229, 46%) (Fig.16D). Evidence for retention of activity of NP-encapsulated biologics

[0300] NGF, TrkA and NRP1 monoclonal antibodies (mAbs) offer superior specificity and areclinically effective. mAbs would not be expected to engage targets in endosomes; they are too large to pass the plasma and endosomal membranes. NP encapsulation of proteins with retention of activity is a challenge. To demonstrate feasibility, a two-step inverse flash nanoprecipitation (iFNP) process was developed in which PEGylated NPs were loaded with the therapeutic protein deoxyribonuclease-1 (DNase-1). NPs had an intensity weight size of 115 nm (DLS) (Fig. 17A). DNase-1 encapsulation efficiency was 96±5% (BCA assay) (Fig. 17B). Zymography gel assay demonstrated retention of DNase-1 activity after NP release (Fig.17C). To demonstrate DNase-1 release and activity in vitro, NPs were incubated with neutrophil extracellular traps comprising extracellular DNA labeled with sytox nuclear dye. Incubation with Dnase-1 NPs resulted in decreased fluorescence indicating DNA degradation by released and active DNase-1 (Fig. 17D, Fig.17E). Example 11. NRP1 inhibitors suppress NGF-induced sensitization of TRPV1 in mouse DRG neurons

[0301] Many algesic receptors, including TrkA, sensitize transient receptor potential vanilloid-1 (TRPV1) on nociceptors58,59. The contribution of NRP1 to NGF-induced sensitization of TRPV1 71 304870344v1Attorney Docket No: 243735.000408 was determined by calcium imaging of mouse DRG neurons. Exposure of neurons to the TRPV1 agonist capsaicin (100 nM) increased [Ca2+]i by 239.3 ± 137.3% (mean ± SD, N=1696 cells) ofbasal, consistent with TRPV1 activation (Fig. 18A). The response to a second capsaicin challenge6 minutes later was reduced to 96.13 ± 23.60% (N=1696 cells) of the first response, indicating TRPV1 desensitization (P<0.001, paired t-test). When neurons were incubated with mouse NGF (100 nM) for 2 minutes before the second challenge, the response to the second capsaicin challenge was amplified to 119.3 ± 45.26% (N=1621 cells) of the first response, denoting TRPV1 sensitization (P<0.001, paired t-test).

[0302] The contribution of NRP1 to NGF-induced sensitization of TRPV1 was examined byincubating DRG neurons with NRP1 antagonists or control reagents before the second capsaicin challenge. EG00229 is a small molecule inhibitor developed to inhibit binding of VEGF-A to the b1 domain of NRP160. EG00229 (3, 10, 30 µM) caused a concentration-dependent inhibition of NGF-induced sensitization of TRPV1 compared to vehicle control (0.1% DMSO) (Figs.-18A- 18B). EG00229 30 µM prevented NGF-induced sensitization of TRPV1, 10 µM EG00229 partially inhibited TRPV1 sensitization, and 3 µM EG00229 was ineffective. To competitively inhibit binding of NGF to NRP1, a peptide fragment of NGF was synthesized that includes the two conserved ‘CendR’ R / KXXR / K motifs within the C-terminus of NGF (underlined, QAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO: 26), corresponds to 96-120 amino acid residues of mature NGF), which were predicted by molecular modeling to interact with the b1 domain of NRP1. A peptide fragment of NGF that was not predicted to interact with NRP1 (ARVAGQTRNITVDPRLFKKRRLRSP (SEQ ID NO: 27), corresponds to 61-85 amino acid residues of immature NGF) was used as a control (Ctrl peptide). The CendR peptide (0.1, 0.3, 1 µM) caused a concentration-dependent inhibition of NGF-induced sensitization of TRPV1 compared to a Ctrl peptide. CendR at 0.3 or 1 µM prevented TRPV1 sensitization whereas 0.1 µM CendR was ineffective (Figs.18C-18D). Vesencumab, a human mAb against the b1b2 domain of NRP161(0.7 µg / ml), prevented NGF-induced sensitization of TRPV1 compared to control IgG (Figs.18E-18F). None of the inhibitors affected the response to capsaicin in neurons that were not treated with NGF. The finding that three mechanistically distinct NRP1 inhibitors reproducibly blocked NGF-induced sensitization of TRPV1 suggests that NRP1 controls the pronociceptive actions of NGF and TrkA, potentially by enhancing the signaling competency of the NGF-TrkA complex that sensitizes TRPV1. 72 304870344v1Attorney Docket No: 243735.000408 Example 12. Designing peptide inhibitors of NGF / TrkA / NRP1 interactions for the treatment of pain

[0303] As evidenced by the above data, NRP1 interacts with both NGF and TrkA and is thus akey mediator of NGF-evoked pain. Biochemical approaches and molecular modeling were used to identify sites of interaction between NGF, TrkA and NRP1. Peptides that mimic interaction sites between NGF and NRP1, and TrkA and NRP1 were synthesized and tested them in assays of pain.

[0304] NRP1 and TrkA Interactions To investigate binding between NRP1 and TrkA, co-immunoprecipitation was applied using lysates of transfected HEK cells and a microarray peptide analysis.

[0305] The extracellular domain of NRP1 binds to TrkA For co-immunoprecipitationexperiments, full-length human TrkA fused to green fluorescent protein (GFP) at the C-terminus was used. This construct was used for immunoprecipitation with an anti-GFP nanobody coupled to agarose resin.

[0306] Potential NRP1 binding partners for TrkA included three different constructs:extracellular domain (ECD) 1 (amino acid residues 22-850), containing the extracellular domains a1a2, b1b2 and c / MAM, with a C-terminal His tag; ECD2 (amino acid residues 22-644), which includes a1a2 and b1b2, with a C-terminal His or Strep tag, and full-length NRP1 fused to mCherry.

[0307] The a1a2 domain, also known as CUB domain, is involved in semaphoring binding andshares homology with complement binding factors C1s / C1r62.

[0308] The b1b2 domain is required for VEGF-A and semaphoring binding and show highstructural similarity to the C1 / C2 domain of coagulation Factors V and VIII. The c or MAM domain is thought to promote NRP1 oligomerization.

[0309] Immunoprecipitation with an anti-GFP nanobody of lysates containing the differentNRP1 ECD constructs showed that NRP1 was present when the TrkA-GFP construct was co- transfected in HEK cells (Fig. 19A, left panel) whereas NRP1 proteins were absent when transfected alone (Fig.19A, right panel), indicating that the NRP1 ECDs specifically bound to TrkA. 73 304870344v1Attorney Docket No: 243735.000408

[0310] Similar results were obtained for the full-length NRP1-mCherry fusion (Fig.19B). NRP1was only present when co-transfected with the TrkA-GFP constructs (first lane vs. last lane in Fig. 19B).

[0311] These results show that TrkA and NRP1 interact and that the a1a2b1b2 domain of NRP1is sufficient for binding to TrkA.

[0312] Because NRP1-mCherry is extremely abundant in the co-immunoprecipitation (Figure19B) a ten-fold lower amount of NRP1-mCherry was also tested (Fig.19B, middle lane).

[0313] However, in this case NRP1 was barely detectable in the lysate (Fig.19B, middle panel).Surprisingly, TrkA also was at very low, a phenomenon that was observed in these cases. It is possible that expression of NRP1 stimulates expression of or stabilizes TrkA (and vice versa).

[0314] TrkA binds to NRP1 via its leucine-rich domain. To determine what part of the TrkAprotein is involved in NRP1 binding, a microarray peptide analysis was employed. A library of 91 15-amino-acid-residue peptides with 11 residues overlap was synthesized and printed on glass microarray slides. As the above results strongly suggested that interaction between NRP1 and TrkA is through their extracellular domains, the peptide library only covered the ECD of TrkA (amino acid #34-407; Figs.20A-20C). For probe a His-tagged NRP1-ECD2 fragment (a.a.# 22- 644) was incubated with the microarray slide at three different concentrations (0.1, 1 and 10 mg / ml). To detect NRP1 binding to peptides, a fluorescently labeled secondary anti-His antibody was used and slides were then scanned using a fluorescence scanner. A heatmap diagram was generated displaying fluorescence intensities wherein binding is equally proportional to the number of units. For examples, higher number of units indicate stronger binding of the peptides to NRP1. (see Material and Methods below for detailed outline).

[0315] As shown in Figs. 20A-20C, two strong “hotspots” (as defined in Material and Methodsbelow) could be detected. They were adjacent and overlapping, with the second hotspot (peptides #18-20) seemingly the stronger of the two (three consecutive peptides showing strong fluorescence even at the lowest dilution of the NRP1 probe).

[0316] The two hotspots both map to the leucine-rich repeats (LRR) of the TrkA protein (Fig.20D). LRRs are a subset of cell adhesion-related motifs and can be found in a variety of transmembrane and secreted proteins involved in axon guidance, target selection, synapse formation etc. (63). LRRs are found in tandem repeats and form curved, horseshoe-like structure which function as excellent protein-protein interaction motifs. 74 304870344v1Attorney Docket No: 243735.000408

[0317] A weaker hotspot was observed in the second IGc2-like domain encompassing peptide#72 and 73 (Figs. 20A-20C). The second IGc2-like domain has been shown to bind nerve growthfactor (NGF) and a high-resolution structure of NGF bound to TrkA found that several amino acid residues in these two peptides are directly involved in binding to NGF (T325, F327, E331, L333 and E334, Fig.24;64). This raises the intriguing hypothesis that NRP1 might be modulating NGF binding to TrkA.

[0318] Effects of Peptide Inhibitors of TrkA and NRP1 interactions on Nociception Theeffects of an NGF fragment (designated CendR peptide), designed to block interactions between NGF and NRP1, on NGF-evoked sensitization of nociceptors and NGF-evoked nociception in mice are described in Example 11. The effects of peptide inhibitors of TrkA and NRP1 interactions were similarly examined.

[0319] Selected peptides Fragments of TrkA were selected from peptide array data and areshown in Figs. 21A-21C.

[0320] NGF-evoked sensitization of transient receptor potential vanilloid 1 (TRPV1)Challenge of mouse DRG neurons with capsaicin, a TRPV1 agonist, evoked an increase inintracellular Ca2+ levels, consistent with TRPV1 activation (Fig. 22). The response to a secondcapsaicin challenge was diminished, indicating TRPV1 desensitization. Incubation with NGF 2 minutes prior to the second capsaicin challenge prevented desensitization, which provides evidence that NGF sensitizes TRPV1, a key mediator of pain.

[0321] Preincubation of neurons with TrkA peptides P15-16, p19-20 or p72-73 all preventedNGF-evoked sensitization of TRPV1 (Fig.22). The control peptide did not prevent NGF-evoked sensitization of TRPV1.

[0322] NGF-evoked mechanical allodynia and thermal hyperalgesia Injection of NGF into thepaw of mice decreased the paw withdrawal threshold to stimulation with von Frey filaments and reduced the withdrawal time to radiant heat, consistent with mechanical allodynia and thermal hyperalgesia, respectively (Fig.23).

[0323] Intraplantar injection of TrkA peptides P15-16 or P18-20 significantly inhibited NGF-evoked mechanical allodynia and thermal hyperalgesia (Fig. 23). The control peptide had no effect. Data and Discussion 75 304870344v1Attorney Docket No: 243735.000408

[0324] NRP1 is identified as a previously unrecognized co-receptor for NGF and TrkA that isnecessary for NGF-induced nociception, demonstrated in both rodent and human tissue. NRP1 enhances NGF / TrkA signaling, where inhibition of NRP1 abrogates the actions of NGF in intact mice, isolated nociceptors and cell lines, while NRP1 overexpression enhances TrkA signaling and trafficking. The results are consistent with evidence that NRP1 is a co-receptor for VEGF- mediated angiogenesis using pharmacological24or genetic37interventions. NRP1 is proposed to promote NGF / TrkA-mediated pain by at least two mechanisms: as a coreceptor that directly binds NGF, and as a chaperone that forms assembles a heterodimeric complex to enhance TrkA trafficking to and from the plasma membrane (Fig.9).

[0325] The coreceptor function of NRP1 is supported by nanomolar affinity bindingmeasurements between recombinant NRP1 and NGF. Supported by molecular modeling, the “CendR” motif of NGF remains exposed when interacting with TrkA28, which could allow assembly of a trimeric NGF / TrkA / NRP1 complex. Despite the glycosylated nature of NRP1, indirect association of NGF with matrix components is unlikely to mediate NGF / NRP1 interactions as NGF does not directly interact with the extracellular matrix38. NRP1 potentiation of VEGFR2 signaling is linked to growth factor binding kinetics26, similar to p75NTRpotentiation of NGF / TrkA binding kinetics39. Further studies of the effects of NRP1 on NGF / TrkA binding kinetics are warranted. Since the NGF / p75NTRbinding site40does not directly conflict with the proposed NGF / NRP1 binding site, a TrkA / p75NTR / NRP1 complex can interact with NGF (e.g., 2:1:1 stoichiometry), although there could be steric clashes.

[0326] The chaperone function of NRP1 is supported by BRET proximity assays showing thatNRP1 and TrkA form a heteromeric complex and that NRP1 re-routes TrkA from the biosynthetic pathway to the plasma membrane and subsequently signaling endosomes. NRP1 colocalizes with TrkA to a greater extent than with VEGFR230. Further studies are required to identify potential NRP1 / TrkA interaction sites. TrkA redistribution to the cell surface would amplify exposure to extracellular NGF. Accordingly, NRP1 potentiates the effects of NGF on TrkA dimerization and endocytosis.

[0327] GIPC1 is another previously unrecognized mediator of NGF / TrkA-evoked pain. GIPC1interacts with TrkA33and NRP132, and can thus scaffold TrkA / NRP1 interactions to facilitate NGF signaling. GIPC1 disruption abrogates NRP1-stimulated translocation of TrkA to the plasma membrane and inhibits NGF-evoked endocytosis of TrkA and ERK signaling, in line with reports 76 304870344v1Attorney Docket No: 243735.000408 in other systems41. Considering GIPC1 regulates the trafficking of many receptors and ion channels, other mechanisms could mediate the antinociceptive effects of GIPC1 knockdown. Crucially, NRP1 and GIPC1 prominently colocalize with TrkA in human nociceptors where that can mediate NGF-induced pain. NRP1 may also serve as a co-receptor for NGF / TrkA signaling from adjacent cells, akin to evidence of VEGFR2 modulation from both ‘cis’ (same cell) and ‘trans’ (neighboring cell) forms42. This intercellular signaling may underlie functional interactions between nociceptors and neighboring cells that contribute to pain (e.g., Schwann cells).

[0328] Electrophysiological and behavioral studies support the essential role of NRP1 andGIPC1 in NGF-induced pain. Two chemically distinct small molecule NRP1 inhibitors and a mAb against the b1 domain of NRP1 prevent NGF-evoked mechanical allodynia and thermal hyperalgesia in mice, providing confidence in specificity. GIPC1 siRNA similarly prevents NGF- induced nociception. NRP1 and GIPC1 inhibition blunts CFA-induced inflammatory nociception, indicating that NRP1 and GIPC1 mediate the nociceptive action of endogenous and exogenous NGF. NRP1 is necessary for NGF-induced action potential firing in both mouse and human DRG neurons, supporting human translation. NRP1 is required for the proximal events of NGF-evoked nociception (TrkA dimerization, phosphorylation, channel activation), as well as sustained components of pain signaling (ERK activation, transcription), both of which mediate nociceptor sensitization43. Although TrkA is the principal pronociceptive NGF receptor, p75NTRalso contributes to NGF-induced pain44and the effect of NRP1 on p75NTRwarrants further study. NGF / TrkA signalosomes transported from nerve terminals to the soma have been studied in compartmentalized sympathetic45and DRG46neuronal cultures, where retrograde NGF / TrkA signaling controls gene expression during the development of sympathetic neurons. Whether endosomal signaling of NGF / TrkA contributes to pain, as observed with G protein-coupled receptors47, deserves study. Further studies are also required to determine whether NRP1 is necessary for NGF-induce expression of pronociceptive transmitters and channels, and whether NRP1 mediates the neurotrophic actions of NGF. NRP1 interacts with semaphorins19as a co- receptor for plexins, which regulate TrkA retrograde signaling48. Accordingly, NRP1 is also highly expressed in axonal growth cones49, as well as small diameter NGF-responsive DRG neurons during neurite sprouting50. Thus, disruption of NRP1 can affect NGF-induced neuronal growth, with implications for the neuroplasticity associated with chronic pain. 77 304870344v1Attorney Docket No: 243735.000408

[0329] NGF is implicated in neuropathic, surgical and cancer pain5, highlighting the need tostudy contributions of NRP1 and GIPC1 across pain pathologies. Upregulation of NRP1 and GIPC1 in painful conditions, such as cancer, can amplify NGF / TrkA signaling. Changes in NRP1 expression have been shown in migraine patients51; though presumed to link to vasculature changes through VEGF signaling, this could mediate migraine pain through NGF52. Given the key role of NGF / TrkA in pain and the challenge of developing TrkA-selective antagonists, NGF mAbs were prioritized for the treatment of pain. While NGF mAbs provided beneficial pain relief to patients with arthritis, rapidly progressing osteoarthritis observed in some patients has precluded FDA approval12. It is unknown whether NRP1 contributes to NGF signaling in bone tissue, considering its expression on osteoblasts53. Similar to the development of partial or biased drugs that ‘fine-tune’ GPCR signaling to reduce adverse effects, targeting NGF signaling via co- receptors / scaffolds can be advantageous for treating patients that suffer from chronic pain without the side effects of systemic NGF sequestration. Identifying the antinociceptive efficacy of vesencumab, in particular, highlights an opportunity to repurpose a biologic developed for cancer for the treatment of chronic pain, facilitating the development of a non-opioid therapeutic targeting NGF signaling through this novel co-receptor. Below are the materials and methods used in the Examples described above.

[0330] Compounds. Recombinant βNGF (R&D Systems or Alomone Labs) and recombinantNRP1 (R&D Systems) were reconstituted according to manufacturer’s instructions. GIPC1 inhibitors (CR1023 or negative control) were from Genscript. Myosin VI inhibitor (2,4,6- triiodophenol) was from Thermo Scientific Chemicals. Unless stated otherwise, in vitro studies used human NGF, whereas ex vivo and in vivo studies used murine NGF. EG00229 (HY-10799) was from MedChemExpress, compound 5 has been described (1), human monoclonal anti-NRP1 antibody (Vesencumab) was from Invitrogen (MA5-41940), and IgG control was from ThermoFisher (02-6502). Fragments of human NGF were from Genscript. The C-endR peptide includes the two conserved ‘CendR’ R / KXXR / K motifs within the C-terminus (underlined, QAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO: 26)) and corresponds to position 96-120 of mature NGF peptide. The control peptide (ARVAGQTRNITVDPRLFKKRRLRSP (SEQ ID NO: 27)) corresponds to position 61-85 of immature NGF peptide. GIPC1 inhibitors (CR1023 or 78 304870344v1Attorney Docket No: 243735.000408 negative control) were from Genscript. Myosin VI inhibitor (2,4,6-triiodophenol) was from Thermo Scientific Chemicals.

[0331] cDNAs. SnapTag-TrkA was cloned with a GSSGAIA spacer (SEQ ID NO: 126) and anN-terminal interleukin-6 signal sequence (IL-6-SnapTag-TrkA). IL-6-NanoLuc-TrkA, IL-6- NanoLuc-p75NTRand TrkA-HA-Rluc8 were cloned using Gibson assembly. HiBiT, a short fragment of NanoLuc54, was cloned on the N-terminus of VEGF165a or C-terminus of βNGF with a GSSG linker (SEQ ID NO: 127). HaloTag-NRP1, SnapTag-NRP1 and NanoLuc-NRP1 were from Dr. Laura Kilpatrick and Prof. Stephen Hill (University of Nottingham). SnapTag-CALCRL was from Alex Thomsen (New York University). RGFP-CAAX (prenylation CAAX box of KRas), tdRGFP-Rab5a, tdRGFP-Rab4a and tdRGFP-Giantin were from M. Bouvier (Université de Montréal). Nuclear and cytosolic EKAR (CFP / YFP) FRET biosensors were from Addgene.

[0332] Cell culture. HEK293T cells were grown in complete Dulbecco’s modified Eagle’smedium GlutaMAXTMsupplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin. CAD.a cells were grown in RPMI 1640 containing 4% FBS, 8% horse serum and 100 U / mL penicillin-streptomycin.

[0333] siRNAs. ONTARGETplus siRNA targeting human GIPC1 (L-019997-00-0010), murineGIPC1 (L-062534-00-0005) or nontargeting control (Ctrl) siRNA (D-001810-10-05) were from Dharmacon (Table 3).

[0334] Transfection of cell lines. HEK293T cells were transfected using polyethylenimine (PEI,Polysciences; 1:6 DNA:PEI) diluted in a 150 mM NaCl solution. CAD.a cells were transfected with Lipofectamine 3000 (according to manufacturer’s instructions) diluted in serum-free DMEM.

[0335] Animals. Experiments were in accordance with the guidelines recommended by theNational Institute of Health, the International Association for the study of Pain, the National Centre for the Replacement, Refinement and Reduction of Animals in Research (ARRIVE) guidelines, and were approved by the New York University Institutional Animal Care and Use Committee. Male C57BL / 6 mice (8-10 weeks, Charles River) were housed four per cage at 22 ± 0.5°C under a controlled 14 / 10 h light / dark cycle with free access to food and water. Mice were randomly assigned to experimental groups; group size was based on previous similar studies. Investigators were blind to treatments.

[0336] Molecular docking. The predicted structure of full-length human NRP1 (UniProt O14786)was obtained from AlphaFold. In the downloaded model, both the transmembrane region and the 79 304870344v1Attorney Docket No: 243735.000408 MAM domains were folded against the a1a2b1b2 globular domains; therefore, domains were removed from the model prior to docking, leaving only the a1a2b1b2 domains. The AlphaFold predicted structure of full-length NGF (UniProt P01138) or the NGF / NRP1 complex was modeled using PIPER55from within the Schrödinger Biologics Suite (Release 2022-1). For PIPER, the NMR structure of the human NGF dimer56contains a C-terminal Arg118. All 20 NMR models were examined and one with the C-terminal residues in an extended conformation was selected. Initial PIPER docking analysis revealed that the orientation of the a1 domain potentially inhibited access of the NGF dimer to the b1 domain. Thus, the position of the NRP1 a1 domain was modified to match that observed in the structure of mouse NRP121. Model rebuilding was performed in COOT with Ramachandran restraints. Models were prepared using the Schrödinger Suite and PIPER docking was repeated using standard mode with a single attractive distance restraint that Arg118 (NGF chain A) and Asp320 (NRP1) sidechains be within 2-4 Å. Other settings were left at default values: standard mode, a maximum of 70,000 ligand rotations to probe, a maximum of 30 poses returned, and output poses were refined. All of the 30 returned clusters had Arg118 in or near to the b1 pocket. Only three clusters had the Arg118 side-chain terminal nitrogens oriented toward the terminal oxygens of Asp320. The largest of these clusters (117 members, PIPER pose energy = 634 kcal / mol, PIPER pose score -66 kcal / mol) was selected for visualization of potential interactions.

[0337] Microscale thermophoresis. Purified NRP1-His (Bio-Techne, 3870-N1) wasfluorescently labeled using the Monolith Protein Labeling kit RED-Tris-NTA (NanoTemper).200 nM of NRP-His in Tris 40 mM, NaCl 250 mM and DTT 1 mM was mixed with NT-647-His- labeling dye (30 minutes, RT). Labeled NRP was centrifugated (15,000 g, 10 minutes, 4°C). NRP1-His (15 nM) was mixed with increasing concentrations of unlabeled NGF (10-10M to 10-5M) in PBS (10 minutes, RT). Thermophoresis was measured using a Monolith NT.115 (NanoTemper), using premium MST capillaries at 100% MST power. Data were analyzed with MO Affinity Analysis software using the Kd model.

[0338] Binding using HiBiT-BRET. HEK293T cells were transfected with SnapTag-NRP1 orSnapTag-NRP1 Y297A (3 µg / 10 cm dish) using PEI. After 24 h, cells were transferred to 96-well plates. The next day, cells were incubated with SNAP-Surface®Alexa Fluor®488 (0.25 µM, 1 h, 37ºC), then washed in HBSS / 0.1% BSA. Separately, HEK293T cells were transfected with IL-6- HiBiT-VEGF165a (3 µg / 10 cm dish) or IL-6-NGF-HiBiT (5 µg / dish). After 48 h, medium was 80 304870344v1Attorney Docket No: 243735.000408 replaced with HBSS / 0.1% BSA to collect secreted growth factor (2 h, 37ºC). Supernatant was diluted (1:2) in HBSS / 0.1% BSA and incubated with recombinant HaloTag-LgBiT protein and furimazine (1:50 final dilution for both; Promega Corporation). NRP1-expressing cells were pre- incubated with vehicle or unlabeled VEGF165a (10 nM, 30 minutes), followed by supernatant with furimazine (1:2, 15 minutes, 37ºC). Luminescence and fluorescence emissions were recorded (Synergy Neo2, Agilent BioTek; BRET1 donor 460 ± 40 nm, acceptor 540 ± 25 nm).

[0339] Information-driven construction of a ternary NGF / TrkA / NRP1 computational model. TheTrkA extracellular domain (ECD) structure from the crystal structure of the NGF / TrkA complex (PDB 2IFG) and the AlphaFold65-derived model of hNRP1 with only a1a2b1b2 domains were used in docking calculations. hNGF has two prospective ‘CendR’ R / KXXR / K motifs within the C-terminus that are important for mediating interactions of other growth factors with NRP1 b1 domains66, 67. The AlphaFold-derived model of soluble hNGF sterically clashed with TrkA when superimposed with the NGF / TrkA crystal structure. Further, the NGF C-terminus CendR118 motifs were missing density in NGF / TrkA crystal structure. Hence, Modeller68was used to add missing structure to NGF in the NGF / TrkA crystal structure and to sample conformations of NGF CendR118 motifs for docking calculations. When modeled on the cell membrane, analysis of the NGF / TrkA crystal structure (PDB 2IFG) revealed that hNGF CendR motif R100FIR103is membrane proximal whereas motif K115AVR118 is membrane distal. hNGF polarity with respect to the membrane was determined by the relative membrane orientation of bound hTrkA ECDs (PDB 2IFG). This analysis identified two probable binding modes of NRP1 to the membrane-tethered NGF / TrkA complex. In mode 1, NRP1 b1 domain is poised to interact with CendR motif R100FIR103. In this mode, a1a2b2 domains are membrane proximal and NRP1 can possibly interact with the membrane. In mode 2, NRP1 b1 domain is poised to interact with CendR motif R115FIR118. This time, the a1a2b2 domains are membrane distal and NRP1 can also possibly interact with the TrkA ECD domains. However, in mode 1, the NGF CendR motif R100FIR103 is at the interface with TrkA Ig-C2 domain and is not accessible for NRP1 binding. Hence, binding mode 2 was computationally explored and analyzed its relevance in a physiological context as outlined below. The NGF / TrkA / NRP1 model was generated in a data-driven approach using HADDOCK 2.4 webserver69. Since TrkA binding to NGF does not utilize CendR motif R115FIR118 of NGF, NRP1 was directly docked to the NGF / TrkA crystal structure. NGF models with solvent-accessible conformations of CendR motif R115FIR118in the NGF / TrkA complex were selected as first docking 81 304870344v1Attorney Docket No: 243735.000408 partners and the AlphaFold-derived NRP1 a1a2b1b2 domain structure as the second docking partner. Knowledge-based restraints in the form of active and passive residues were used as restraints in the docking protocol. In HADDOCK, active residues are solvent-exposed residues directly involved in the interaction between the two proteins followed by solvent-exposed passive residues close to the active residues that may be at the interface70. From NGF / TrkA, residues of CendR motif R115FIR118 and from NRP1, b1 domain residues Y297, D320, S346, T349 and Y353 were specified as active residues. These NRP1 b1 domain residues constitute a C-terminal arginine binding pocket that is known to bind C-terminal arginine in NRP-binding proteins and peptides66,67, 71, 72. All residues within 6.5 Å of active residues were specified as passive residues in both docking partners. Standard docking parameters were used during the rigid-body energy minimization followed by semi-flexible refinement where residues at the interface are allowed to move. Besides docking score, several factors were considered while selecting the docked models. This was to ensure the selection of sterically feasible binding modes of NRP1 to NGF / TrkA complex modeled on the membrane without clashing with either the membrane or TrkA. Although non-standard residues were excluded from docking calculations, compatibility of glycosylated TrkA with NRP1 binding in cellular context was considered while selecting docked models (Fig. 25A). Further, suitable orientations of the NRP1 b2 domains in connection with NRP1 MAM domain were also considered during selection to ensure membrane-tethered NGF / TrkA / NRP1 complex formation were also possible (Fig. 25D). In the different docked poses derived from docking calculations, the NRP1 b1 domain still binds to the NGF C-terminus in a similar manner while NRP1 a1, a2 and b2 domains are oriented differently resulting in poses where one NRP1 molecule can bind to one TrkA monomer (Fig. 25C) or two TrkA monomers at once thereby bridging TrkA dimers (Fig.25B). Explicit water refinement was then performed on the selected docked models using the refinement interface of HADDOCK 2.4 webserver69. The refined ternary complexes were analyzed for molecular interactions at the interfaces using PISA73. Structural superposition of structural complexes for analysis and preparation of figures was made using PyMol74and UCSF ChimeraX75.

[0340] Collection of mouse tissue. Mice were anesthetized (5% isoflurane) and perfused throughthe ascending aorta with PBS and then 4% paraformaldehyde in PBS. DRG (vertebrae L4-L5) were removed, fixed in 4% paraformaldehyde in PBS (1 h, 4°C), cryoprotected in 30% sucrose 82 304870344v1Attorney Docket No: 243735.000408 (24 h, 4°C), and embedded in Optimal Cutting Temperature compound (Tissue Tek). Frozen sections (10-12 μm) were mounted, dried (15 minutes) and stored (-20°C).

[0341] Collection of human tissue. For RNAScope® studies, the collection of DRG fromdeidentified organ donors was reviewed by the Institutional Review Board of the University of Cincinnati (#00003152, Study ID 2015-5302) and deemed to be human subjects exempt. Donor information has been provided57. DRG (L4-L5) were collected in the operating room within 90 minutes of aortic cross clamp and after removal of vital organs. DRG were placed in N-methyl-D- glucamine-artificial cerebrospinal fluid (4°C), dissected and immersion fixed in 4% paraformaldehyde in PBS (overnight, 4°C), cryoprotected in 30% sucrose (24 h, 4°C), and embedded in Optimal Cutting Temperature compound. Frozen sections (14-18 μm) were mounted, dried (15 minutes) and stored (-80°C).

[0342] Immunofluorescence. Mouse tissue was blocked (2% bovine serum albumin, BSA, 0.2%Triton X-100 in PBS; 1 h, room temperature (RT)). Sections were incubated (4ºC, overnight) with monoclonal rabbit anti-TrkA (1:50; Millipore-Sigma, SU0354), and monoclonal mouse anti-NRP1 (1:200; Santa Cruz, A-12) or monoclonal human anti-NRP1 vesencumab (1:500; Invitrogen, MA5- 41940). Slides were washed and incubated with donkey anti-rabbit Alexa Fluor®488 (1:1000) and either donkey anti-mouse or anti-human Alexa Fluor®647 (1:1000, 1 h, RT). Slides were incubated with DAPI (1 μg / ml, 5 minutes) and mounted (ProLong®Gold Antifade). Sections were imaged using a Leica SP8 confocal microscope with HCX PL APO 10x air objective (Leica Microsystems).

[0343] RNAScope® in situ hybridization in mouse and human tissue. RNAScope MultiplexFluorescent Reagent v2 Assay (Advanced Cell Diagnostics Inc.) was carried out for fresh-frozen tissue as recommended by the manufacturer, except for omission of the initial on-slide fixation step with mouse tissue. Probes to Mm-Nrp1(#471621-C3), Mm-Ntrk1 (#435791-C2), Mm-Gipc1 (#1232971-C1), Hs-Nrp1 (#424361-C1) Hs-Ntrk1 (#402631-C2) and Hs-Gipc1 (#1232961-C3) were used. Mouse tissue was incubated with Opal 620 (1:1000, FP1495001KT, Akoya Biosciences) and Opal 520 (FP1487001KT) reagents for fluorescence detection. For human tissue, Tyramide Signal Amplification Plus Cyanine 3 (1:1500) and Cyanine 5 (1:1000) were used to detect the hybridized signals. To detect neurons in mouse tissue, hybridized slides were blocked and incubated with guinea pig anti-NeuN antibody (1:500, overnight, 4°C; EMD Millipore, N90;). Slides were washed and incubated with goat anti-guinea pig Alexa Fluor®647 (1:1000, 45 minutes, 83 304870344v1Attorney Docket No: 243735.000408 RT). Alternatively, neurons were detected using Nissl staining. Slides were washed, incubated with DAPI and mounted. For mouse tissue, sections were imaged using a Leica SP8 confocal microscope with HCX PL APO 40x (NA 1.30) oil objective. The percentage of hybridized positive neurons were quantified and normalized by the total number of neurons. For human tissue, images were captured as stitched Z stacks using Keyence BZ-X800E fluorescence microscope. Cells positive for Ntrk1, Nrp1 and Gipc1 were counted to mark positive cells, where 10+ fluorescent puncta were considered to be positive.

[0344] Human DRG cultures. Human DRG suspension cells were obtained from AnaBiosCorporation. Cells were recovered with a gentle centrifugation at room temperature (3 minutes) and resuspension with DMEM / F12 media containing 10% horse serum, 1% penicillin- streptomycin, 25 ng / ml hNGF and 25 ng / ml GDNF. Cells were seeded on poly-D-lysine (0.1 mg / ml) and laminin (1 mg / ml)-coated 12-mm glass coverslips. Half of the culture medium was replaced with fresh medium every 3 days. All cultures used within 96 h.

[0345] Mouse DRG cultures. C57BL / 6 mice (~3-4 weeks) were anaesthetized (5% isoflurane)and decapitated. Thoracic and lumbar DRG were excised, then ganglia dissociated by collagenase type I (1 mg / ml) and neutral protease (0.62 mg / ml; 45 minutes, 37°C) under gentle agitation. Dissociated cells were pelleted and resuspended in DMEM containing 10% FBS and 1% penicillin-streptomycin. For siRNA transfection, cells were resuspended in Nucleofector® transfection reagent containing GIPC1 or Ctrl siRNA (500 nM) and a plasmid encoding the enhanced green fluorescent protein (eGFP, 1 µg). Cells were electroporated using Amaxa Biosystem (Lonza) with protocol O-003. Neurons were plated on glass coverslips pretreated with poly-D-lysine (0.1 mg / ml) and laminin (0.01 mg / ml; 30 minutes). Neurons were used within 48 h and siRNA transfection verified by eGFP fluorescence.

[0346] Mouse DRG cultures for calcium imaging, C57BL / 6 mice (~4-6 weeks) wereanaesthetized (5% isoflurane). All DRGs were excised and dissociated with collagenase type I (1 mg / ml; 30 min, 37°C), followed by papain (0.125 mg / ml; 30 min, 37°C). Dissociated cells were pelleted and resuspended in DMEM containing 10% FBS and 1% penicillin-streptomycin. Neurons were plated on MatTEK 35 mm dishes (P35G-1.5-14-C) coated with poly-D-lysine (0.1 mg / ml; 20 min; Sigma-Aldrich; P7280) and imaged within 24 h.

[0347] Calcium imaging on mouse DRG neurons. DRG neurons were incubated with Fluo-4AM(1 µM; Tocris; 6255) in a calcium buffer (in mM: 150 NaCl, 2.5 KCl, 2.2 CaCl2.2H20, 1.18 84 304870344v1Attorney Docket No: 243735.000408 MgCl2.6H2O, 10 D-Glucose, 10 HEPES, 0.5% BSA, 1X Probenecid, pH 7.4; 1 h at 37°C). Neurons were continuously perfused (10 ml / minute) with calcium buffer and maintained at 37°C using the Tokai Hit incubation system (ZILCS S / N 172288). Fluorescence was recorded in individual neurons using a Leica DMi8 microscope equipped with a HC PL FLUOTAE 10x (NA 0.30) air objective (Leica Microsystem) and a Leica DFC9000GTC camera. Images were collected every 5 seconds to minimize photobleaching and phototoxicity. After basal recording for 1 minute, neurons were challenged with capsaicin (100 nM) at 1 minute and at 7 minutes. Depolarization was evoked with 50 mM KCl at 13 minutes. Neurons were exposed to mouse NGF (100 nM) or vehicle (control) at 5 minutes. In some experiments, neurons were preincubated with EG00229 (3, 10 or 30 µM) or vehicle (0.1% DMSO, control) for 1 minute before capsaicin. CendR or control peptide (0.1, 0.3 or 1 µM) and Vesencumab mAb or control IgG (0.7 µg / ml) were co-administered with NGF at 5 minutes. Changes in fluorescence were calculated by subtracting the background from the fluorescence intensity at a specified time point (ΔF) and then normalized to the initial fluorescence intensity (F) resulting in ΔF / F.

[0348] Whole-cell patch-clamp recordings of evoked action potentials in mouse DRG neurons.For current-clamp recordings, the external solution contained (mM): 154 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 10 D-Glucose, and 8 HEPES (pH 7.4 adjusted with KOH, and mOsm / L= 300). The internal solution comprised (mM): 137 KCl, 10 NaCl, 1 MgCl2, 1 EGTA, and 10 HEPES (pH 7.3 adjusted with KOH, and mOsm / L= 277). Recordings of action potentials were made at RT in whole-cell patch clamp configuration and current-clamp mode. DRG neurons with a resting membrane potential (RMP) more hyperpolarized than −40 mV, stable baseline recordings, and evoked spikes that overshot 0 mV were used for experiments and analysis. The action potentials were evoked by a ramp pulse from 0-1000 pA in 1 sec for human DRG, or 0-250 pA in 1 sec for mouse DRG. Rheobase was measured by injecting currents from 0 pA with an increment of 50 pA in 100 ms (human) or 10 pA in 50 ms (mouse). Analyses were performed by using Fitmaster software (HEKA) and Origin 9.0 software (OriginLab).

[0349] Whole-cell patch-clamp recordings of Ca2+ and Na+ currents in mouse DRG neurons.Currents were recorded using an EPC 10 Amplifier-HEKA (HEKA Elektronik) with Patchmaster software. DRG neurons were preincubated with EG00229 (30 µM, 30 minutes) or vehicle (0.1% DMSO) and then challenged with NGF (50 nM, 30 minutes). Ca2+currents (ICa2+) and Na+(INa+) currents were recorded as described17,18. In brief, peak Ca2+current was acquired by applying 200- 85 304870344v1Attorney Docket No: 243735.000408 millisecond voltage steps from −70 to +70 mV in 10-mV increments from a holding potential of −90 mV to obtain the current-voltage (I-V) relation. Peak Na+current was acquired by applying 150-millisecond voltage steps from −70 to +60 mV in 5-mV increments from a holding potential of −60 mV to obtain the current-voltage (I-V) relation. Capacitive artifacts were fully compensated, and series resistance was compensated by ∼70 %. Recordings made from cells with greater than a 20% shift in series resistance compensation error were excluded from the analysis. All recordings were made at RT (∼23 °C).

[0350] NGF-evoked nociception. Mouse NGF (50 ng / 10 μl) was administered by i.pl. injectioninto the right hindpaw of mice. EG00229 (MedChemExpress, HY-10799), compound 527, a human monoclonal anti-NRP1 antibody (Vesencumab, Invitrogen, MA5-41940), or IgG control (Ctrl, ThermoFisher 02-6502) was administered (i.pl.) concomitantly with NGF.

[0351] Inflammatory pain. CFA (1 mg / ml) or vehicle (0.9% NaCl) was administered (10 µl,i.pl.) into the right hindpaw of sedated mice (2% isoflurane). NRP1 inhibitors were injected 48 h after CFA.

[0352] Intrathecal administration of siRNA. Mouse GIPC1 or Ctrl siRNA (1.25 µg) was mixedwith in vivo-jetPEI transfection reagent (8:1 PEI:DNA; Polyplus, 201-50G) and administered to conscious mice (5 µl, i.t., L4-L5), 48 h before NGF (i.pl.) or 48 h after CFA (i.pl.). Gipc1 mRNA in DRG (L4-L5) was analyzed by RNAScope® in situ hybridization, 48 h after siRNA injection.

[0353] Nociception assays. Investigators were blinded to treatments. Mechanical allodynia wasassessed by measuring hindpaw withdrawal response to VFF stimulation using the up-and-down method57. The Hargreaves apparatus was used to evaluate hypersensitivity to heat (Ugo Basile)57.

[0354] Phosphorylated TrkA assays. Acutely dispersed mouse DRG neurons were plated ontocoverslips pre-coated with Cell-Tak (30 minutes, 37°C; CB-40240) in DMEM containing 10% FBS. After 18 h, neurons were incubated with a monoclonal NGF antibody (1:500, 4 h, 37°C; MA5-41968) in serum-free DMEM to reduce interference from endogenous NGF. Neurons were washed in HBSS / 0.1% BSA, pre-incubated with EG00229 (30 µM, 30 minutes) or control, then stimulated with vehicle or murine NGF (100 nM, 15 minutes, 37°C). Neurons were fixed with 4% paraformaldehyde in PBS (20 minutes, 4°C), blocked (2% BSA, 0.2% Triton-X-100 in PBS), and incubated with a rabbit mAb to phosphorylated TrkA Y785 / TrkB Y816 (1:500, overnight, 4°C; Cell Signaling, 4168). Neurons were washed in PBS and incubated with donkey anti-rabbit Alexa Fluor®488 (1:1:000, 1 h, RT). Slides were incubated with DAPI, mounted then imaged using a 86 304870344v1Attorney Docket No: 243735.000408 Leica SP8 confocal microscope with HCX PL APO 40x (NA 1.30) oil objective. Mean fluorescence intensity was quantified from regions of interest drawn using a phase contrast image with the maximum projection from four Z planes (0.5 µm thickness). Images were quantified from 34-44 individual cells across three independent experiments using separate mice.

[0355] ERK activity FRET biosensor assays. CAD.a cells were serum-starved and transfectedwith EKAR FRET biosensors (300 ng / well) and human SnapTag-TrkA (150 ng / well). HEK293T- FLPN cells were transfected with FRET biosensors (1 µg / 10 cm dish), SnapTag-TrkA (1 µg), and pcDNA3.1 or HaloTag-NRP1 (2 µg). After 24 h, cells were transferred to 96-well plates and serum-starved overnight. On the day of the assay, cells were washed and incubated in Hanks’ buffered saline solution (HBSS, pH 7.4) containing 0.1% BSA. After 30 minutes (37°C), FRET was measured at 60 s intervals (CLARIOstar, BMG Labtech). After 5 baseline reads, cells were stimulated with NGF (0.1 pM-10 nM) or phorbol 12,13-dibutyrate (PDBu, 10 µM). ^^FRET represents the ratio (YFP / CFP), minus the mean from 5 initial reads and baseline-corrected to vehicle. Area under the curve (AUC) was determined for each replicate.

[0356] ERK transcriptional assays. HEK293T-FLPN cells were transfected with SRE-Luc2P (3µg / 10 cm dish), SnapTag-TrkA (1 µg) and either pcDNA3.1 or HaloTag-NRP1 (2 µg). After 24 h, cells were transferred to 96-well plates. CAD.a cells were transfected with SRE-Luc2P (250 ng / well, 96-well plate), SnapTag-TrkA (150 ng / well) and 100 nM siRNA. After 24 h, cells were serum-starved overnight. Cells were then incubated in HBSS / 0.1% BSA and stimulated with NGF (1 pM-10 nM) or PDBu (10 µM). After 5 h stimulation (37ºC), cells were incubated with luciferin (1:2) and lysis buffer (1:5). After 5 minutes to enable the substrate reaction, luminescence emissions were recorded (CLARIOstar).

[0357] Phosphorylated ERK assays. Mouse DRG neurons were plated onto coverslips pre-coated with poly-D- lysine (0.1 mg / ml; 20 minutes) in DMEM containing 10% FBS. After 18 h, neurons were incubated with a monoclonal NGF antibody (1:500, 4 h, 37°C; MA5-41968) in serum-free DMEM. Neurons were incubated with EG00229 (30 µM), CendR (1 µM), or controls, and vehicle or mouse NGF (100 nM) for 30 minutes at 37°C. Neurons were washed with PBS and fixed with 4% paraformaldehyde in PBS (15 minutes, 4°C), blocked (10% normal donkey serum in PBS) for 1 h, and incubated with a rabbit mAb to phosphorylated 44 / 42 Thr202 / Tyr204 ERK1 / 2 (1:400; Cell Signaling; 4168) and a guinea pig polyclonal anti-NeuN (1:800; Sigma- Aldrich; ABN90) in blocking solution for 2 h at RT. Neurons were washed in PBS and incubated with 87 304870344v1Attorney Docket No: 243735.000408 donkey anti-guinea pig Alexa Fluor®488 (1:1,000) and donkey anti-rabbit Alexa Fluor®568 (1:1,000, 45 min, RT). Slides were incubated with DAPI, mounted, and stored at 4°C. Neurons were imaged using a Leica SP8 confocal microscope with HC PL APO 20x (NA 0.75) air objective (Leica Microsystem), employing identical illumination exposure parameters for all groups. The FIJI-Image J cell counter plug-in was utilized to count the number of NeuN-positive cells and p- ERK / NeuN double-positive cells. Images were quantified from 15-432 individual cells across four independent experiments using separate mice.

[0358] Live imaging. HEK293T cells were transfected with SnapTag-TrkA (1 µg / 10cm dish),HaloTag-NRP1 (2 µg) or both TrkA and NRP1. After 24 h, cells were transferred to 35 mm dishes. CAD.a cells were seeded in 35 mm dishes, serum-starved for 24 h, and then transfected with SnapTag-TrkA (250 µg / 35 mm), HaloTag-NRP1 (500 µg) or both. The next day, proteins were labeled with SNAP-Surface®Alexa Fluor®488 and membrane-impermeant HaloTag® Alexa Fluor® 660 (0.5 µM, 30 minutes, 37ºC). Cells were washed in HBSS and imaged on the Leica SP8 confocal.

[0359] Receptor-receptor BRET. For endpoint studies, HEK293T cells were transfected with afixed concentration of NanoLuc-NRP1, NanoLuc-p75NTRor NanoLuc-TrkA (10 ng / well, 96-well plate) and increasing concentrations of SnapTag-TrkA or SnapTag-CALCRL (0-50 ng / well). For kinetic studies, cells were transfected with NanoLuc-TrkA (10 ng / well), SnapTag-TrkA (25 ng / well) and either pcDNA3.1 or HaloTag-NRP1 (50 ng / well). After 48 h, cells were incubated with SNAP-Surface® Alexa Fluor® 488 (0.5 µM, 30 minutes, 37ºC), washed in HBSS before fluorescence emissions were measured (Synergy Neo2). In some conditions, cells were incubated with NGF (30 nM, 30 minutes, 37ºC) or vehicle. Cells were then incubated with furimazine (10 µM, 5 minutes, Promega). For kinetic studies, cells were stimulated with increasing NGF concentrations (1 pM-100 nM) following 5 baseline reads. Luminescence and fluorescence emissions were quantified (Synergy Neo2; BRET1 filter).

[0360] Quantifying SNAP-Surface® Alexa Fluor® 488. HEK293T cells were transfected withSnapTag-TrkA (25 ng / well, 96-well plate) and pcDNA3.1 or HaloTag-NRP1 (50 ng / well). CAD.a cells were transfected with SnapTag-TrkA (150 ng / well) and either pcDNA3.1 or HaloTag-NRP1 (300 ng / well). Cells were labeled using SNAP-Surface® Alexa Fluor® 488 and HaloTag® Alexa Fluor®660 (0.5 µM, 30 minutes, 37ºC), washed in HBSS / 0.1% BSA and fluorescence emissions were recorded (CLARIOstar). 88 304870344v1Attorney Docket No: 243735.000408

[0361] BRET trafficking. HEK293T cells were transfected with TrkA-HA-Rluc8 (15 ng / well, 96-well plate) and a fluorescent marker (15 ng / well) for the plasma membrane (RGFP-CAAX), early endosome (tdRGFP-Rab5a), recycling endosome (tdRGFP-Rab4a) or cis-Golgi apparatus (tdRGFP-Giantin). In some conditions, cells were co-transfected with pcDNA3.1 or HaloTag- NRP1 (30 ng / well), or 100 nM siRNA. Alternatively, CAD.a cells were transfected with TrkA- HA-Rluc8 (125 ng / well) in the absence or presence of HaloTag-NRP1 (250 ng / well), as well as RGFP-CAAX, tdRGFP-Rab5a, tdRGFP-Rab4a or tdRGFP-Giantin (150 ng / well). After 48 h, cells washed in HBSS / 0.1% BSA were pre-incubated (30 minutes, 37ºC) with hypertonic sucrose (0.45 M), Pitstop 2 (30 µM; Abcam), an inhibitor of GIPC1 (CR1023, 300 µM) or its negative control (CR2055), or an inhibitor of myosin VI (TIP, 50 µM). Cells were incubated with coelenterazine purple (25 µg / ml, 10 minutes, Nanolight). For kinetic studies, 5 baseline reads were measured, then cells were stimulated with NGF (1 pM-100 nM), using 30 nM NGF for endocytic inhibitors. BRET ratios were recorded every 45 s (Synergy Neo2; BRET2 filter: donor 410 ± 80 nm, acceptor 515 ± 30 nm). ^^BRET represents BRET in the presence of agonist, minus the vehicle BRET over time. For endpoint studies lacking agonist stimulation, BRET ratios were baseline-corrected to TrkA-HA-Rluc8 expression alone (100%).

[0362] Quantitative PCR (qPCR). RNA was isolated from cells or snap-frozen tissues usingDirect-zol RNA MiniPrep Kit (Zymo Research, R2050). cDNA was prepared using MultiScribe Reverse Transcriptase (Thermo Fisher, 4311235). cDNA was amplified for 40 cycles by qRT-PCR using the QuantStudio 3 Real-Time PCR System and TaqMan Fast Advanced PCR Mastermix (4444556). Primers to hTrkA (Hs01021011_m1), mTrkA (Mm01219406_m1), hNgfr (Hs00609976_m1), mNgfr (Mm00446296_m1), hGIPC1 (Hs00991802_m1), mGIPC1 (Mm00457561_m1), or GAPDH (Mn99999915_g1) were used. The relative abundance of mRNA was calculated as described57.

[0363] Data analysis. Data presented as mean ± SEM using GraphPad Prism (8.0). Imagesprocessed using ImageJ. Illustrations were produced using Adobe Illustrator, Mol*Viewer or BioRender. Concentration-response data fit using non-linear regression analysis as log vs. response (three parameters) to determine EC50values. Differences were assessed using paired or unpaired t-test for two comparisons, and 1- or 2-way ANOVA and Tukey’s, Dunnett’s or Šidák’s post-hoc test for multiple comparisons. P<0.05 was considered significant at the 95% confidence level. 89 304870344v1Attorney Docket No: 243735.000408

[0364] Reagents used in Example 12. Fugene 4K Transfection Reagent (#E5911) was fromPromega Corporation. Pierce IP Lysis Buffer (#87787), rabbit polyclonal anti-TrkA antibody(#PA5-120786) and SuperSignal™ West Femto Maximum Sensitivity Substrate Kit (#34095)were from ThermoFisher Scientific. Secondary horseradish peroxidase-conjugated anti-mouse (#NA9310) and anti-rabbit (#9340) were from Cytiva Lifesciences. Mouse monoclonal anti-NRP1 antibody (#60067) and ChromoTek GFP-Trap®Agarose (#gta) was from Proteintech Group, Inc. Expression vectors pRB30-GFP (#165481), pRB30 (#165480) and pcDNA-mCherry (#128744) were from Addgene.

[0365] DNA constructs. Full-length human TrkA (UniProt #P04629-1) was cloned into pRB30-GFP by ligation-independent cloning (LIC) to generate a TrkA-GFP fusion protein. The extracellular domain (ECD) of human NRP1 (UniProt #O14786-1) from amino acid residues 22- 850 was inserted into pRB30 by LIC to generate NRP1-ECD1 fused to a C-terminal 10xHis tag (SEQ ID NO: 131). The ECD region from amino acid residues 22-644 was similarly cloned into pRB30 to form a C-terminal His tag fusion (NRP1-ECD2) or a C-terminal STREP tag fusion (NRP1-ECD2S). Full-length NRP1 was cloned into pcDNA-mCherry by seamless cloning (Gibson) to generate a NRP1-mCherry fusion protein.

[0366] Co-immunoprecipitation. Adherent HEK cells in 30 mm dishes (400,000 cells / dish) weretransfected with a total of 2 µg DNA (1 µg for each construct for co-immunoprecipitation) using Fugene transfection reagent according to the manufacturer’s instructions. Two days post- transfection cells were lysed with 400 µl of lysis buffer. After centrifugation (17,000x g, 20 minutes, 4°C) supernatant was removed and a sample was dissolved in SDS gel loading buffer (120 mM Tris-Cl, pH=7; 20% glycerol; 4% SDS;0.04% bromphenolblue; 5% ^-mercaptoethanol). The balance was incubated with 20 µl of ChromoTek anti-GFP resin overnight at 4°C. Resin was washed twice with 1 ml wash buffer (10 mM Tris-Cl, pH=8; 150 mM NaCl; 0.05% NP-40) and resuspended with 100 µl of SDS gel loading buffer. Protein samples were run on 4-20% gradient gels, transferred to PVDF membranes using an iBlot 2 transfer device (ThermoFisher Scientific) and then incubated overnight with primary antibody. Protein bands were detected using secondary horseradish peroxidase-conjugated antibody and a chemiluminescent kit.

[0367] Peptide microarray binding studies. Peptide microarray binding studies were performedby JPT Peptide Technologies (Germany). Ninety-one overlapping 15-amino-acid-residues(overlap of 11 residues) covering the TrkA extracellular domain from residue 34 to 407 (Fig. 20)90 304870344v1Attorney Docket No: 243735.000408 were printed on a glass microarray slide. In addition, full-length human and mouse IgG and His- tag peptides were included as negative and positive controls, respectively. The microarray slide was then incubated with a His-tagged NRP1 (ECD2, as described in the section of DNA constructs above) at three different concentrations (10, 1 and 0.1 µg / ml) for one hour at 30°C. Following the sample incubation, a fluorescently labeled secondary antibody specific to the His-tag was added at a concentration of 1 µg / ml and left to react for 1 hour. Additionally, control incubation using only the detection antibody, without any samples, was performed in parallel on the same microarray slide to assess false-positive binding to the peptides. After washing and drying, microarrays were scanned using a high-resolution fluorescence scanner (Axon Genepix Scanner 4300 SL50). Laser settings and applied resolution were identical for all performed measurements. The resulting images were analyzed and quantified using the spot-recognition software GenePix (Molecular Devices). For each spot, the mean signal intensity was extracted (ranging from 0 to 65,535 arbitrary units). To compare binding regions across the individual incubations, a heatmap diagram for all proteins of interest was generated, displaying fluorescence intensities in a manner wherein < 1,000 units, indicating no binding to >60,000 units, indicating very strong binding; Fig. 20A). “Binding hotspots” were defined as regions with at least two consecutive peptides that showed moderately strong binding (>30,000 units) at two or three dilutions of the antigen (NRP1- His; Fig.20A).

[0368] NGF-evoked sensitization of mouse dorsal root ganglion (DRG) neurons. Mice (~4-6weeks) were anaesthetized (5% isoflurane). All DRGs were excised and dissociated with collagenase type I (1 mg / ml; 30 min, 37°C), followed by papain (0.125 mg / ml; 30 min, 37°C). Dissociated cells were pelleted and resuspended in DMEM containing 10% FBS and 1% penicillin-streptomycin. Neurons were plated on MatTEK 35 mm dishes (P35G-1.5-14-C) coated with poly-D-lysine (0.1 mg / ml; 20 min; Sigma-Aldrich; P7280) and imaged within 24 h. DRG neurons were incubated with Fluo-4AM (1 µM) in a calcium buffer (in mM: 150 NaCl, 2.5 KCl, 2.2 CaCl2.2H20, 1.18 MgCl2.6H2O, 10 D-Glucose, 10 HEPES, 0.5% BSA, 1X Probenecid, pH 7.4; 1 h at 37°C). Neurons were continuously perfused (10 ml / min) with calcium buffer and maintained at 37°C using the Tokai Hit incubation system (ZILCS S / N 172288). Fluorescence was recorded in individual neurons using a Leica DMi8 microscope equipped with a HC PL FLUOTAE 10x (NA 0.30) air objective (Leica Microsystem) and a Leica DFC9000GTC camera. Images were collected every 5 s to minimize photobleaching and phototoxicity. After basal recording for 1 min, 91 304870344v1Attorney Docket No: 243735.000408 neurons were challenged with capsaicin (100 nM) at 1 minute and at 7 minutes. Depolarization was evoked with 50 mM KCl at 13 min. Neurons were exposed to mouse NGF (100 nM) or vehicle (control) at 5 minutes. In some experiments, neurons were preincubated with peptide inhibitors or control peptides for 30 minutes before NGF challenge. Changes in fluorescence were calculated by subtracting the background from the fluorescence intensity at a specified time point (ΔF) and then normalized to the initial fluorescence intensity (F) resulting in ΔF / F.

[0369] NGF-evoked nociception. Peptide inhibitors or control peptides were administered byintraplantar (i.pl.) injection into the right hindpaw of mice. Thirty minutes later, mouse NGF (50 ng / 10 μl) injected into the right hindpaw. Investigators were blinded to treatments. Mechanical allodynia was assessed by measuring hindpaw withdrawal response to VFF stimulation using the up-and-down method. The Hargreaves apparatus was used to evaluate hypersensitivity to heat (Ugo Basile).

[0370] Peptide inhibitors. Peptides corresponding to sequences of canonical TrkA(uniprot.org / uniprotkb / P04629 / entry#sequences) determined to interact with NRP1 by microarray analysis were synthesized by Thermofisher. Figure 21 shows the selected sequences. The following peptides were synthesized: Peptide TrkA 15,16: LRGLGELRNLTIVKSGLRFVAPDAF (corresponds to human TrkA a.a. # 87-111) (SEQ ID NO: 28) Peptide TrkA 18, 20: KSGLRFVAPDAFHFTPRLSRLNLSF (corresponds to human TrkA a.a. # 100-124) (SEQ ID NO: 29) Peptide TrkA 72, 73: WLFNGSVLNETSFIFTEFLEPAANE (corresponds to human TrkA a.a. # 315-339) (SEQ ID NO: 30) Control peptide: NPFEFNPEDPIPVSFSPVDTNSTSG (corresponds to human TrkA a.a. # 381- 405) (SEQ ID NO: 31)

[0371] The control peptide is a sequence of TrkA not found to interact with NRP1.

[0372] The peptide sequences are highly conserved between species. Peptide TrkA15, 16 andPeptide TrkA 18, 20 differ by one amino acid between human and mouse. Peptide TrkA 72, 73 differs by 3 amino acids between human and mouse. 92 304870344v1Attorney Docket No: 243735.000408 Table 1. Gating properties of Ca2+and Na+currents recorded from mouse DRG neurons Total Ca2+DMSO (0.1%) EG00229 (30 μM) NGF (50 nM) EG00229 + NGF Activation ) )* ) idualcells (in parentheses) to the Boltzmann equation; V1 / 2midpoint potential (mV) for voltage- dependent of activation or inactivation; k, slope factor. Data were analyzed with 1-way ANOVA with Tukey’s multiple comparisons. † p=0.0115 for V1 / 2 activation of EG00229 vs DMSO (0.1%); †† p=0.0131 for V1 / 2 activation of NGF vs EG00229; * p= 0.0175 for V1 / 2 inactivation of EG00229 + NGF vs DMSO (0.1%). Table 2. Pharmacological parameters (pEC50) of NGF derived in at human TrkA, in the absence or presence of NRP1 modulation CAD.a cells Control NRP1 inhibition93 304870344v1Attorney Docket No: 243735.000408 Cytosolic ERK (FRET) 10.64 ± 0.41 (5) 10.53 ± 0.17 (5) Nuclear ERK (FRET) 11.09 ± 0.33 (5) 10.01 ± 0.38 (5) * n uman TrkA.aues are mean cacuae rom non- near regressons o e aa rom independent replicates (in parentheses), each with triplicate wells. Quantified as the negative antilog of the half- maximal effective concentration (pEC50 = -logEC50). * p<0.05, *** p<0.001. Paired t-test. Table 3. Summary of siRNA sequences used in this study Target Sequence Control UGGUUUACAUGUCGACUAA (SEQ ID NO: 14)94 304870344v1Attorney Docket No: 243735.000408 References 1. Levi-Montalcini, R. & Hamburger, V. Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J Exp Zool 116, 321-361 (1951). doi.org:10.1002 / jez.1401160206 2. 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[0373] The present invention is not to be limited in scope by the specific embodiments describedherein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. 101 304870344v1Attorney Docket No: 243735.000408

[0374] All patents, applications, publications, test methods, literature, and other materials citedherein are hereby incorporated by reference in their entirety as if physically present in this specification. 102 304870344v1

Claims

Attorney Docket No: 243735.000408 Claims1. An isolated peptide comprising a fragment of nerve growth factor (NGF) comprising twomotifs each having the amino acid sequence R / KXXR / K wherein X is any amino acid, or a pharmaceutically acceptable salt, fragment or derivative thereof.

2. The isolated peptide of claim 1, wherein the isolated peptide comprises the amino acidsequence QAAWRFIRIDTACVCVLSRKAVRRA (SEQ ID NO: 26) or QAAWRFIRIDTACVCVLSRKAVRR (SEQ ID NO: 124), or a sequence having at least 80% identity thereto.

3. The isolated peptide of claim 1 or 2, wherein the isolated peptide consists essentially of theamino acid sequence of SEQ ID NOs: 26 or 124.

4. The isolated peptide of any one of claims 1-3, wherein the isolated peptide consists of theamino acid sequence of SEQ ID NOs: 26 or 124.

5. The isolated peptide of any one of claims 1-4, wherein the isolated peptide comprises oneor more of the following characteristics: a. binds to neuropilin-1 (NRP1);b. inhibits the interaction between NRP1 and NGF; andc. inhibits NGF-induced sensitization of transient receptor potential vanilloid 1(TRPV1).

6. An isolated peptide comprising the amino acid sequenceLRGLGELRNLTIVKSGLRFVAPDAF (SEQ ID NO: 28), KSGLRFVAPDAFHFTPRLSRLNLSF (SEQ ID NO: 29), or WLFNGSVLNETSFIFTEFLEPAANE (SEQ ID NO: 30), or an amino acid sequence having at least 80% identity to any of SEQ ID NOs: 28-30, or a pharmaceutically acceptable salt, fragment or derivative thereof. 103 304870344v1Attorney Docket No: 243735.0004087. The isolated peptide of claim 6, wherein the isolated peptide consists essentially of anamino acid sequence of any one of SEQ ID NOs: 28-30.

8. The isolated peptide of claim 6 or 7, wherein the isolated peptide consists of an amino acidsequence of any one of SEQ ID NOs: 28-30.

9. The isolated peptide of any one of claims 6-8, wherein the isolated peptide comprises oneor more of the following characteristics: a. binds to neuropilin-1 (NRP1);b. inhibits the interaction between NRP1 and tropomyosin receptor kinase A (TrkA);c. prevents NGF activation of TrkA;d. inhibits NGF-induced sensitization of transient receptor potential ion channelsincluding TRPV1, vanilloid 1 (TRPV1); e. prevents NGF-induced sensitization of pain sensing nerves; andf. inhibits NGF-evoked mechanical allodynia and thermal hyperalgesia.

10. The isolated peptide of any one of claims 1-9, wherein the isolated peptide comprises oneor more modification to improve affinity and / or metabolic stability.

11. The isolated peptide of claim 10, wherein the isolated peptide comprises one or more Damino acids or unnatural amino acids.

12. A pharmaceutical composition comprising one or more isolated peptides of any one ofclaims 1-11, and a pharmaceutically acceptable carrier or excipient.

13. A method for treating chronic pain in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of an inhibitor of neuropilin-1 (NRP1).

14. The method of claim 13, wherein the inhibitor of NRP1 inhibits expression or function ofNRP1 protein. 104 304870344v1Attorney Docket No: 243735.00040815. The method of claim 13 or claim 14, wherein the inhibitor of NRP1 inhibits the interactionbetween NRP1 and tropomyosin receptor kinase A (TrkA).

16. The method of claim of any one of claims 13-15, wherein the inhibitor of NRP1 inhibitsthe interaction between NRP1 and nerve growth factor (NGF).

17. The method of any one of claims 13-16, wherein the inhibitor of NRP1 reduces NGF-evoked nociception.

18. The method of any one of claims 13-17, wherein the inhibitor of NRP1 is a small molecule,an siRNA, an shRNA, an antisense oligonucleotide, an antibody or antigen-binding fragment thereof, a peptide inhibitor, or a site-specific nuclease.

19. The method of claim 18, wherein the peptide inhibitor comprises one or more isolatedpeptide of any one of claims 1-11.

20. The method of claim 18, wherein the small molecule that inhibits NRP1 is N2-[[3-[(2,1,3-Benzothiadiazol-4-ylsulfonyl)amino]-2-thienyl]carbonyl]-L-arginine, or an analog or derivative thereof.

21. The method of claim 18, wherein the antibody or antigen-binding fragment is a humanantibody, a humanized antibody, a chimeric antibody, a murine antibody, a monoclonal antibody, a single chain antibody, a bispecific antibody or antigen-binding fragment thereof, a bi-epitopic antibody or antigen-binding fragment thereof, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a VH domain, or a nanobody.

22. The method of claim 21, wherein the antibody or antigen-binding fragment is a bispecificantibody.

23. The method of claim 21 or claim 22, wherein the bispecific antibody comprises a firstantigen-binding domain that binds to NRP1 and a second antigen-binding domain that binds to TrkA. 105 304870344v1Attorney Docket No: 243735.00040824. The method of claim 21 or claim 22, wherein the bispecific antibody comprises a firstantigen-binding domain that binds to NRP1 and a second antigen-binding domain that binds to NGF.

25. The method of claim 21, wherein the antibody or antigen-binding fragment that inhibitsNRP1 is Vesencumab, or an antigen-binding fragment thereof.

26. The method of claim 18, wherein the siRNA that inhibits NRP1 comprises a nucleotidesequence GAAUUGCUGUGGAUGAUAU (SEQ ID NO: 1), AGUAAGAGGUGUCAUCAUU (SEQ ID NO: 2), CCACAAGGUUCAUCAGGAU (SEQ ID NO: 3), GGAAUGUUCUGUCGCUAUG (SEQ ID NO: 4), CGAUAAAUGUGGCGAUACU (SEQ ID NO: 22), GGACAGAGACUGCAAGUAU (SEQ ID NO: 23), GUAUACGGUUGCAAGAUAA (SEQ ID NO: 24), AAGACUGGAUCACCAUAAA (SEQ ID NO: 25), or a modified version, a fragment, or a combination thereof.

27. The method of any one of claims 13-26, further comprising administering to the subject atherapeutically effective amount of an inhibitor of G Alpha Interacting Protein (GAIP) Interacting Protein C-terminus 1 (GIPC1).

28. The method of claim 27, wherein the inhibitor of GIPC1 inhibits expression or function ofGIPC1 protein.

29. The method of claim 27 or claim 28, wherein the inhibitor of GIPC1 reduces NRP1-induced plasma membrane expression of TrkA.

30. The method of any one of claims 27-29, wherein the inhibitor of GIPC1 reduces NGF-evoked nociception.

31. The method of any one of claims 27-30, wherein the inhibitor of GIPC1 is a small molecule,an siRNA, an shRNA, an antisense oligonucleotide, a peptide inhibitor, or a site-specific nuclease. 106 304870344v1Attorney Docket No: 243735.00040832. The method of claim 31, wherein the siRNA that inhibits GIPC1 comprises a nucleotidesequence GCACUCGGGCUCACCAUCA (SEQ ID NO: 5), GGCCGUACCUUC ACGCUGA (SEQ ID NO: 6), GCAAGGCCUUCGACAUGAU (SEQ ID NO: 7), CUGGAGAGUUACAUGGGUA (SEQ ID NO: 8), GCAUCGAGGGCUUCACUAA (SEQ ID NO: 9), CGUCGGCCUUUGAGGAGAA (SEQ ID NO: 10), GUGGAUGACUUGCUAGAGA (SEQ ID NO: 11), GCUGAGGCCUUCCGACUAC (SEQ ID NO: 12), or a modified version, a fragment, or a combination thereof.

33. The method of claim 31, wherein the peptide inhibitor that inhibits GIPC comprises theamino acid sequence N-myristoyl-PSQSSSEA (SEQ ID NO: 13), or a modified version or a fragment thereof.

34. The method of any one of claims 13-33, wherein the chronic pain is an inflammatory pain,a neuropathic pain, a cancer pain, or a postoperative pain.

35. The method of claim 34, wherein the inflammatory pain is inflammatory bowel disease,irritable bowel syndrome, pancreatitis, arthritis, or migraine.

36. The method of claim 34, wherein the neuropathic pain is neuropathic pain secondary tonerve injury and trauma, diabetic neuropathy, viral neuropathy (e.g., trigeminal neuralgia), or chemotherapy-induced peripheral neuropathy.

37. The method of claim 34, wherein the cancer pain is associated with oral cancer, non-smallcell lung cancer, mesothelioma, melanoma, head and neck cancer, breast cancer, ovarian cancer, prostate cancer, renal cancer, liver cancer, or colorectal cancer.

38. The method of any one of claims 13-37, wherein the administration of the inhibitor is viasystemic or local delivery.

39. The method of claim 38, wherein the administration of the inhibitor is via systemicdelivery.

40. The method of claim 38, wherein the administration of the inhibitor is via local delivery.107 304870344v1Attorney Docket No: 243735.00040841. The method of claim 40, wherein the local delivery is via oral, intraocular, intrathecal,intranasal, intracolonical, intraluminal, intraintestinal, intracisternal, intraventricular, epidural, intratumoral, or intraarticular delivery, or any combination thereof.

42. The method of any one of claims 13-41, wherein the inhibitor is formulated in ananoparticle or a liposome.

43. The method of any one of claims 13-42, further comprising monitoring a pain levelexperienced by the subject before, during, and / or after the administering of the inhibitor.

44. The method of claim 43, wherein the monitoring comprises measuring one or more painparameters associated with chronic pain in the subject.

45. The method of claim 44, wherein the one or more pain parameters are selected from painduration, pain radiation pattern, pain severity, pain quality, degree of pain level fluctuation, frequency of pain remissions, and level of function of the subject.

46. The method of claim 44 or claim 45, wherein the measuring comprises assessing the painlevel experienced by the subject using a pain intensity scale and / or a pain questionnaire.

47. The method of any one of claim 27-46, wherein the inhibitor of NRP1 is administeredbefore, simultaneously with, or after the administration of the inhibitor of GIPC1.

48. The method of any one of claims 13-47, further comprising administering an additionaltreatment to the subject.

49. The method of claim 48, wherein the additional treatment comprises administering anonsteroidal anti-inflammatory drug (NSAID), acetaminophen, a local anesthetic, a benzodiazepine, capsaicin, an antidepressant, an anti-seizure medication, an anti-epileptic medication, a Cox-2 inhibitor, an opioid, a muscle relaxant, a steroid, an anticonvulsant, a triptan, dihydroergotamine, a beta blocker, a calcium channel blocker, a calcitonin gene- related peptides antagonist, a serotonin and norepinephrine reuptake inhibitor (SNRI), onabotulinumtoxinA, Lasmiditan, or an anti-nausea drug, or a combination thereof. 108 304870344v1Attorney Docket No: 243735.00040850. The method of any of claims 13-49, wherein the subject is a mouse.

51. The method of any of claims 13-49, wherein the subject is a human.109 304870344v1

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